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Virus-Mediated Delivery of the Fmr1 Gene as a Tool for the
Treatment of Fragile X Syndrome
by
Shervin Gholizadeh Moghaddam
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Department of Pharmaceutical Sciences University of Toronto
© Copyright by Shervin Gholizadeh Moghaddam, 2016
ii
Virus-Mediated Delivery of the Fmr1 Gene as a Tool for the
Treatment of Fragile X Syndrome
Shervin Gholizadeh Moghaddam
Doctor of Philosophy
Department of Pharmaceutical Sciences
University of Toronto
2016
Abstract
Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a trinucleotide
repeat expansion in the FMR1 gene that codes for fragile X mental retardation protein (FMRP).
The goal of the present study was to determine whether restoring FMRP expression in the brains
of the Fmr1 knockout (KO) mouse model of FXS could correct the disorder. Initially, we
investigated major factors affecting tropism, expression level, and cell-type specificity of adeno-
associated viral vectors (AAV), including encapsidation of different AAV serotypes, promoter
selection, and the timing of vector administration, using intra-cerebroventricular injections of
AAV vectors expressing enhanced green fluorescent protein. Our results favored the use of
AAV serotype 9 (AAV9) vectors containing the neuron-selective synapsin-1 promoter, injected
into the lateral ventricles of neonatal mice. To determine if FMRP expression in the central
nervous system could reverse phenotypic deficits in the Fmr1 KO mouse model of FXS, we
used a single-stranded AAV9 that contained a major isoform of FMRP. The vector was
delivered to the brain via a single bilateral intra-cerebroventricular injection into neonatal Fmr1
KO mice on postnatal day 5 (in the first phase of the study) or 0-1 (in the second phase).
Transgene expression and behavioral assessments were conducted either 22-26 or 50-56 days
post-injection. Western blotting and immunocytochemical analyses of AAV-FMRP-injected
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mice revealed neuron-specific FMRP expression in the striatum, hippocampus, retrosplenial
cortex, and cingulate cortex. AAV-FMRP injections reversed the pathologically elevated
repetitive behavior and the deficit in social dominance behavior seen in Fmr1 KO mice in phase
1, as well as the elevated startle response and lower anxiety in phase 2. These results provide
the first proof-of-principle that gene therapy can correct specific behavioral abnormalities in the
mouse model of FXS.
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Table of Contents
Chapter 1. Introduction .................................................................................................................. 1
1.1. Fragile X syndrome ............................................................................................................. 1
1.2. FMRP expression in the brain of the wild-type mouse ....................................................... 3
1.3. FMRP: Structure and molecular functions ......................................................................... 4
1.4. The mGluR theory of fragile X syndrome .......................................................................... 7
1.5. Prospects of gene therapy application for FXS ................................................................... 9
1.6. Viral vectors for gene delivery into the CNS .................................................................... 10
1.7. Adeno-associated viral vectors ......................................................................................... 13
1.8. Subsequent steps in AAV cell entry and implications for gene delivery into the CNS .... 15
1.9. Routes of AAV administration into the CNS ................................................................... 15
1.9.1. Intra-cranial Administration ....................................................................................... 17
1.9.2. Intra-vascular Administration..................................................................................... 19
1.9.3. Intra-CSF .................................................................................................................... 21
1.9.4. Retrograde transport delivery ..................................................................................... 22
1.10. Timing of AVV administration ....................................................................................... 22
1.11. Promoter selection .......................................................................................................... 24
1.12. AAV transport within the CNS ....................................................................................... 26
1.13. Hypotheses and objectives .............................................................................................. 26
Chapter 2. Materials and Methods .............................................................................................. 28
2.1. Animals ............................................................................................................................. 28
2.2. AAV vectors ..................................................................................................................... 28
2.3. Vector injections ............................................................................................................... 29
2.3.1. i.c.v. injections in neonatal mice ................................................................................ 29
2.3.2. Stereotaxic injections in juvenile mice ....................................................................... 30
2.4. Behavioral analysis ........................................................................................................... 30
2.4.1. Locomotor activity measurements ............................................................................. 30
2.4.2. Marble Burying for stereotypic behavior ................................................................... 31
2.4.3. Ultrasonic vocalizations ............................................................................................. 31
2.4.4. Tube test of social dominance .................................................................................... 31
2.4.5. Elevated plus maze test of anxiety ............................................................................. 32
2.4.6. Pre-pulse inhibition for sensorimotor gating .............................................................. 32
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2.4.7. Audiogenic Seizures ................................................................................................... 33
2.5. Tissue preparation, immunocytochemistry and confocal microscopy .............................. 34
2.5.1. Semi-quantitative and quantitative analysis of transgene expression in the brain ..... 35
2.6. Quantitative western blotting ............................................................................................ 36
2.7. Statistical analyses ............................................................................................................ 37
Chapter 3. Expression of FMRP protein in neurons and glia of the developing and adult mouse
brain ............................................................................................................................................. 38
3.1. Specific hypotheses, objectives and rationale ................................................................... 38
3.2. Gradual reduction in the number of FMRP-positive cells from the early postnatal period
to adulthood ............................................................................................................................. 38
3.3. Predominant neuronal expression of FMRP throughout development ............................. 39
3.4. Developmental expression of FMRP in astrocytes, microglia and oligodendrocyte
precursor cells .......................................................................................................................... 41
Chapter 4. Transduction efficiency of adeno-associated viral vectors expressing eGFP after
intra-cerebroventricular administration in neonatal and juvenile mice ....................................... 47
4.1. Specific hypotheses, objectives and rationale ................................................................... 47
4.2. AAV9-CMV-eGFP targets astrocytes in neonatal mice but neurons in juvenile mice .... 48
4.3. Use of the synapsin-1 promoter to achieve neuron-specific transduction ........................ 50
4.4. Low transduction efficiency of AAV5-GFP vectors ........................................................ 54
Chapter 5. FXS gene therapy-Phase 1: Reduced phenotypic severity following adeno-associated
virus mediated Fmr1 gene delivery in postnatal day 5 Fmr1 mice.............................................. 57
5.1. Specific hypotheses, objectives and rationale ................................................................... 57
5.2. Quantification of FMRP expression levels following neonatal i.c.v. injection of AAV-
FMRP in postnatal day 5 mice ................................................................................................. 59
5.3. Distribution and cellular selectivity of transgene expression ........................................... 60
5.4. Phase 1 - Behavioral analyses ........................................................................................... 63
Chapter 6. FXS gene therapy-Phase 2: ........................................................................................ 71
Fmr1 transgene delivery in postnatal day 0 Fmr1 mice .............................................................. 71
6.1. Specific hypotheses, objectives and rationale ................................................................... 71
6.2. Quantification of FMRP expression levels following neonatal i.c.v. injection of AAV-
FMRP in postnatal day 0 mice ................................................................................................. 73
6.2.1. Levels of FMRP substrates in AAV-FMRP-treated mice .......................................... 77
6.3. Distribution and cellular selectivity of transgene expression ........................................... 79
6.4. Phase 2 - Behavioral Analyses .......................................................................................... 86
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Chapter 7. Discussion ................................................................................................................. 93
7.1. FMRP expression in the developing and adult brain ........................................................ 93
7.2. Gene therapy as a potential therapeutic avenue for the treatment of Fragile X syndrome 96
7.3. Major factors affecting cellular tropism, efficiency and distribution of AAV-induced
transduction .............................................................................................................................. 97
7.4. FMRP transgene expression in the brains of Fmr1 KO mice ........................................... 99
7.5. Behavioral effects of FMRP restoration in the brains of Fmr1 KO mice ....................... 101
7.6. Possible contributions of aberrant MeCP2 expression to pathological abnormalities in
FXS ........................................................................................................................................ 104
7.7. Concluding remarks and future directions ...................................................................... 107
References .............................................................................................................................. 111
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List of Publications
1. Hampson DR, Gholizadeh S, Pacey LK. Pathways to drug development for autism
spectrum disorders. Clin Pharmacol Ther. 2012 Feb;91(2):189-200. Review.
2. Gholizadeh S, Tharmalingam S, Macaldaz ME, Hampson DR. Transduction of the
central nervous system after intra-cerebroventricular injection of adeno-associated viral
vectors in neonatal and juvenile mice. Hum Gene Ther Methods. 2013 Aug;24(4):205-
13.
3. Gholizadeh S, Arsenault J, Xuan IC, Pacey LK, Hampson DR. Reduced Phenotypic
Severity Following Adeno-Associated Virus-Mediated Fmr1 Gene Delivery in Fragile X
Mice. Neuropsychopharmacology. 2014 Jul 7.
4. Gholizadeh S, Halder SK, Hampson DR. Expression of fragile X mental retardation
protein in neurons and glia of the developing and adult mouse brain. Brain Research.
2015 Jan 30;1596:22-30.
5. Arsenault J, Gholizadeh S, Pacey LK, Halder SK, Koxhioni E, Hampson DR. Viral
vector-mediated FMRP transgene expression: from phenotypic rescue in Fragile X mice
to pathological over-expression (Manuscript submitted, 2016)
viii
List of Tables
Table 1. Comparison between fragile X patients and Fmr1 KO mice .......................................... 2
Table 2. Clinical trials for gene therapy using AAV vectors in neurological disorders. ............. 11
Table 3. Comparison of AAV viral vectors against the ideal vector for gene delivery to the CNS.
..................................................................................................................................................... 11
Table 4. Primary receptors and gene delivery tropism of different AAV serotypes in the
mammalian CNS .......................................................................................................................... 16
Table 5. Promoters commonly used for gene delivery into the CNS. ......................................... 25
Table 6. Quantitative analysis of cell type-specific expression of FMRP in the cingulate cortex
and corpus callosum. ................................................................................................................... 39
Table 7. Semi-quantitative analysis of cell type-specific FMRP expression in the developing and
adult mouse brain. ........................................................................................................................ 40
Table 8. Analysis of AAV-eGFP vector transduction patterns in discrete areas of the brain after
i.c.v. delivery in PND 5 or PND 21 mice. ................................................................................... 56
Table 9. Summary of the main differences in the experimental design between Phase 1 and
Phase 2 ......................................................................................................................................... 73
Table 10. Quantitative analysis of AAV-FMRP transduction in cingulate cortex. ..................... 82
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List of Figures
Fig. 1. FMR1 gene expansion and FMRP protein structure .......................................................... 5
Fig. 2. FMRP shuttling between the nucleus and cytoplasm ......................................................... 7
Fig. 3. Major factors affecting tropism, transduction and transgene expression properties of
AAV ............................................................................................................................................. 17
Fig. 4. The brain barriers in the developing and adult brain........................................................ 20
Fig. 5. AAV9 tropism in the CNS at different developmental stages ......................................... 24
Fig. 6. Expression of FMRP in neurons of the WT mouse brain. ............................................... 41
Fig. 7. Expression of FMRP in the astrocytes of mouse brain. ................................................... 44
Fig. 8. Expression of FMRP in oligodendrocyte precursor cells in the mouse brain. ................. 45
Fig. 9. Expression of FMRP in microglia. ................................................................................... 46
Fig. 10. Schematic diagrams depicting (A) the AAV-eGFP vector constructs used and (B)
bilateral intra-cerebroventricular injections on postnatal days 5, and 21. ................................... 48
Fig. 11. AAV-eGFP transgene expression in the mouse striatum. .............................................. 51
Fig. 12. AAV-eGFP transgene expression in the retrosplenial cortex. ....................................... 52
Fig. 13. AAV-eGFP transgene expression in hippocampus. ....................................................... 53
Fig. 14. AAV-eGFP transgene expression in the cerebellum. ..................................................... 55
Fig. 15. Fmr1 gene delivery (Phase 1): Overview of viral vector construction, experimental plan
for injections, and behavioral analyses. ....................................................................................... 58
Fig. 16. Western blots of FMRP expression in control and AAV-FMRP-treated mice. ............. 60
Fig. 17. Immunocytochemical analysis of AAV-FMRP transduction in discrete brain regions . 62
Fig. 18. FMRP transgene expression in the striatum, hippocampus, cingulate cortex, and
retrosplenial cortex 61 days after i.c.v. administration of AAV-FMRP on PND 5. .................... 65
Fig. 19. Quantitative analysis of AAV-FMRP transduction in cingulate cortex. ........................ 66
Fig. 20. Summary of behavioral analyses of the short arm of the study. .................................... 67
Fig. 21. Summary of the behavioral results from the long arm of the study. .............................. 68
Fig. 22. Correlation between the number of marbles buried and FMRP transgene expression
levels in the cerebral cortex. ........................................................................................................ 70
Fig. 23. Fmr1 gene delivery (Phase 2): Overview of experimental plan for injections, and
behavioral analyses ...................................................................................................................... 73
Fig. 24. Analysis of GFAP following AAV-FMRP injections in WT animals. .......................... 74
Fig. 25. Western blots of FMRP expression in AAV-FMRP-treated KO and WT mice. ........... 76
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Fig. 26. Characterization of MeCP2 as an mRNA substrate for FMRP and quantification of
MeCP2 levels in AAV-treated WT and KO mice. ...................................................................... 79
Fig. 27. Immunocytochemical analysis of AAV-FMRP transduction in discrete brain regions of
AAV-treated KO mice. ................................................................................................................ 80
Fig. 28. Immunocytochemical analysis of FMRP over-expression in different brain regions of
AAV-SYN-Fmr1-treated WT mice. ............................................................................................ 84
Fig. 29. Quantitative analysis of FMRP over-expression in AAV-SYN- Fmr1- treated WT mice.
..................................................................................................................................................... 85
Fig. 30. Analysis of locomotor activity. ...................................................................................... 88
Fig. 31. Analysis of sensorimotor gating. .................................................................................... 90
Fig. 32. Analysis of anxiety, stereotypical behavior, and courtship behavior. ............................ 92
Fig. 33. Overlapping characteristics of FXS, Rett syndrome, FMR1 duplication syndrome, and
MeCP2 duplication syndrome. .................................................................................................. 106
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Abbreviations
AAV Adeno-associated virus
BBB blood brain barrier
CBA chicken β-actin
CMV cytomegalovirus
CNS central nervous system
CSF cerebrospinal fluid
eGFP enhanced green fluorescent protein
FMRP fragile X mental retardation protein
FXS fragile X syndrome
GFAP glial fibrillary acidic protein
ITR inverted terminal repeat
KO knockout
LTD long-term depression
mGluR metabotropic glutamate receptors
PBS phosphate-buffered saline
PND postnatal day
SYN synapsin
WPRE woodchuck hepatitis post-transcriptional regulatory element
WT wild-type
1
Chapter 1. Introduction
1.1. Fragile X syndrome
Fragile X syndrome (FXS) is a genetic disorder and a leading cause of cognitive impairment
and autism (Bagni et al., 2012). The disorder is caused by a pathological CGG trinucleotide
triplet repeat expansion in the 5’ untranslated region of the FMR1 gene, located on the X
chromosome. The CGG repeat range in unaffected individuals is 5-55, while expansions of 200
or more result in hyper-methylation of the FMR1 gene and FXS. The highly expanded CGG
repeat also causes the transcribed mRNA to form RNA-DNA heteroduplexes (Colak et al.,
2014), which together with gene hyper-methylation severely reduces or abrogates the expression
of the encoded protein, fragile X mental retardation protein (FMRP). Trinucleotide expansion
in the intermediate range of 55 – 199 repeats results in the formation of toxic intra-nuclear
inclusions, a mild reduction of FMRP, and is associated with the neurodegenerative disorder
Fragile X-associated Tremor and Ataxia Syndrome (Bagni et al., 2012; LaFauci et al., 2013;
Ludwig et al., 2014). Reduced FMRP expression has also been reported in other mental
disorders including autism, schizophrenia, bipolar disorder, and major depression (Fatemi and
Folsom, 2011).
Patients with FXS experience a wide range of symptoms including cognitive deficits, social
anxiety, attention deficit and hyperactivity disorder, repetitive stereotyped behaviors, seizures,
and sensory hypersensitivity (Wijetunge et al., 2013). Parallel to human FXS, Fmr1 knockout
(KO) mice also display deficits consistent with an “autistic phenotype”. Fmr1 KO mice display
hyperactivity, reduced ultrasonic vocalizations during courtship, increased repetitive/stereotypic
behaviors, increased susceptibility to audiogenic seizures, and reduced social interactions (Pacey
et al., 2009) (Table 1). Furthermore, numerous studies have demonstrated altered synaptic
plasticity in animal models of the disorder. Studies in mice lacking FMRP revealed disrupted
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ocular dominance plasticity in visual cortex (Dolen et al., 2007) and impaired synaptic plasticity
in barrel cortex (Harlow et al., 2010), and auditory cortex (Kim et al., 2013a). Hypersensitivity
to auditory stimuli in humans with FXS and mice lacking FMRP suggests that the synaptic
plasticity in the auditory system may also be perturbed by the loss of FMRP (Chen and Toth,
2001; Kim et al., 2013a; Rotschafer and Razak, 2014). Fmr1 KO mice also show deficient fear
memory (Zhao et al., 2005; Neuwirth et al., 2015), impaired cerebellar eyeblink
conditioning (Koekkoek et al., 2005; Tobia and Woodruff-Pak, 2009; Reeb-Sutherland and Fox,
2015), inhibitory avoidance learning (Dolen et al., 2007; Yuskaitis et al., 2010b; Michalon et al.,
2014). Drosophila and zebrafish mutants lacking FMRP also have an impairment in long-term
memory (Bolduc et al., 2008; Bolduc et al., 2010; Kanellopoulos et al., 2012) and avoidance
learning (Ng et al., 2013), respectively.
Table 1. Comparison between fragile X patients and Fmr1 KO mice Fragile X patients Fmr1 Knockout mice
Mutation Expansion CGG repeat in 5′
untranslated region of exon 1
Insertion neomycin cassette in
exon 5
FMRP expression Very low or absent Absent
Physical features Elongated face with
prominent ears
Craniofacial abnormalities
(Heulens et al., 2013)
Macro-orchidism Macro-orchidism
Cognitive and
behavioural features
Intellectual disability
(IQ < 70)
Mild learning and memory
deficits
Autistic features Repetitive, perseverative
digging; Impaired social
behaviour
Hyperactivity Increased locomotor activity
Anxiety Decreased non-social anxiety
(e.g. in elevated plus maze) and
increased social anxiety
Epileptic seizures Increased susceptibility to
audiogenic seizures
3
Deficit in sensorimotor gating Altered sensorimotor gating
(acoustic startle response and
pre-pulse inhibition)
Neuroanatomical
features
Increased density and
immature morphology of
dendritic spines
Age- and brain-region-
dependent spine abnormalities
Comparison of the physical, neuro-anatomical and behavioral features in fragile X patients
and Fmr1 KO mice.
1.2. FMRP expression in the brain of the wild-type mouse
FMRP is ubiquitously expressed, most abundantly in brain, testes, and ovaries (Devys et al.,
1993; Hinds et al., 1993; Ascano et al., 2012). In the adult mouse brain FMRP is abundantly
expressed in most neurons throughout the CNS. In neurons, the protein is primarily located in
the cytoplasm, including in the cell body, dendrites and axons and in synaptic spines where it
plays a role in spine maturation (Cruz-Martín et al., 2010). A portion of total cellular FMRP is
also present in the nucleus, where its role is less well-characterized. FMRP is highly abundant
in “fragile X granules” in neuronal axons and pre-synaptic terminals where it apparently
regulates recurrent neuronal activity (Akins et al., 2012). FMRP is also present in neural stem
cells (Luo et al., 2010) where it has been shown to control hippocampal-dependent neurogenesis
and learning in the mature brain (Guo et al., 2011).
In contrast to the expression of FMRP in neurons of the adult brain, relatively little is known
about the types of glial cells that express FMRP during CNS development. FMRP was reported
in primary cultures of rodent astrocytes (Yuskaitis et al., 2010a), and in the mouse hippocampus,
FMRP is expressed in astrocytes within the first week of birth and then its expression declines to
low or undetectable levels (Pacey and Doering, 2007). These findings highlight the important
role for FMRP expression in astrocytes during early postnatal weeks, which coincide with the
peak of synaptogenesis. It is also present in oligodendroglia (Wang et al., 2004) and
4
oligodendrocyte precursor cells in the immature cerebellum where it appears to be a factor in the
proper progression of myelination (Pacey et al., 2013).
1.3. FMRP: Structure and molecular functions
The primary transcript of the mouse Fragile X Mental Retardation-1 gene (Fmr1) spans 39
kb, and is composed of 17 exons (Eichler et al., 1993). Similar to many transcripts in the CNS,
it undergoes alternative splicing and possesses alternative transcription start sites such that at
least 12 isoforms are generated (Tassone et al., 2011; Brackett et al., 2013). Isoform 1 is the
longest form and codes for a protein of 632 amino acids and a molecular weight of 71
kilodaltons. Isoform 1 is also known to account for about 40% of total FMRP in the human
brain (Huang et al., 1996). FMRP isoform 1 is the full-length protein which contains both a
nuclear localization signal (NLS) and a nuclear export signal (NES), and sites for post-
translational modification through phosphorylation and methylation (Sittler et al., 1996; Ceman
et al., 2003). Furthermore, nuclear localization of FMRP is isoform dependent (Dury et al.,
2013). Lack of the NES signal, in FMRP isoforms 4,6,10 and 12, results in predominant nuclear
localization of these FMRP isoforms, indicating its functional significance (Sittler et al., 1996;
Dury et al., 2013). Interestingly, isoforms 6 and 12 are also associated with Cajal bodies in the
nucleus (Dury et al., 2013). In contrast, the most common FMRP isoforms (isoforms 1 and 7)
which are associated with the translation machinery (Ascano et al., 2012), are mainly
cytoplasmic (Dury et al., 2013). These observations suggest that the nuclear FMRP isoforms
might have independent functions from the dominant cytoplasmic FMRP isoforms.
5
Fig. 1. FMR1 gene expansion and FMRP protein structure
Expansion of CGG repeats in the 5' UTR region of the fragile X mental retardation 1 (FMR1)
gene that encodes FMRP underlies FXS. Repeats that contain >200 copies (full mutation) lead
to loss of FMRP expression.
FMRP is an mRNA binding protein that controls the expression of hundreds of genes in the
CNS (Darnell et al., 2011). FMRP has three RNA-binding domains, including two K homology
domains (KH1 and KH2) and an arginine-glycine-glycine (RGG) box, and binds a subset of
neuronal mRNAs (Darnell et al., 2005) (Fig. 1). KH domains bind tertiary motifs in mRNAs
which are generally known as the “kissing-complexes” (Darnell et al., 2005). Furthermore, the
RGG boxes recognize stem-G-quartet loops, potentially in a methylation-dependent mechanism
(Blackwell et al., 2010). The presence of nuclear localization and export signals suggests that
FMRP is a nucleo-cytoplasmic shuttling protein involved in transport of a subset of RNAs from
the nucleus to ribosomes (Eberhart et al., 1996; Fridell et al., 1996; Feng et al., 1997), however
it has been shown that FMRP binds its cargo mRNAs in the nucleus (Kim et al., 2009).
FMRP binds to more than 800 different identified gene transcripts in mouse brain tissue and
in human cell lines (Darnell et al., 2011; Ascano et al., 2012). This is equivalent to
6
approximately 4% of total mRNAs that are expressed in human brain (Darnell et al., 2011). For
many of these substrates, FMRP has been shown to act as a translational suppressor (Wang et
al., 2012). However, FMRP has been shown to also act as a positive modular of protein
translation for some of its mRNA substrates (Bechara et al., 2009), possibly by enhancing
mRNA stability (Zalfa et al., 2007) or through potentiating the actions of the translational
activating gene Sod1 (Bechara et al., 2009). Interestingly, many of the mRNA targets of FMRP
encode pre- and post-synaptic proteins, including several proteins that are implicated in other
autism spectrum disorders, indicating molecular communalities between FXS and other
neurodevelopmental disorders (Darnell et al., 2011; Ouwenga and Dougherty, 2015).
Despite FMRP is commonly acknowledged as a regulator of mRNA translation, the precise
mechanism by which FMRP influences the translational machinery remains to be further
identified. Considering the fact that FMRP co-sediments with polyribosomes, it was initially
believed that FMRP might inhibit translation by blocking elongation (Tamanini et al., 1996;
Ceman et al., 2003). This hypothesis received strong support in a more recent study by the
Darnell group (Darnell et al., 2011). It was further shown that FMRP binds the vast majority of
its 842 mRNA substrates within the coding sequence, instead of the 5′ or 3′ untranslated regions.
Ribosomal run-off assays confirmed that FMRP forms a complex with its mRNA targets and
stalled ribosomes (Darnell et al., 2011). Furthermore, mounting evidence suggests that FMRP
stalls ribosomes and represses translation via interaction with the RNA-induced silencing
complex including several micro-RNAs (Kelley et al., 2012).
7
Fig. 2. FMRP shuttling between the nucleus and cytoplasm
FMRP enters the nucleus by its nuclear localization signal and forms a messenger
ribonucleoprotein (mRNP) complex together with its target mRNAs. FMRP also interacts with
members of the RNA-induced silencing complex, including micro-RNAs and this messenger
ribonucleoprotein complex is transported to the synapses transporting dendritically localized
mRNAs. After synaptic stimulation, regulated by mGluR signalling pathway, the transported
mRNA targets get detached from FMRP and are locally translated in dendrites. FMRP interacts
with its cargo mRNAs either directly or through non-coding RNAs (ncRNA), and micro-RNAs
(miRNA) (Fig. adapted from Bagni and Greenough, 2005).
1.4. The mGluR theory of fragile X syndrome
The mGluR theory is one of the most prominent theories of the underlying cause for fragile
X symptoms. This theory postulates that increased activation of group 1 metabotropic
glutamate receptors (mGluRs) in FXS, and its aberrant downstream molecular consequences
contribute to many molecular and symptomatic abnormalities of FXS. Local dendritic protein
synthesis is essential for many forms of long-term changes in synaptic strength or synaptic
plasticity, mechanisms that are believed to underlie learning and memory (Santoro et al., 2012)
8
Activation of mGluRs triggers long term depression in the CA1 region of hippocampus
(Huber et al., 2002), which is dependent on the rapid de novo synthesis of new proteins in
synapses. FMRP serves as a brake on the synthesis of synaptic mRNAs that encode long-term
depression (LTD) proteins, including proteins that mediate α-amino-3-hydroxyl-4-isoxazole
propionic acid receptor (AMPA-R) internalisation and stabilise LTD. In Fmr1 mice, mGluR-
LTD is exaggerated and no longer dependent on new protein synthesis, suggesting that the
LTD-related proteins are already expressed at adequate levels (Hou et al., 2006; Nosyreva and
Huber, 2006). Interestingly, postnatal FMRP expression in Fmr1 KO mouse reduces the
mGluR-LTD magnitude and retrieves its dependence on new protein synthesis.
Numerous studies in mouse, zebrafish and drosophila models of FXS have supported the
validity of mGluR theory (for a review see Bhakar et al., 2012). Furthermore, the presumption
that reducing mGluR activity by pharmacological and genetic interventions can normalize FXS
behaviors has been validated in a wide range of animal studies (reviewed in Berry-Kravis, 2014;
Pop et al., 2014). Administration of a potent and selective mGluR5 antagonist, 2-methyl-6-
(phenylethynyl)-pyridine (MPEP) in Fmr1 mice, normalized susceptibility to audiogenic seizure
(Yan et al., 2005; Thomas et al., 2012), impaired eyelid conditioning (Koekkoek et al., 2005),
motor hyperactivity (Yan et al., 2005; Min et al., 2009), defective pre-pulse inhibition and
increased repetitive behavior (Thomas et al., 2012). Furthermore, MPEP corrected several
molecular phenotypes in Fmr1 mice including increased AMPA receptor internalization
(Nakamoto et al., 2007), increased protein synthesis (Osterweil et al., 2010), and increased
dendritic spine density (Su et al., 2011).
The promising results from the use of mGluR antagonists in animal models of FXS strengthened
the rationale and paved the way for the use of these agents in clinical trials. However, the
compelling pre-clinical evidence for the therapeutic benefits of mGluR antagonists in rodent
9
models of the disease did not translate in human patients in clinical trials. A number of phase 2
clinical trials of mGluR5 antagonists such as fenobam demonstrated some phenotypic
improvement including reduced anxiety and hyper-arousal, improved pre-pulse inhibition, and
higher accuracy in a performance task (Berry-Kravis et al., 2009) and improved adaptive skills
(Berry-Kravis et al., 2008). From these trials, only a few advanced to phase 3, including the
studies investigating the efficacy of selective mGluR5 antagonists such as mavoglurant
(AFQ056), RG7090, and STX107. However, these trials were all terminated in 2014, mostly
due to lack of efficacy. There are a number of reasons why the clinical trials may have failed,
including inappropriate inclusion criteria, inadequate outcome measures, inadequate drug dosing
or development of drug tolerance (Mullard, 2015). The failure of these clinical trials of
mGluR5 antagonists, together with the failure of the GABAB agonist R-baclofen, has prompted
consideration of other therapeutic approaches.
1.5. Prospects of gene therapy application for FXS
Considering the extensive expression of FMRP in the developing brain, it is obvious that
FXS is not merely a bi-product of aberrant mGluR signalling. Accordingly it can be presumed
that certain defects in FXS pathophysiology might be less reversible in response to mGluR
antagonists (Thomas et al., 2011; Thomas et al., 2012). Given the plethora of genes whose
expression is regulated by FMRP, a priori, it may be expected that restoring FMRP expression
in the CNS could provide a more comprehensive reversal of the disorder compared to targeting
single molecules (e.g. mGluR5). The possibility of restoring normal brain function after
introduction of exogenous FMRP into the brain has previously been attempted. Using an AAV5
vector coding for FMRP, Zeier et al, (2009) reported that injections directly into the
hippocampus of five week old Fmr1 mice resulted in correction of the abnormally enhanced
hippocampal long-term synaptic depression (Zeier et al., 2009); however, no other analyses of
10
phenotypic rescue were carried out and immunocytochemical analysis showed localized
transgene expression only in the hippocampus.
When weighing the possibility of developing a gene therapy approach for FXS, significant
barriers need to be overcome before achieving sufficient therapeutic benefits. In FXS there is
no recognizable, circumscribed locus of pathology, necessitating global delivery of the
transgene in the brain. Furthermore, it is reasonable to presume that cell-specific transgene
expression in KO mice must mimic the WT FMRP expression pattern to provide the maximum
therapeutic benefit. Although FMRP is primarily expressed in neurons of adult WT mice, it is
also present in astrocytes, oligodendrocyte precursor cells, and microglia during the early and
mid-postnatal developmental stages of brain maturation (Gholizadeh et al., 2015). Furthermore,
identifying the therapeutic window for essential levels of FMRP protein in the brain which
translates to rescue of behavioral abnormalities without causing deleterious effects is essential.
1.6. Viral vectors for gene delivery into the CNS
Viruses are among the most efficient biological agents naturally capable of entering cells
through transfection and delivering genetic cargo into the nucleus and possess properties that are
crucial for gene delivery. Recent advancements in viral vector technology have been
encouraging for the development of treatments for disorders of the central nervous system
(CNS). Gene therapy offers promise for treating monogenic neurological disorders, as it can
target the fundamental cause of the loss of functionality in affected cell types within the CNS
through gene replacement. Several viral vectors have been utilized for CNS gene delivery,
including vectors derived from adeno-associated virus (AAV), retrovirus, adeno-
virus and herpes simplex virus. Each of these vectors offers unique solutions to the challenges
of CNS gene delivery. The properties that an ideal vector should possess for gene delivery into
11
the CNS include high transduction efficiency in the target tissue together with cell type specific
tropism and minimal transduction of “off-target” cells, tissues or organs (Table 3).
Table 2. Clinical trials for gene therapy using AAV vectors in neurological
disorders.
Disease Transgene Trial
Number Phase Status
Published
Results
Parkinson’s
disease
GDNF NCT01621581 1 Recruiting N/A
Parkinson’s
disease
hAADC NCT02418598 2 Recruiting N/A
Parkinson’s
disease
GAD NCT00643890 2 Terminated (LeWitt et al.,
2011)
Parkinson’s
disease
Neurturin NCT00985517 2 Ongoing (Bartus et al.,
2013)
Alzheimer’s
disease
NGF NCT00087789 1 Terminated N/A
Spinal Muscular
Atrophy Type 1
SMN NCT02122952 1 Recruiting N/A
Batten disease CLN2 NCT01414985 2 Recruiting N/A
Data obtained from http://clinicaltrials.gov/. GDNF, Glial cell-derived neurotrophic factor;
hAADC, human aromatic l-amino acid decarboxylase; GAD, Glutamic acid decarboxylase;
NGF, nerve growth factor; SMN, survival motor neuron.
Table 3. Comparison of AAV viral vectors against the ideal vector for gene
delivery to the CNS. Properties of an ideal vector for CNS gene delivery AAV vectors
Ability to transduce dividing and non-dividing cells neurons, astrocytes, glial and
ependymal cells
Minimal pathogenesis no associated pathologies
12
Long-lasting expression after a single injection 6 months in brain, 6 years in
other tissues of primates
Limited host immunological response Very low or no
immunological response
Ability to pass through the blood brain barrier AAV8, AAV9, AAVrh8,
AAVrh10
Scalable production large scale production of
highly pure vector
Wide distribution in the CNS AAV9, AAVrh10
Main features of AAV vectors in comparison to the ideal vector for gene delivery to the CNS
. (Table adapted from Lentz et al., 2012)
Furthermore, depending on the particular goals of the gene delivery required, the transgene
expression needs to be maintained at a specific level, usually for a protracted period of time to
provide maximal therapeutic impact. Transgene over-expression of specific genes in the CNS
can have cytotoxic effects on target cells and consequently detrimental ramifications on
behavior. High expression levels may also cause a stronger immune response against the
therapeutic transgene in the host body.
Another challenge of targeting the brain for gene delivery, and perhaps the most important is
identifying vectors that are able to cross the blood brain barrier (BBB) which prevents global
transduction of the CNS following non-invasive peripheral delivery of the vectors (Fig. 4).
Furthermore, different viral vectors have various abilities to spread within the brain parenchyma
which may affect their application in brain disorders where a widespread global transgene
delivery is required. .
13
The efficiency of a virus for gene delivery into the CNS is mainly determined by the natural
properties of the virus. These properties include the packaging capacity, the tissue tropism and
immunogenicity of the virus. Furthermore, the transgene expression level and duration may rely
on the type of the viral genome and its cellular stability. Alterations in the natural biology of
vectors, such as trans-encapsidation or pseudotyping can result in changes in the tropism of viral
vectors, and consequently improvements in the efficiency of transgene delivery in the CNS
(Ojala et al., 2015).
1.7. Adeno-associated viral vectors
Adeno-associated viruses (AAV) are the most common viral vectors now used for CNS gene
delivery, because they possess many properties of an ideal vector described in the previous
section (Table 3). AAVs are non-enveloped, helper-virus dependent parvoviruses with an
icosahedral capsid architecture of approximately 25 nm in diameter. AAVs package ~4.7 kb
genome flanked by ~145 bp inverted terminal repeats (ITRs) on the 5′ and 3′ ends. The WT
AAV genome is a linear single-stranded DNA with two open reading frames. These open
reading frames encode four replication proteins (78, 68, 52 and 40, named by their molecular
weight in kilodaltons), three capsid proteins (VP1, VP2 and VP3) and an assembly activating
protein (Agbandje-McKenna and Kleinschmidt, 2011). The capsid proteins are vital in binding
of the virus to the cell surface receptor which is different for various serotypes, whereas
replication proteins are crucial for the replication and packaging of the viral genome (Ojala et
al., 2015).
Integration of AAV vectors into host chromosome occurs infrequently, at a specific site on
chromosome 19 in most cases; however, this integration is highly dependent on the presence of
Rep 78/68 (McCarty et al., 2004). Recombinant AAV vectors appear to integrate randomly at
an infrequent rate while most genomes are maintained as episomes. It is critical to prevent the
14
formation of WT AAV vectors with chromosome 19 integration, when making recombinant
AAV vectors. This is done by separating the rep and cap coding sequences from the vector
plasmid DNA.
Importantly, the duration of AAV-delivered transgene expression is essentially permanent in
non-dividing cells following a single administration. AAV vectors can also result in long-term,
stable transgene expression after a single administration in rodents and non-human primates.
AAV-delivered transgenes express for more than 18 months in the mouse brain (Miyake et al.,
2011) and can persist in the serum of rhesus monkey for at least 6 years (Rivera et al., 2005) and
in dogs for over 8 years (Niemeyer et al., 2009; Stieger et al., 2009). Interestingly, a recent
long-term follow-up report from a gene therapy trial of Canvan disease has shown that the
therapeutic effects of AAV-delivered transgenes can persist in the human brain for more than
10 years after intra-parenchymal administration (Leone et al., 2012).
Despite these advantages, the use of AAV vectors for CNS gene delivery is hindered by
several limitations. One of the challenges for the use of AAV vectors in clinical trials is the
presence of humoral responses to the wild-type (WT) AAV, which is common among humans
and the possibility of immune clearance of the vector and vector-infected cells (Boutin et al.,
2010). Approximately 70% of the human population carries neutralizing antibodies against
AAV2, the most studied serotype (Boutin et al., 2010). However, the prevalence of neutralizing
factors against other AAV serotypes such as AAV8 and AAV9 is lower (38% and 47%
respectively) indicating a higher therapeutic potential for human gene therapy trials using these
serotypes (Boutin et al., 2010).
Another constraining factor for single stranded AAV vectors is dependence of transgene
expression on second-strand synthesis (Ferrari et al., 1996). This rate-limiting step in transgene
expression has been circumvented after the development of self-complementary viral vectors
15
(McCarty et al., 2001). With these vectors earlier and higher expression can be achieved
compared to single-stranded AAV vectors. However, limited coding capacity has restricted the
use of self-complementary AAV for the delivery of transgenes larger than 5 kb. To circumvent
this, dual hybrid trans-splicing vectors have been developed with transgene capacities up
to 8 kb, in which the transgene cassette is divided between two vectors and assembled into the
full-length transgene only when the two vectors infect a single cell together (Duan et al., 2001).
1.8. Subsequent steps in AAV cell entry and implications for gene
delivery into the CNS
Efficient transduction by AAV vectors relies on several essential steps including cell surface
binding, endocytosis, trafficking to the nucleus, nuclear entry, capsid uncoating, genome
release, second strand synthesis, and finally transcription. The capsid proteins (VP1, VP2, VP3)
determine the interactions with the host cell surface receptors (Huang et al., 2014). Cell surface
glycans serve as the most common receptors for the majority of natural AAVs (Asokan et al.,
2012) (Table 4). Among different AAV serotypes, AAV9 is one of the most effective vectors
for gene delivery into the CNS (Weinberg et al., 2013). AAV9 is able to induce extensive
neuronal and glial transduction through different routes of administration in a variety of animal
models (for a review see Murlidharan et al., 2014). Apart from the important features of the
capsid proteins, AAV vector mediated gene delivery can also be affected by multiple trafficking
factors within the CNS, which will be discussed further in sections 1.9 to 1.12.
1.9. Routes of AAV administration into the CNS
One of the main factors affecting the efficiency and cellular tropism of AAV is the route of
administration. Four of the most commonly used routes for CNS gene delivery are discussed
below. Different routes of administration can affect interactions of AAV vectors with different
16
receptors, vector dissemination from the site of injection, directional transport of the vector and
eventually efficiency and cellular tropism of transfection (Fig. 3).
Table 4. Primary receptors and gene delivery tropism of different AAV
serotypes in the mammalian CNS Serotype Primary
receptor
Intra-CSF or intra-
parenchymal administration
Intra-vascular
administration
Axonal
transport
Neuronal
transduction
Glial
transduction
Neuronal
transduction
Glial
transduction
AAV1 α2,3/α2,6 N-
linked Sialic
acid
++ + + +
A–,R+
AAV2 Heparan
sulfate + – – –
A+,R–
AAV4 α2,3 O-linked
Sialic acid – + – –
?
AAV5 α2,3 N-linked
Sialic acid ++ + – –
?
AAV6 α2,3/α2,6 N-
linked Sialic
acid/heparan
sulfate
++ – + +
A–,R+
AAV8 ? ++ ++ ++ ++ A+, R+
AAV9 N-linked β1,4-
Galactose +++ ++ +++ +++
A+,R+
AAVRh.8 ? ++ ++ +++ +++ ?
AAVRh.10 ? +++ + +++ +++ ?
? Receptor usage/axonal transport has not been characterized; + low levels of transduction;
++ moderate levels of transduction; +++ high levels of transduction; – no transduction; ? A+ or
R+ (AAV vector undergoes axonal transport in the anterograde (A) or retrograde (R) direction
during in vivo characterization). Table adopted from (Murlidharan et al., 2014).
17
Fig. 3. Major factors affecting tropism, transduction and transgene
expression properties of AAV
A. Anterograde gene transport can result in transgene expression distal to the AAV injection
site. This occurs through axonal transport of the protein product. Retrograde viral transport
(e.g. from muscle tissue to motor neuron cell bodies) requires viral transport from the delivery
site to the neuronal nucleus. B. Three major CNS delivery routes including intra-cranial, intra-
cerebroventricular, and intra-venous delivery. C. Encapsidation of different AAV serotypes can
dramatically alter both overall tissue transduction volume and specific cellular expression levels.
(Fig. adapted from Weinberg et al., 2013).
1.9.1. Intra-cranial Administration
Intra-cranial administration of AAV has been the most common route for vector
administration to the brain parenchyma (Gelfand and Kaplitt, 2013; Kantor et al., 2014;
Murlidharan et al., 2014; Ojala et al., 2015). Compared to intravenous systemic delivery, intra-
cranial administration circumvents the challenging requirement of passing through the BBB
18
(Fig. 4), and minimizes the risk of vector neutralization by anti-AAV antibodies, since the levels
of neutralizing antibodies in brain parenchyma are less than 1% of their levels in the blood
(Treleaven et al., 2012).
However, intra-cranial vector delivery has significant down sides. It is perhaps the most
invasive route of vector administration to the CNS, with a risk of hemorrhage, edema and
bacterial infection. Limited vector dissemination from the injection site is another major
drawback in brain disorders where efficient transgene delivery is essential in large brain regions
such as Parkinson’s disease or Alzheimer’s disease, or the entire brain, such as Fragile X
syndrome, Rett syndrome or lysosomal storage disorders. Multiple intracranial injections
spaced throughout the brain have been used with limited success in clinical trials of lysosomal
storage disorders such as Canavan disease (Leone et al., 2000; McPhee et al., 2006) and late
infantile Batten disease (Crystal et al., 2004; Worgall et al., 2008). It is estimated that 50-350
intracranial injection tracts are required for sufficient gene delivery to the entire human brain in
these disorders, which is technically impossible (Cunningham et al., 2008).
AAV diffusion in brain parenchyma can be enhanced using a technique referred to as
convection-enhanced delivery (Hadaczek et al., 2006; Carty et al., 2010; Barua et al., 2013),
which takes advantage of the convective flow in the interstitial fluid, in addition to simple
diffusion, to accelerate the spread of viral vectors away from cannula tip at the injection site.
This technique was safely used for intracranial delivery of AAV2 in a phase 1 clinical trials of
patients with Parkinson’s disease (Mittermeyer et al., 2012), and is currently being assessed in
two ongoing large-scale double-blinded clinical trials of Parkinson’s disease (ClinicalTrials.gov
Identifiers: NCT01621581 and NCT01973543).
19
1.9.2. Intra-vascular Administration
Intravascular delivery provides a non-invasive route to deliver viral vectors into the whole
brain with a single injection, by taking advantage of the dense network of capillaries in the
brain. However, the application of this approach is obviously limited by the BBB, which
consists of endothelial cells, with tight junctions that preclude the transfer of most AAV
serotypes to brain parenchyma. A few serotypes such as AAV9 and AAVrh10 have the ability
to bypass through BBB, possibly through receptor-mediated transcytosis (Di Pasquale and
Chiorini, 2006; Duque et al., 2009), but the exact para-cellular or trans-cellular mechanisms
remain to be identified (Zhang et al., 2011).
Peripheral delivery of the vector often results in off-target undesirable transduction which
might be detrimental, depending on the endogenous expression pattern of the transgene, and at
high doses can lead to liver toxicity(Chandler et al., 2015). Furthermore, as this route delivers
only a small number of virus particles to the brain and spinal cord, it is necessary to administer
higher doses of the vectors compared to direct intracranial or i.c.v. routes. This entails a higher
risk of immunological response from the body (Samaranch et al., 2014), given that almost 50%
of individuals are sero-positive for both AAV9 (Mingozzi et al., 2007) and AAVrh10 viruses
(Thwaite et al., 2015).
20
Fig. 4. The brain barriers in the developing and adult brain
A. The blood-CSF barrier. A barrier between choroid plexus blood vessels and the cerebro-
spinal fluid (CSF). B. The blood-brain barrier. A barrier between the lumen of cerebral blood
vessels and the brain parenchyma. C. The inner CSF-brain barrier, present only during early
development. A barrier between the CSF and the brain parenchyma. D. The outer CSF-brain
barrier. A barrier between the CSF-filled subarachnoid space (sas) and overlying structures.
Abbreviations: arach, arachnoid membrane; cpec, choroid plexus epithelial cells; dura, dura
mater; nu., nucleus; pia, pia mater; sas, subarachnoid space (Fig. adapted from Liddelow, 2011).
21
1.9.3. Intra-CSF
Direct delivery to the CSF reduces peripheral off-target transduction, and lowers vector
neutralization by circulating serum antibodies. Direct AAV administration into ventricles,
cisterna magna, or intra-vertebral lumbar puncture has resulted in extensive transduction in
multiple areas of the CNS (for a review see Murlidharan et al., 2014).
Intra-cerebroventricular (i.c.v.) administration provides widespread transduction of CNS
through transporting the viral vectors in the CSF, as well as transduction of the ependymal cell-
lining of the ventricles, from which transgene can be expressed into the CSF, and distributed
throughout the brain. Transduction of the ependymal cells, which consist of adult neural stem
cells with the ability of lifelong migration and differentiation (Alvarez-Buylla and Lim, 2004;
Lim and Alvarez-Buylla, 2014), makes i.c.v. a viable route for more widespread transduction in
the brain compared to intra-cranial injections. Among different AAV serotypes, AAV9 is one
of the most efficacious serotypes for i.c.v. delivery both in neonatal and adult mice (Haurigot et
al., 2013; Dirren et al., 2014; McLean et al., 2014; Gong et al., 2015).
Intra-thecal AAV vector delivery has also been broadly investigated, as it provides
advantages conferred by the CSF connectivity between the brain and the spinal cord. These
injections are typically performed by exposing either the sub-arachnoid space at the sub-
occipital cisterna magna region or the intra-vertebral space at lumbar region. The lumbar
puncture has been utilized in studies where efficient transduction of motor, sensory or
nociceptive neurons were required (e.g., within dorsal root ganglia). Preclinical studies utilizing
an intra-thecal route of administration for AAV vectors have been encouraging in rodents and
non-human primates (Gray et al., 2013; Hirai et al., 2014; Dirren et al., 2015; Hinderer et al.,
2015; Hordeaux et al., 2015). From different AAV serotypes, AAV6 and AAV9 yield the most
efficient transduction in the mouse brain (Snyder et al., 2011). Consistent with this, intra-thecal
AAV9 administration provided widespread transduction in the brain and spinal cord in mice
22
(Hirai et al., 2014; Dirren et al., 2015), as well as dogs and rhesus monkeys (Hinderer et al.,
2015) and pigs (Federici et al., 2012). The successful scaling of the intra-CSF approach to
larger non-human primates suggests the potential for translation to human therapy.
1.9.4. Retrograde transport delivery
Retrograde axonal transport of vectors (i.e. transport of vectors from axonal terminals back to
cell bodies) can provide a non-invasive gene delivery via peripheral intra-muscular vector
administration and subsequent vector transfer to cell bodies in the spinal cord. This approach is
particularly favorable for transducing motor neurons affected in disorders such as amyotrophic
lateral sclerosis and spinal muscular atrophy. Using this approach, intra-muscular AAV9
vectors have been used to deliver the survival motor neuron gene into motor neurons in neonatal
(Foust et al., 2010) and adult (Benkhelifa-Ziyyat et al., 2013) mouse model of spinal muscular
atrophy. In both studies, more than 40% of motor neurons were transduced which resulted in
improvements in motor function, and survival (Foust et al., 2010; Benkhelifa-Ziyyat et al.,
2013).
1.10. Timing of AVV administration
In addition to capsid serotype and route of administration, other factors such as age of
animals when receiving injections also influences efficiency and CNS bio-distribution of AAV
transduction from systemic i.v. or i.c.v. injections. Injection of viral vectors in neonatal mice
(postnatal day 0 – 2) provides a wider distribution in the brain and a higher chance of passing
through the BBB (Miyake et al., 2011; Yang et al., 2014), which is more permeable due to its
immature status (Ek et al., 2012). AAV2/1 i.c.v. injections into mouse pups on their day of birth
results in widespread transduction (Passini et al., 2003; Levites et al., 2006), but vector spread
was attenuated on postnatal day 2 injections (Chakrabarty et al., 2010).
23
The timing of AAV administration also governs cellular tropism of transduction after global
CNS delivery. Foust et al. (2009) demonstrated that intra-venous administration of self-
complimentary AAV9 resulted in extensive transduction of neuronal cells in neonatal mice,
while expression was predominantly restricted to astrocytes in adults (Foust et al., 2009). These
differences in transduction tropism might be explained by the ongoing development of
astrocytes in neonatal mice and the interactions between astrocytic endfeet and endothelial cells.
Considering the important contributions of astrocytic processes to the BBB biology, there are
significant alterations in the mouse BBB composition within the first postnatal week. Follow-
up studies using i.c.v. injections of AAV serotypes 1, 8, and 9 performed on postnatal day 0
mice demonstrated preferential neuronal tropism. However, from injections performed on
postnatal day 1 or later (24–84 hours postnatal), the tropism changes remarkably towards
astrocytes (Chakrabarty et al., 2013). These studies highlight how the important effects of
developmental age on AAV9 tropism and efficiency, which should be considered when
assessing the potential of these vectors for human translation.
24
Fig. 5. AAV9 tropism in the CNS at different developmental stages
Transduced cells are shown in green, non-transduced cells in gray. In neonates (left), AAV9
can easily cross an immature endothelium and diffuses through a lax extracellular matrix to
transduce neurons, astrocytes and microglia. In adults (right), the brain endothelium has
become a tight barrier surrounded by a cuff of closely packed astrocyte end feet. AAV9 can still
cross the brain endothelium but does not seem able to get beyond the glial barrier, thus
transducing mainly astrocytes (presumably by infecting end feet) and endothelial cells, and
possibly pericytes and perivascular macrophages, but only very few neurons or microglia. (Fig.
taken from Lowenstein, 2009).
1.11. Promoter selection
Based on specific goals of the gene delivery, gene expression can be regulated by employing
a cell type specific promoter to restrict expression to specific transduced cell types, or a
ubiquitous promoter of the desired strength to adjust the overall level of the target transgene
expressed. However, promoter strength can also be vastly affected by the choice of 5' UTR, 3'
UTR, enhancer, and poly-adenylation signal (see Table 5 for a list of common promoters and
the elements affecting their strengths). One challenge in choosing the most appropriate
25
promoter for AAV vectors is that the promoter and transgene must be small enough to fit within
the restricted packaging capacity of the AAV virus.
Two of the most commonly used ubiquitous promoters are the cytomegalovirus (CMV)
promoter and the chicken beta actin (CBA) promoter, both with an approximate size of 800 bp
(including the CMV enhancer and 5' UTR sequence) (Gray et al., 2011). The CMV promoter
is stronger than CBA, however it is more prone to silencing over time in the brain. To provide
ubiquitous long-term sustainable expression at high levels, CBh hybrid promoters have been
developed by the incorporation of MVM introns in the CBA promoter (Gray et al., 2011).
Furthermore, translation may be additionally enhanced utilizing heterologous viral mRNA
stabilization elements in AAV cassettes, such as the woodchuck hepatitis virus post-
transcriptional response element (WPRE). For neuronal transgene expression, the neuron-
specific enolase (NSE), synapsin, MeCP2 or the platelet-derived growth factor (PDGF)
promoters can be used, with sizes of 2.2 kb, 470 bp, 229 bp, and 225 bp respectively.
Astrocytic expression can be achieved using a truncated glial fibrillary acidic protein (GFAP)
promoter (Lee et al., 2008), and oligodendrocytes can be targeted using myelin basic protein
promoters.
Table 5. Promoters commonly used for gene delivery into the CNS.
Enhancer Promoter 5'UTR/intron Strength Size Specificity
CMV CMV SV40 High 800bp Ubiquitous
CMV CBA SV40 High 800bp Ubiquitous
CMV CBA CBA-MVM High 800bp Ubiquitous
None UBC None Weak 430bp Ubiquitous
None GUSB None Weak 378 bp Ubiquitous
None NSE None Strong 2.2 kb Neuron
None Synapsin None Medium 470bp Neuron
None MeCP2 None Weak 229bp Neuron
None GFAP None Medium 681 bp Astrocyte
CMV CBA CBA-MVM High 800bp Ubiquitous
CMV: cytomegalovirus; CBA: chicken Beta actin; UBC: ubiquitin C; GUSB: beta
glucuronidase; NSE: neuron-specific enolase; GFAP: glial fibrillary acidic protein; MVM:
26
minute virus of mice (Table taken from Gray et al., 2011).
1.12. AAV transport within the CNS
Following vector administration and interactions with cell surface receptors, AAV vectors
undergo interstitial and intra-cellular transport throughout the CNS. The spread of AAV vectors
throughout the CNS are mainly affected by two major mechanisms, namely para-vascular CSF
transport, which is responsible for the spread of interstitial fluid within the CNS, and the axonal
transport. Recent findings by Iliff et al. (2013) showed that para-vascular movement of CSF by
the “glymphatic pathway” clears accumulations of metabolites from the brain parenchyma (Iliff
et al., 2013). These results indicate that CSF transport mechanisms can affect the extent of virus
dispersion within the CNS.
Once viruses enter the host neurons, axonal transport serves as another established pathway
affecting virus diffusion within the CNS. In case of AAV vectors, both unidirectional and
bidirectional (anterograde or retrograde transport or both) axonal transport have been
established depending on the strain of the virus (table 4). AAV9, for instance, is believed to be
able to travel in both anterograde and retrograde directions (Castle et al., 2014).
1.13. Hypotheses and objectives
There is no pharmacological cure for FXS and the currently prescribed medications only
partially alleviate selected symptoms and are associated with deleterious side effects.
Considering the plethora of genes whose expression is regulated by FMRP, a priori, it may be
expected that restoring FMRP expression in the CNS could provide a more comprehensive
reversal of the disorder compared to targeting single molecules (e.g. mGluR5). Our overarching
hypothesis was that introducing the Fmr1 gene into the brains of neonatal Fmr1 KO mice, using
i.c.v. injection of AAV vectors serotype 2/9 would provide widespread transgene dispersion
27
throughout the brain, and will prevent or ameliorate the neurodevelopmental impairments in the
Fmr1 KO mice.
The specific objectives of the present study were:
1. Analysis of FMRP cell type expression: To investigate FMRP expression in neurons,
astrocytes, microglia and oligodendrocyte precursor cells in the developing and adult brain of
WT mice by immunocytochemistry (Chapter 3, Gholizadeh et al., 2015).
2. Efficiency of AAV-mediated transgene delivery: To determine the most effective route(s) of
administration, age of injection and promoter that would lead to wide-spread transduction in the
brains of neonatal mice using AAV5 and AAV9 vectors expressing enhanced green fluorescent
protein (eGFP) (Chapter 4, Gholizadeh et al., 2013).
3. FXS gene therapy-Phase 1: To assess the efficacy of administering an AAV9 vector
containing the Fmr1 gene driven by the neuron- specific human synapsin promoter directly into
the CNS of Fmr1 KO mice via i.c.v. administration to PND 5 Fmr1 KO mice (Chapter 5,
Gholizadeh et al., 2014).
4. FXS gene therapy-Phase 2: To assess level and distribution of FMRP and cell-type
specificity of transduction, after i.c.v. injection of a viral vector coding for FMRP, in PND-0
Fmr1 KO mice and also to extend the behavioral analyses to include additional tests not
employed in phase 1 (e.g. anxiety and pre-pulse inhibition) (Chapter 6).
5. To study the potential effects of FMRP over-expression on mouse behavior by i.c.v.
injections of AAV-FMRP in WT mice at PND 0-1 followed by behavioral analysis starting 60
days post-injection (Chapters 6).
28
Chapter 2. Materials and Methods
2.1. Animals
All injections were carried out using C57/BL6 wild-type (WT) or Fmr1 knockout (KO) mice
on postnatal day (PND) 0-1, 5 or 21. Mice were kept in a room maintained at constant
temperature (21 ± 2 °C) and humidity (55 ± 5%) with an automatic 12 h light/dark cycle and
free access to standard laboratory diet and tap water. All animal experiments were carried out in
accordance with the guidelines set out by the Canadian Council on Animal Care and were
approved by the University of Toronto Animal Care Committee.
2.2. AAV vectors
Various single-stranded AAV vectors were used with different promoters or cDNA transgene
sequences. AAV vector containing the inverted terminal repeat DNA sequences from the
genome of serotype type 2 (AAV2), and the capsid protein genes from serotype 5 (AAV5) or
serotype 9 (AAV9) were used. The AAV9 vectors contained the cDNA for the enhanced green
fluorescent protein (eGFP), driven by either the ubiquitous cytomegalovirus (CMV) promoter,
referred to here as “AAV-CMV-eGFP”, or the human synapsin-1 (SYN) promoter, defined here
as “AAV-SYN-eGFP”. We also used AAV9 vectors containing the cDNA for the major murine
isoform of Fmr1 (isoform 1) (Brackett et al., 2013), driven by the neuron-specific human
synapsin (SYN), referred to here as “AAV-FMRP”, or empty control vectors containing no
transgene, here referred to as “AAV-null”.
Single stranded AAV5 vectors were constructed and packaged at the University of Florida
Powell Gene Therapy Center. The AAV5 vectors contained an eGFP reporter gene driven by
the chicken-β-actin (CBA) promoter (AAV5-CBA-eGFP) and the titer was 1.24 × 1013
genomes/ml. AAV9 vectors were supplied by the University of Pennsylvania Vector Core
facility (Philadelphia, PA, USA) and were titer-matched to 1 × 1013
genomes/ml. A woodchuck
29
hepatitis post-transcriptional regulatory element (WPRE) sequence, was inserted in the AAV9
expression cassette downstream of the eGFP or Fmr1 gene to enhance stabilization of the
mRNAs and to eventually increase transgene expression (Loeb et al., 1999; Cederfjäll et al.,
2012). All AAV vectors were suspended in sterile phosphate-buffered saline (PBS) and stored
at -80ºC.
2.3. Vector injections
2.3.1. i.c.v. injections in neonatal mice
For injection of PND 0 or PND 5 mouse neonates, the pups were immobilized via cryo-
anesthesia for 2 minutes and the immobilized pup was grasped by the skin behind the head and
placed on a fiber-optic light to illuminate the midline and transverse sutures that were used as a
guide for injections. A 30-gauge needle attached to a 5 µl Hamilton syringe (Hamilton, Reno,
NV) through long polyethylene tubing was used. For PND 0 injections the needle was inserted
2 mm deep, perpendicular to the skull surface, at a location approximately 0.1 mm lateral to the
sagittal suture and 0.50 mm rostral to the neonatal coronary suture. PND 5 injections were
performed 2 mm deep, 0.25 mm lateral to the sagittal suture and approximately 0.75 mm rostral
to the neonatal coronary suture. 0.75 - 1 µl of the vector or vehicle (PBS or AAV-null vector)
was injected using a syringe pump (NE-1002X, New Era Pump Systems, Farmingdale, NY) at
the rate of 1 µl/min into each lateral ventricle. The needle was left in place for 1 minute after
discontinuation of plunger movement to prevent backflow. The pups were allowed to recover in
a warmed container in order to return to normal temperature and were placed back into the cage
with the dam after normal movement and general responsiveness were restored.
30
2.3.2. Stereotaxic injections in juvenile mice
Juvenile PND 21 animals were anesthetized with isofluorane and secured in a stereotaxic
frame (David Kopf instruments, Tujunga, CA). The skull was exposed and holes the size of the
30 gauge injection needle were drilled into the skull. AAV5 or AAV9 vectors expressing eGFP
were injected bilaterally into the lateral ventricles with 1 μl of vector or PBS per side at the rate
of 0.5 μl/minute. Coordinates for injections were 0.22 mm caudal to bregma, ±1 mm lateral to
the midline, and 1 mm ventral to pial surface. Following infusion, the needle was left in place
for 5 minutes prior to being slowly retracted from the brain. The incision was cleaned with
sterile saline and closed with taper point non-absorbable 5-0 surgical sutures (Syneture,
Mansfield, MA). After surgery, the mice were housed individually and were monitored every
day for signs of stress or infection until euthanasia.
2.4. Behavioral analysis
All behavioral tests were conducted on aged and sex-matched WT and KO animals. All mice
were naive to the tests and tested only once in each test. The experimenters were blinded to the
treatment groups during the time of testing.
2.4.1. Locomotor activity measurements
Locomotor activity was assessed using an automated open field locomotor monitor system
(Accuscan Images, Salt Lake City, USA). Mice were acclimated to the testing room for 5
minutes, then placed in the open field and monitored for 20 minutes under low-light conditions.
The total distance covered (horizontal movement) was recorded using the Fusion software
(Fusion software, Johannesburg, South Africa). Total distance travelled was compared between
groups.
31
2.4.2. Marble Burying for stereotypic behavior
Approximately 10 cm of laboratory animal bedding (Bed-o'Cobs combination bedding;
Andersons Inc, Maumee, USA) was added to empty cages and blue marbles were placed
equidistant from each other in a 4–by-5 grid covering two thirds of the surface. Mice were
acclimated to the testing room for 5 minutes, and then placed in the cage on the side devoid of
marbles. Mouse activity was left undisturbed for 30 min and then the number of buried marbles
was counted; a buried marble consisted of any marble where less than 50% of its surface was
left uncovered by bedding.
2.4.3. Ultrasonic vocalizations
Virgin adult female mice were placed in a new cage that contained only bedding for two
minutes and the cage was fitted with a customized polystyrene cover. Subsequently, a virgin
male was placed into the same cage and ultrasonic vocalizations (USV) were recorded using an
Ultrasound Detector D 1000X (Pettersson Elektronik AB, Uppsala, Sweden) for 4 minutes as
described previously (Wang et al., 2008). The distinct waveform patterns seen in the
spectrograph rendering, indicative of vocalizations, were counted. The first 5 seconds of
recording following the introduction of the male was also ignored. The total number of
vocalization contained within 4 minutes of recording was compared between animals.
2.4.4. Tube test of social dominance
The mice were tested using the tube test to measure social dominance. Each match involved
two mice of different genotype (WT or Fmr1 KO) or injection groups (PBS, AAV-null or AAV-
FMRP) that were not housed together. The experimenter was blind to the category of the
groups being tested. One mouse was placed into each end of a transparent PVC tube (2.5 cm
inner diameter, 30.5 cm length) and the mice were released simultaneously. The match ended
when one mouse placed at least two paws outside the tube with the mouse remaining inside the
32
tube being deemed the “winner”. The number of wins for each group or genotype was tallied
and a chi-square analysis was used to determine whether the percent of wins in each group was
significantly different from the 50:50 win/loss outcome expected by chance. Each animal was
tested three to five times, depending on pairing availability, against animals of opposing
injection group and/or genotype.
2.4.5. Elevated plus maze test of anxiety
The elevated plus maze consisted of four arms (two open without walls and two enclosed by
15.25 cm high walls) 30 cm long and 5 cm wide. Each arm of the maze was attached to sturdy
legs such that it was elevated 40 cm off from the floor. The maze was placed in the testing
room in a way that there were similar levels of illumination on both open and closed arms. The
legs of the maze were adjusted so that the maze was perpendicular to the ground and each arm
was level. Each mouse was placed at the junction of the open and closed arms, facing the open
arm opposite to where the experimenter was. The mouse was allowed to move freely in the
maze for 5 minutes while the total number of entries in both the open and closed arms, as well
as the total time spent in each arm was measured by an observer. The observer was blind to the
experiment groups and made minimal movements and no noise during the experiment. An arm
entry was counted when all four paws of the mouse were on the arm. At the end of the 5-min
test, the mouse was removed from the plus maze and placed into its home cage. The maze was
cleaned with Virox and dried with paper towels before testing with another mouse.
2.4.6. Pre-pulse inhibition for sensorimotor gating
Sensorimotor gating was measured by pre-pulse inhibition (PPI) of the startle response, as
previously described (Thomas et al., 2011). PPI of acoustic startle responses was measured
using the SR-Lab System (San Diego Instruments, San Diego, CA). The apparatus comprised
of a sound attenuating chamber, which contained a cylindrical tube. A test session began by
33
placing a mouse in the Plexiglas cylinder where it was left undisturbed for 5 min. Each test
session consisted of 48 trials comprising of 6 blocks of eight trial types each presented in a
pseudo random order such that each trial type was presented once within a block of seven trials.
One trial type was a 40 millisecond, 120 dB sound burst used as the startle stimulus. There were
five different acoustic pre-pulse plus acoustic startle stimulus trials. The pre-pulse sound was
presented 100 milliseconds before the startle stimulus. The 20 milliseconds pre-pulse sounds
were 74, 78, or 82 dB. Finally, each block had a trial where no stimulus was used to measure
baseline movement in the cylinders where no sound was presented. The inter-trial interval
ranged from 10 s to 20 s, and the startle response was recorded every 1 millisecond for
65 millisecond following the onset of the startle stimulus. The startle response was recorded for
65 milliseconds (measuring the response every 1 millisecond) starting at the onset of the startle
stimulus. The background noise was 70 dB in each chamber. The maximum startle amplitude
during the 65 milliseconds was utilized as the dependent variable. The following formula was
used to calculate percent PPI of a startle response: 100 – [(startle response on acoustic pre-pulse
+ startle stimulus trials / startle response alone trials) × 100]. Thus, a high percent PPI value
indicates that the subject showed a reduced startle response when a pre-pulse stimulus was
presented compared to when the startle stimulus was presented alone. Conversely, a low
percent PPI value indicates that the startle response was similar with and without the pre-pulse.
2.4.7. Audiogenic Seizures
For audiogenic seizure testing, the apparatus consisted of a Plexiglas mouse cage (28 × 17 ×
14 cm) with a 135-dB sound source (Piezo siren, Electrosonic; Piezo Technologies,
Indianapolis, IN) attached to the lid and extending 5 cm down into the cage. Mice (30-31 days
old) were placed individually into the testing apparatus and were allowed to explore for 2 min,
after which the bell was rung for 2 min. Seizure activity was observed and scored using a
34
seizure severity score as follows: wild running = 1; clonic seizure = 2; tonic seizure = 3; status
epilepticus/respiratory arrest/death = 4 (Musumeci et al., 2000). Animals were considered to
have had a seizure if the seizure severity score was greater than 1. Animals were tested only
once. Seizure testing was carried out between 1:00 PM and 5:00 PM. All mice were
immediately euthanized at the end of the test.
2.5. Tissue preparation, immunocytochemistry and confocal microscopy
PND 10, PND 20, PND 30 and adult (PND 55-65) WT or KO mice were anesthetized with a
mixture of ketamine and xylazine (intra-peritoneal, 80 mg/kg and 8 mg/kg respectively) and
perfused transcardially with a solution of PBS (pH = 7.4) followed by 4% paraformaldehyde
(pH = 7.4). For PND 0, pups were decapitated, rinsed with PBS and the skull was fixed in 4%
PFA at room temperature for 2-4 hours. The brain was then removed from the skull and further
incubated in 4% PFA at 4 °C for 20-24 hours. Perfused brains were then removed and placed in
4% paraformaldehyde overnight at 4 °C, then transferred to 30% sucrose for cryo-protection.
Once the brains sank in the sucrose, they were mounted in optimum cutting temperature solution
(Sakura, Torrance, CA) and frozen at −20 °C until sectioning. Serial coronal or sagittal
sectioning sectioning of the entire brain was performed at a thickness of 25 μm using a cryostat
(Leica Microsystems, Wetzlar, Germany). Free-floating sections were rinsed with Tris buffered
saline and antigen retrieval was performed as described previously (Gabel et al., 2004).
Monoclonal mouse anti-FMRP (2F5; 1:1000; gift from Dr. Jennifer Darnell, or 5c2, 1:1000; gift
from Richard Kascsak) was used along with one of the following monoclonal rabbit primary
antibodies: NeuN (1:1000; Abcam) to immunolabel neurons and anti-S100β (1:1000; Abcam) to
mark astrocytes. For analyzing the cell-type specific expression of FMRP in the WT mouse
brain, sections were also stained with rabbit polyclonal anti-Iba-1 (1:2000; WAKO, Richmond,
VA, USA) to immunolabel microglia, and rabbit polyclonal anti-NG2 (1:500; Millipore,
35
Temecula, CA, USA) to stain oligodendrocyte precursor cells. To assess the transduction
efficiency of AAV-eGFP vectors, polyclonal rabbit anti-GFP (1:2000; Abcam, Cambridge,
USA) was used along with each of the following monoclonal mouse primary antibodies: NeuN
(1:200; Millipore, Temecula, CA, USA) to mark neurons and anti-S100β to mark astrocytes.
Cerebellar sections were also co-incubated with a polyclonal rabbit anti-calbindin antibody
(diluted 1:10,000; Swant, Marly, Switzerland) to label Purkinje neurons. After overnight
incubation, 5 washes with Tris buffered saline for 10 minutes each were carried out and
secondary antibodies diluted in Tris buffered saline containing 5% goat serum were applied.
The sections incubated with anti-FMRP were labeled with goat anti-mouse Alexa Fluor 488 and
goat anti-rabbit Alexa Fluor 594 (1:1000; Jackson ImmunoResearch Laboratories, Inc. West
Grove, PA). The images were captured using a laser-scanning confocal microscope (Nikon A1,
Tokyo, Japan) at 10, 40, 60 or 100X magnifications and analyzed using the NIS-Elements
software (Nikon Instruments, Tokyo, Japan). The images for sagittal sections were captured
using a Zeiss Mirax slide scanner at 20X magnification.
2.5.1. Semi-quantitative and quantitative analysis of transgene expression in the
brain
For a comparative analysis of the transduction pattern in the brain, two scoring methods
were used: a semi-quantitative scoring system to analyze transduction in different brain regions
and a quantitative cell-count of transduced cells in the retrosplenial cortex. We first used a
semi-quantitative scoring system to estimate transduction efficiency of different AAV vectors in
discrete regions of the mouse brain including the prefrontal cortex, striatum, cingulate cortex,
hippocampus, retrosplenial cortex, thalamus, piriform cortex, inferior colliculus, superior
colliculus, brainstem and cerebellum. The semi-quantitative scoring system was used to analyze
the cellular tropism of the AAV-eGFP or AAV-FMRP in different brain regions. Scoring was
done by classifying the number of transduced cells in four categories: regions with no detectable
36
transgene expression, regions with 0 - 100 transgene expressing cells/mm2, regions with 100 -
200 transgene expressing cells, and regions with more than 200 transgene expressing cells.
Because individual cells were most easily resolved and distinguished in the retrosplenial
cortex and cingulate cortex, these brain regions were targeted for quantitative analysis of
neuronal vs. astroglial virus transduction in photomicrographs captured at 10X or 40X
magnifications. The boundaries of the retrosplenial cortex and cingulate cortex were delineated
according to the Paxinos atlas of the mouse brain (Paxinos and Franklin, 2013) in 3 consecutive
25 µm sections stained for transduced cells via GFP or FMRP immunoreactivity. Quantification
was carried out by an individual masked to the time and type of AAV vector administration.
The total number of transgene-positive cells in each group as well as the percentage of
transgene-positive cells that co-localized with the neuronal marker NeuN or the astrocytic
marker S100β was recorded.
2.6. Quantitative western blotting
Samples of the inferior colliculus, cerebellum, cortex, frontal cortex, hippocampus and
striatum were collected and stored at – 80 oC. After electrophoresis the proteins were
transferred onto nitrocellulose membranes and then incubated overnight with primary antibodies
including mouse anti-FMRP 5c2 (1:250), (LaFauci et al., 2013) and mouse anti-GAPDH
(1:40,000; Clone GAPDH-71.1; Sigma-Aldrich, Saint Louis, Missouri, USA). The sections
were incubated with horseradish peroxidase-conjugated goat anti-mouse (Jackson
ImmunoResearch Laboratories, Inc.; West Grove, PA, USA); after washing, SuperSignal West
Pico Chemiluminescent Substrate (Thermo Scientific; Rockford, IL, USA) was added to the
membranes and the bands were revealed by exposure under Alpha Innotec Fluorochem gel
imager (Protein Simple; Toronto, ON, Canada). Images were analyzed using AlphaEase SA.
Quantification of protein expression was normalized to GAPDH expression. Results are
37
presented as averages ± S.E.M. Statistical significance was determined by using one-way
ANOVA and Tukey's multiple comparison tests.
2.7. Statistical analyses
Fisher's exact test was used for statistical analysis of audiogenic seizure incidence between
the groups. In the tube test, a chi-square analysis was used to determine whether the percent of
wins were significantly different from the 50:50 win/loss outcome expected by chance. For
motor activity, marble burying, ultrasonic vocalization, elevated plus maze and pre-pulse
inhibition tests, one-way ANOVA test was performed followed by Bonferroni post-hoc test.
The GraphPad prism software (version 6) was used to perform the statistical analyses.
38
Chapter 3. Expression of FMRP protein in neurons and glia of the developing and adult mouse brain
Acknowledgements: Immunocytotochemical analyses and imaging were performed with
assistance from Dr. Sebok Kumar (Post-doctoral fellow).
3.1. Specific hypotheses, objectives and rationale
The purpose of this phase of the study was to fully document the cell type expression of
FMRP in neurons and glia in the developing and mature brain of WT C57/BL6 mice. In
contrast to the expression of FMRP in neurons of the adult brain, relatively little is known about
the types of glial cells that express FMRP during CNS development. Because fragile X
syndrome is a neurodevelopmental disorder, a comprehensive analysis of its cell type
expression in the developing brain can provide a better understanding of the molecular functions
of FMRP, and the pathogenesis of the syndrome. Moreover, this information was used in the
generation of our AAV vectors. Our analysis encompassed the cerebral cortex, striatum,
hippocampus, cerebellum, and the corpus callosum, and extended from postnatal days (PNDs) 0
(day of birth), to PNDs10, 20, and adult (8-12 weeks old) WT mice. We investigated FMRP
expression in the brain by double-labeling immunocytochemistry using an anti-FMRP antibody
and cell type markers for neurons (NeuN), astrocytes (S100β), microglia (Iba-1) or
oligodendrocyte precursor cells (NG2).
3.2. Gradual reduction in the number of FMRP-positive cells from the
early postnatal period to adulthood
FMRP+ cell counts in the cingulate cortex and corpus callosum revealed that the number of
FMRP+ cells in both brain regions was highest at PND 0 (see “Total FMRP+” column in Table
6). In the cingulate cortex there was a gradual decline over time such that the adult level (8-12
weeks old) was approximately 57% of that at birth (P0). In the corpus callosum there was a
relatively minor decline whereby adult mice showed about 85% of the level at P0. Overall,
39
these observations are consistent with previous reports indicating peak FMRP expression over
the first 1-2 postnatal weeks and declining thereafter (Lu et al., 2004).
Table 6. Quantitative analysis of cell type-specific expression of FMRP in the
cingulate cortex and corpus callosum. Brain region Age Total FMRP+ % NeuN+ % S100β % Iba-1 % NG2
Cingulate
cortex
PND 0 154 ± 5 85 ± 2 5 4 6
PND 10 123 ± 5 89 ± 1 2 5 3
PND 20 86 ± 5 94 ± 1 2 2 4
Adult 88 ± 7 94 ± 1 3 2 5 ± 1
Corpus
callosum
PND 0 82 ± 3 2 27 ± 2 20 ± 2 18 ± 2
PND 10 75 ± 3 2 33 ± 3 19 ± 3 14 ± 2
PND 20 76 ± 3 2 33 ± 22 10 ± 1 11 ± 2
Adult 70 ± 3 2 22 ± 3 3 7
The analysis was performed by counting the FMRP-positive neurons (NeuN), astrocytes
(S100β), microglia (Iba-1) and oligodendrocyte precursor cells (NG2) in the cingulate cortex
and corpus callosum. The results are reported as the average number of total FMRP-positive
cells per visual field, and the percentage of FMRP-positive cells that co-localized with NeuN,
S100β, Iba-1, and NG2. Data are presented as mean ± S.E.M. Standard error values smaller
than 0.5 are not reported.
3.3. Predominant neuronal expression of FMRP throughout
development
At all postnatal times examined, FMRP expression was predominantly neuronal in all of the
brain regions analyzed, except for corpus callosum (Fig. 6, Tables 6 and 7) where only a few
NeuN+ cells were present and most FMRP expression was observed in S100β+ cells (Fig. 7,
Table 6). Quantitative analysis of cell type-specific expression of FMRP revealed 85% - 94%
co-expression with NeuN in FMRP+ cells in the cingulate cortex. In stark contrast only 2%
NeuN/FMRP co-expression was detected in the corpus callosum at all of the time points studied.
40
Table 7. Semi-quantitative analysis of cell type-specific FMRP expression in
the developing and adult mouse brain. Brain
region
Age of
mice
FMRP+/NeuN
+ FMRP+/S100β+
FMRP+
/Iba-1+ FMRP+/NG2+
Cingulate
cortex
PND 0 +++ + + +
PND 10 +++ + + +
PND 20 +++ + + +
Adult +++ + + +
Corpus
callosum
PND 0 + +++ +++ +++
PND 10 + +++ +++ ++
PND 20 + +++ ++ ++
Adult + +++ + +
Striatum
PND 0 +++ ++ ++ ++
PND 10 +++ + + +
PND 20 +++ + + +
Adult +++ + - +
Hippocamp
us
PND 0 +++ ++ ++ +
PND 10 +++ + + +
PND 20 +++ - - +
Adult +++ - - -
Cerebellum
PND 0 +++ + + +
PND 10 +++ + + ++
PND 20 +++ + - +
Adult +++ + - +
The level of co-localization of FMRP with NeuN-positive neurons, S100β-positive
astrocytes, Iba-1-positive microglia, and NG2-positive oligodendrocyte precursor cells in mouse
brain was assessed in the brain regions indicated at PND 0, PND 10, PND 20, and adults. The
extent of co-localization is reported as: - , none; + low; ++ moderate; and +++ high.
41
Fig. 6. Expression of FMRP in neurons of the WT mouse brain.
Coronal sections from postnatal days 0, 10, 20, and adult mice were double immunolabeled
using anti-FMRP and anti-NeuN, and counterstained with DAPI (A–T). Co-localization of
FMRP in NeuN-positive neurons (FMRP, green; NeuN, red; DAPI, blue) is shown in the
cingulate cortex, hippocampus, striatum, corpus callosum, and cerebellum. Arrows indicate
examples of co-localization. The heavy dashed lines delineate the boundaries of the corpus
callosum. Scale bar = 20 μm.
3.4. Developmental expression of FMRP in astrocytes, microglia and
oligodendrocyte precursor cells
We examined FMRP expression in astrocytes by double labeling with anti-FMRP and anti-
S100β antibodies. FMRP and S100β were co-localized at PND 0 in cells in the cingulate cortex,
striatum, hippocampus, corpus callosum and cerebellum (Fig. 7, Tables 6 and 7). By PND 10,
PN
D 1
0Cingulate cortex Hippocampus Striatum Corpus callosum Cerebellum
NeuN FMRP DAPIP
ND
20
PN
D 0
Ad
ult
DA CB
N
HG
ML
SP RQ
O
J
E
K
IF
T20 μm
42
PND 20 and adult mouse brain, the population of FMRP/S100β -positive cells had declined
drastically in cingulate cortex and hippocampus, (less than 3%). Compared to the cingulate
cortex and hippocampus, the decline in FMRP/S100 β co-expression occurred more slowly in
the striatum where substantial co-expression was seen at PND 10, PND 20, but not in adult
mice. Persistent co-expression of FMRP/S100β was also observed in the cerebellum at PND0,
10, 20 and adult mice, although the number of FMRP/S100β cell gradually decreased in adult
mouse brain.
Interestingly, FMRP was co-expressed in many S100β+ cells in the corpus callosum. Of the
brain regions analyzed, the corpus callosum showed by far the largest percentage of cells co-
expressing of FMRP and S100β (Fig. 7, Table 7); more than 75% of S100β positive cells in the
corpus callosum co-expressed FMRP at all time points. This is an intriguing finding as this
brain region is mainly composed of white matter. Approximately 30% of FMRP-expressing
cells in the corpus callosum also expressed S100β at all developmental stages analyzed (Tables
6 and 7). The percent of S100β/FMRP-positive cells was maintained at a moderate level from
PND 0 to adult mice, indicating a continual expression of FMRP in astrocytes from early
postnatal stages through adulthood in this brain region.
In addition to the expression of S100β in astrocytes, S100β is also expressed by a population
of oligodendrocyte precursor cells and oligodendrocytes (Hachem et al., 2005; Steiner et al.,
2008). Therefore, two population of S100β immune-positive cells may exist, one representing
astrocytes and one dedicated to oligodendrocyte precursor cells. To better characterize these
two populations of cells, we also used NG2 staining as a specific marker for oligodendrocyte
precursor cells. The NG2 proteoglycan is expressed in oligodendrocyte precursor cells, but not
in mature oligodendrocytes, both in vitro and in developing rodent brain (Polito and Reynolds,
2005). FMRP was expressed in many NG2+ oligodendrocyte precursor cells in the cingulate
43
cortex, striatum, hippocampus, corpus callosum and cerebellum at PND 0 (Fig. 8, Tables 6 and
7). Thereafter, from PND 10 to adult mouse brain, FMRP expression in NG2-positive cells
progressively declined in all of the brain regions examined, reaching low co-localization in adult
mice in the cingulate cortex, corpus callosum, striatum and cerebellum and undetectable co-
localization in the hippocampus (Table 7).
FMRP expression in microglia was examined by assessing co-expression with Iba-1. FMRP/
Iba-1 double labeled cells were present in all brain regions analyzed at PND 0 (Fig. 9). At PND
0, more than 75% of the total NG2+ cells and Iba-1+ cells co-expressed FMRP in the all the
brain regions analyzed. At PND 10 and PND 20, the number of FMRP/Iba-1 co-localized cells
remained relatively constant in the cingulate cortex, corpus callosum, and striatum, but
decreased in the hippocampus and cerebellum by PND 20. In the brain regions studied here,
there was a gradual decrease in FMRP/Iba-1 co-expression after PND 20 with no detectable co-
localization in adult mice.
44
Fig. 7. Expression of FMRP in the astrocytes of mouse brain.
Coronal sections from postnatal days 0, 10, 20, and adult mice were labeled using anti-
FMRP, anti-S100β and DAPI. FMRP in S100β -positive astrocytes (FMRP, green; S100β, red;
DAPI, blue) are shown in the cingulate cortex, hippocampus, striatum, corpus callosum, and
cerebellum. Arrows indicate examples of co-localization of FMRP in S100β-positive
astrocytes, whereas dashed-squares indicate the absence of FMRP in astrocytes. The heavy
dashed lines delineate the boundaries of the corpus callosum. Scale bar = 20 μm.
Cingulate cortex Hippocampus Striatum Corpus callosum Cerebellum
S100B FMRP DAPI
PN
D 1
0P
ND
20
PN
D 0
Ad
ult
IF HG
K ML
P RQ
A B C
O
T
J
N
S
D E
20 μm
45
Fig. 8. Expression of FMRP in oligodendrocyte precursor cells in the mouse
brain.
Coronal sections were labeled using anti-FMRP, anti-NG2 and DAPI. Co-localization of
endogenous FMRP in NG2-positive oligodendrocyte precursor cells (FMRP, green; NG2, red;
DAPI, blue) is shown in the cingulate cortex, hippocampus, striatum, corpus callosum, and
cerebellum. Arrows indicate the co-localization of FMRP in NG2-positive oligodendrocyte
precursor cells, whereas dashed-squares indicate the absence of FMRP in oligodendrocyte
precursor cells. The heavy dashed lines delineate the boundaries of the corpus callosum. Scale
bar = 20 μm.
Cingulate cortex Hippocampus Striatum Corpus callosum Cerebellum
NG2 FMRP DAPIP
ND
10
PN
D 2
0P
ND
0A
du
lt
IF HG
NK OM
SP TR
DBA C
J
E
L
Q20 μm
46
Fig. 9. Expression of FMRP in microglia.
Coronal sections were labeled with anti-FMRP and anti-Iba-1. Co-localization of FMRP in
Iba-1-positive microglia (FMRP, green; Iba1, red; DAPI, blue) is shown. Arrows indicate
examples of co-localization of FMRP in Iba-1-positive microglia, whereas dashed-squares
indicate the absence of FMRP in microglia. Scale bar = 20 μm.
Cingulate cortex Hippocampus Striatum Corpus callosum Cerebellum
Iba1 FMRP DAPIP
ND
10
PN
D 2
0P
ND
0A
du
lt
A CB
IF HG
NK ML
SP RQ T
O
J
ED
20 μm
47
Chapter 4. Transduction efficiency of adeno-associated viral vectors expressing eGFP after intra-cerebroventricular
administration in neonatal and juvenile mice
Acknowledgements: Immunocytotochemical analyses and imaging were performed with some
assistance from Dr. Sujeenthar Tharmalingam (Ph.D. student) and Margarita Macaldaz
(summer student).
4.1. Specific hypotheses, objectives and rationale
We hypothesized that intra-cerebroventricular (i.c.v.) administration of single stranded AAV
vectors leads to robust widespread gene delivery to multiple regions of the brain and the
transduction efficiency and cellular tropism is affected by the AAV serotype used, time of
administration and the promoter selected in the AAV construct. The transport of viral vectors
directly in the CSF as well as transducing the ependymal cells lining the ventricles has made
i.c.v. delivery an established route of AAV administration, particularly where a global transgene
delivery in the brain is required. The main objective of this study was to evaluate the ability of
single-stranded AAV 2/9 (AAV9) and 2/5 (AAV5) encoding the enhanced green fluorescent
protein (eGFP) reporter to transduce the brain and target gene expression to specific cell types
following i.c.v. injection in mice. Major factors affecting tropism, expression level, and cell-
type specificity of AAV-mediated transgenes include encapsidation of different AAV serotypes,
promoter selection, and the timing of vector administration. Titer-matched AAV9 vectors
encoding eGFP, driven by the cytomegalovirus (CMV) promoter, or the neuron-specific
synapsin-1 promoter, were injected bilaterally into the lateral ventricles of C57/BL6 mice at
postnatal day 5 (neonatal) or 21 (juvenile). Brain sections were analyzed 25 days after injection
using immunocytochemistry and confocal microscopy.
48
4.2. AAV9-CMV-eGFP targets astrocytes in neonatal mice but neurons
in juvenile mice
To evaluate virus transduction of the brain at early stages prior to CNS maturation, we
investigated transgene expression after i.c.v. injections in neonatal mice on PND 5, or juvenile
mice on PND 21. One group of mice received i.c.v. injections of 2 × 1010
particles of a single
stranded AAV9-CMV-eGFP (Fig. 10A). The animals were euthanized 25 days post-injection,
and brains were evaluated for transgene expression (Fig. 10B).
Fig. 10. Schematic diagrams depicting (A) the AAV-eGFP vector constructs
used and (B) bilateral intra-cerebroventricular injections on postnatal days 5,
and 21.
The vectors were single stranded, contained ITR elements from AAV serotype 2, and were
packaged in either serotype 5 or serotype 9 capsids. A WPRE element was inserted into the
AAV9 vectors downstream of the eGFP cDNA to enhance transcription. Twenty-five days after
AAV administrations (i.e., on postnatal day 30 or 46), animals were sacrificed and brains were
49
analyzed for GFP immunoreactivity. CBA, chicken β-actin; CMV, cytomegalovirus; ITR,
inverted terminal repeat; WPRE, woodchuck hepatitis post-transcriptional regulatory element;
eGFP, enhanced green fluorescent protein; hSynapsin-1, human synapsin-1.
We observed remarkable differences between PND 5 and PND 21 injections both in the
distribution and cell type specificity of transduction. Injection of AAV-CMV-eGFP at PND 5
resulted in an extensive distribution in all brain regions examined: prefrontal cortex, striatum,
hippocampus, piriform cortex, cingulate cortex, retrosplenial cortex and cerebellum. The
transduction was primarily observed in astrocytes in several of the forebrain regions examined
including the striatum (Fig. 11), retrosplenial cortex (Fig. 12), and prefrontal, piriform, and
cingulate cortices (results not shown), as assessed by double labeling with anti-GFP and anti-
S100β antibodies. In contrast, in the hippocampus transduction was mainly neuronal, as
assessed by double labeling with anti-GFP and anti-NeuN antibodies (Fig. 13, A-F). In the
cerebellum, transgene expression was predominantly present in the dendrites and cell bodies of
Purkinje neurons as determined by double labeling of the sections with anti-GFP and anti-
calbindin antibodies (Fig. 14, A-F).
In contrast, i.c.v. injections of titer-matched AAV9-CMV-eGFP vector on PND 21 resulted
in almost exclusively neuronal transduction but with a more restricted distribution compared to
neonatal injections on PND 5. Robust eGFP expression was found in many regions in the
forebrain including the prefrontal cortex, striatum, and hippocampus (Table 8). Co-labeling for
NeuN and GFP expression in the striatum (Fig. 11, D-F), retrosplenial cortex (Fig. 12, D-F),
hippocampus (Fig. 13, D-F) and cingulate cortex (not shown) revealed many NeuN-positive
cells expressing GFP throughout all examined sections, indicating widespread neuronal
transduction. Unlike PND 5 injections, which resulted in transduction of the cerebellum, PND
21 administration of AAV9-CMV-eGFP produced virtually no GFP-positive cells in the
50
cerebellum and showed very limited transduction in the piriform cortex (Table 8), suggesting a
lower vector diffusion after injection at PND 21 compared to PND 5.
In summary, the pattern of eGFP expression observed after i.c.v. administration of AAV9-
CMV-eGFP on PND 5 showed a preferential astrocytic transduction with an extensive
distribution in forebrain regions and the cerebellum, while injections on PND 21 gave a nearly
exclusive neuronal transgene expression and a more restricted distribution in forebrain regions
and no expression in the cerebellum.
4.3. Use of the synapsin-1 promoter to achieve neuron-specific
transduction
We next assessed CNS expression after i.c.v. injection of single stranded AAV9-SYN-eGFP
on PND 5 and PND 21 to compare the cell type specificity and level of transgene expression
using this vector with the same construct driven by the CMV promoter. The use of the
synapsin-1 promoter resulted in extensive neuron-specific transduction regardless of the age of
injection. Intra-cerebroventricular injections of AAV9-SYN-eGFP on PND 5 or PND 21 both
resulted in robust neuron-specific eGFP expression in the prefrontal and cingulate cortex,
striatum (Fig. 11, G-L), retrosplenial cortex (Fig. 12, G-L), hippocampus (Fig. 13, G-L).
However, transgene expression in the piriform cortex and cerebellum (Fig. 14, G-L) was only
observed in mice injected on PND 5. The fact that there was no transgene expression in the
cerebellum after i.c.v. delivery of vectors on PND 21, suggests a wider dispersion of AAV9
after i.c.v. delivery at PND 5. In the cerebellum, unlike the AAV9-CMV-eGFP which resulted
in transgene expression only in the Purkinje cells (Fig. 14, A-F), transduction of AAV9-SYN-
eGFP extended to neurons in the granule cell layer as well (Fig. 14, G-L).
51
Fig. 11. AAV-eGFP transgene expression in the mouse striatum.
eGFP transgene expression in the mouse striatum. AAV9 vectors were injected on PND 5
(A-C, G-I) or on PND 21 (D-F, J-L). Different patterns of cell-specific gene expression were
observed; AAV9-CMV-eGFP predominantly transduced protoplasmic astrocytes in PND 5
injections, as assessed by double labeling with anti-GFP with the protoplasmic astrocyte-
specific anti-S100β antibody (A-C). In contrast, AAV9-CMV-eGFP vectors injected on PND
21 and AAV9-SYN-eGFP injected at either PND 5 or PND 21 resulted in primarily neuronal
transduction (D-L), as confirmed by co-staining with NeuN. Insets: cells of interest at higher
magnification. Scale bars = 50 µm.
52
Fig. 12. AAV-eGFP transgene expression in the retrosplenial cortex.
In mice injected with AAV9-CMV-eGFP on PND 5, the majority of eGFP positive cells in
the retrosplenial cortex were confirmed to be protoplasmic astrocytes by double labeling with
anti-S100β (A-C). In contrast, AAV9-CMV-eGFP injected on PND 21 and AAV9-SYN-eGFP
injected at either PND 5 or PND 21 resulted in transduction of almost exclusively neurons (D-
L), as confirmed by co-staining with anti-NeuN. Insets: cells of interest at higher magnification.
Scale bars = 50 µm.
53
Fig. 13. AAV-eGFP transgene expression in hippocampus.
Transgene expression in the CA1 region of the hippocampus after administration of AAV9-
CMV-eGFP (A-F) or AAV9-SYN-eGFP (G-L) vectors on PND 5 or PND 21. Brain sections
from the hippocampus of AAV-injected mice were double labeled using anti-GFP and anti-
NeuN. Insets: cells of interest shown at higher magnification. Scale bars = 50 µm.
54
4.4. Low transduction efficiency of AAV5-GFP vectors
We also sought to compare AAV9 vectors with an AAV5 vector. The AAV5-eGFP vector
contained the chicken beta actin (CBA) promoter. The CBA promoter has previously been used
in AAV vectors to transduce neurons (Gray et al., 2010; Gray et al., 2011). We observed
however that unlike the AAV9 vectors, i.c.v. injections of titer-matched AAV5-CBA-eGFP
vector on PND 5 resulted in transduction of very few cells in the striatum, most of which were
located close to the lateral ventricles (data not shown). No GFP immunoreactivity was detected
in other brain regions in mice injected at PND 5 with AAV5-CBA-eGFP. Administration of
AAV5-GFP at PND 21 resulted in transduction of a few cells in the hippocampus, striatum,
prefrontal cortex, cingulate cortex and retrosplenial cortex, but no transduction in the piriform
cortex and cerebellum (Table 8). Taken together, these results indicate (a) higher transduction
efficiency was obtained with AAV5-CBA-eGFP injected on PND 21 compared to PND 5, and
(b) at both PND 5 and 21, the two AAV9 vectors showed higher diffusion in the CNS compared
to the AAV5-based vector.
55
Fig. 14. AAV-eGFP transgene expression in the cerebellum.
eGFP transgene expression in the cerebellum after injection of mice with AAV9-CMV-eGFP
or AAV9-SYN-eGFP on PND 5 (A-L). Co-staining with ant-calbindin was used to confirm the
presence of GFP positive cells in Purkinje neurons. M: molecular layer; G: granular layer; P:
Purkinje cell layer. Insets: selected Purkinje neurons shown at higher magnification. Scale bars
= 200 µm (A-C, G-I), 50 µm (D-F, J-L).
56
Table 8. Analysis of AAV-eGFP vector transduction patterns in discrete
areas of the brain after i.c.v. delivery in PND 5 or PND 21 mice. PND 5 PND 21
AAV5-
CBA-eGFP
AAV9-
CMV-
eGFP
AAV9-
SYN-
eGFP
AAV5-
CBA-
eGFP
AAV9-
CMV-
eGFP
AAV9-
SYN-
eGFP
N 3 5 3 2 3 3
Prefrontal cortex _ +++ +++ + ++ ++
Striatum + ++++ ++++ ++ ++++ ++++
Cingulate cortex + ++ ++++ + ++ ++++
Hippocampus _ ++++ ++++ + ++++ ++++
Retrosplenial
cortex
_ ++ ++++ + ++ ++++
Piriform cortex _ +++ +++ _ + +
Cerebellum _ ++ +++ _ _ _
AAV vector Age of
administration
GFP+ / mm2
(mean ± s.e.m.)
%
GFP+/NeuN+
(mean± s.e.m.)
%GFP+/S100β
+ (mean±
s.e.m.)
AAV9-CMV-eGFP PND 5 261 ± 91 ND 89 ± 1
AAV9-SYN-eGFP PND 5 1027 ± 91 95 ± 1 ND
AAV9-CMV-eGFP PND 21 195 ± 23 81 ± 6 ND
AAV9-SYN-eGFP PND 21 856 ± 85 92 ± 2 ND
A. Semi-quantitative analysis of vector transduction patterns in discrete areas of the brain.
The levels of transduction were graded as: (-) no transduction, (+) a few transduced cells, (++) a
moderate number of transduced cells, (+++) many transduced cells and (++++) a region that was
saturated with transduced cells. B. Quantitative analysis of eGFP transduction in the
retrosplenial cortex. The total number of eGFP-positive cells in both hemispheres was counted
in three sections from each brain and reported as average number of cells per square millimetre.
Percentages of neuronal and astrocytic transduction were calculated based on the number of
eGFP-positive cells co-localized with antibodies to NeuN and S100β.
Predominant neuronal transduction Predominant astrocytic transduction
B.
A.
57
Chapter 5. FXS gene therapy-Phase 1: Reduced phenotypic severity following adeno-associated virus mediated Fmr1 gene
delivery in postnatal day 5 Fmr1 mice
Acknowledgements: Vector injections, Western blotting and behavioral experiments were
performed with assistance from Dr. Jason Arsenault (Post-doctoral fellow), Ingrid Xuan
(Graduate student) and Laura Pacey (Research Associate).
5.1. Specific hypotheses, objectives and rationale
There is no pharmacological cure for FXS and the currently prescribed medications, such as
anti-psychotics, anti-depressants, and stimulants, only partially alleviate selected symptoms and
are associated with deleterious side effects. Newer second generation drugs to treat FXS,
including metabotropic glutamate receptor 5 (mGluR5) antagonists, are being investigated in
clinical trials (Castren et al., 2012; Hampson et al., 2012; Pop et al., 2014). However,
considering the plethora of genes whose expression is regulated by FMRP, a priori, it may be
expected that restoring FMRP expression in the CNS could provide a more comprehensive
reversal of the disorder compared to targeting single molecules (e.g. mGluR5).
FMRP is widely distributed throughout most regions of the CNS; therefore a major goal of
this study was to identify conditions for AAV-FMRP administration that would provide
widespread transgene dispersion in the brain. We also sought to attain a level of expression that
was as close to WT levels as possible to achieve the most comprehensive behavioral rescue after
AAV delivery. All analytical assessments were carried out during two periods post-injection:
the first began 22 days post-injection (on PND 27) and is hereafter referred to as the “short
arm”, and the second began 50 days post-injection (on PND 55) and is referred to as the “long
arm” (see Fig. 15b).
58
Fig. 15. Fmr1 gene delivery (Phase 1): Overview of viral vector construction,
experimental plan for injections, and behavioral analyses.
(a) Schematic depiction of the AAV-FMRP vector construct. The single-stranded AAV
viral vector contained the human synapsin-1 promoter and inverted terminal repeat (ITR)
elements from AAV serotype 2 and packaged in serotype 9 capsids. A woodchuck hepatitis
post-transcriptional regulatory element (WPRE) was inserted downstream of the mouse Fmr1
cDNA to induce elevation of transcripts. (b) Timelines for the short and long arms of the study
for mice injected with PBS or viral vector on postnatal day 5. The total number of mice (N)
injected for each treatment group is indicated.
59
5.2. Quantification of FMRP expression levels following neonatal i.c.v.
injection of AAV-FMRP in postnatal day 5 mice
To quantify total transgene expression, samples of the cerebellum, inferior colliculus,
cerebral cortex, striatum and hippocampus were subjected to quantitative western blotting. In
samples from WT mice, three bands corresponding to FMRP isoforms were detected (Fig. 16).
In Fmr1 KO mice injected with AAV-FMRP, as expected, only isoform 1 was observed (Fig.
16). Fmr1 KO mice injected with AAV-FMRP and sacrificed at PND 31 (Fig. 16a and c),
revealed 52 ± 9% of WT FMRP expression in the hippocampus, 41±13% in the striatum, and 71
± 20% in the cerebral cortex (Fig. 16c). At PND 60 (Fig. 16b and c), the AAV-FMRP-injected
Fmr1 mice displayed a mean 47 ±15% of WT expression in the cerebral cortex, 48 ± 20% of
WT expression in the hippocampus and 18 ±5% of WT expression in the striatum (Fig. 16c).
The results shown in the western blots in Fig. 16a, b, and d, and in the error bars shown in the
summary graph in Fig. 16c clearly reveal variable expression of the transgene from mouse-to-
mouse in the brain regions examined. The variable range of FMRP transgene expression levels
in forebrain regions from mouse-to-mouse was likely the result of the ability of the vector to
diffuse from the site of the injection in the lateral ventricles. FMRP was not detected in brain
regions more distal from the lateral ventricles such as the inferior colliculus and the cerebellum;
these brain regions are likely located too far from the ventricles to acquire sufficient vector
uptake for FMRP transgene expression and detection. Importantly, transgene expression levels
in the brain regions where it was detected remained relatively constant for at least 7 months
post-injection, showing 77 ± 28% of WT expression in the cortex, 40 ±14% in the hippocampus
and 14 ± 3% in the striatum (Fig. 16c and d).
60
Fig. 16. Western blots of FMRP expression in control and AAV-FMRP-
treated mice.
(a) Representative western blots of the cerebral cortex, hippocampus, and striatum from
individual mice injected with AAV-FMRP in the short arm where brain samples were collected
at PND 31. (b) FMRP transgene expression in the long arms of the study at PND 60. (c)
Quantification of samples from the short and long arms of the study as well as long term (7
months) expression. Expression in injected Fmr1 KO mice was normalized to the GAPDH
signal intensity and compared with age and sex-matched PBS-injected WT brain regions (100%
FMRP expression). The results are presented as the mean ± S.E.M. (d) Representative western
blots of Fmr1 KO mice 7 months after i.c.v. injection with AAV-FMRP.
5.3. Distribution and cellular selectivity of transgene expression
Immunocytochemical analysis revealed expression of the FMRP transgene in the striatum,
hippocampus, retrosplenial cortex and cingulate cortex at both the end of the short arm (26 days
post-injection) and the long arm (56-62 days post-injection) of the study (Figs. 17a and 18).
61
Representative sagittal images of FMRP expression from PBS-injected WT and KO mice and
AAV-FMRP-injected KO mice are shown in Fig. 17b. No FMRP expression was detected in
regions more distant and caudal to the site of injection in the lateral ventricles including the
piriform cortex, cerebellum, inferior colliculus, and brainstem. The highest transduction
efficiencies were observed in the retrosplenial cortex and the cingulate cortex, while lower
FMRP expression was observed in the striatum (Figs. 17a, b and 18).
Double-labeling experiments showed that FMRP was predominantly observed in NeuN-
positive cells within the striatum, hippocampus, retrosplenial cortex and cingulate cortex,
suggesting strong preferential expression in neuronal populations (Figs. 17c, 18, and 19).
Quantitative analysis of FMRP positive cells co-expressing NeuN in the cingulate cortex
revealed over 90% neuronal transduction (Fig. 19). In contrast, we did not detect transduced
cells that were immune-positive for the glial selective marker S100β in any of the brain regions
examined (Fig. 17c), indicating no or undetectable levels of FMRP transduction in astrocytes.
The latter observation is consistent with the use of the neuron-selective synapsin promoter used
to drive transgene expression. Analysis of the cellular localization of FMRP in AAV-FMRP
transduced cells revealed predominant expression in the cytoplasm of neurons (Figs. 17c, d and
18). This cytoplasmic localization of the FMRP transgene was observed in all brain regions
where transgene expression was detected and was similar to that observed previously in neurons
of WT mice (Pacey et al., 2013; Sidorov et al., 2013).
62
Fig. 17. Immunocytochemical analysis of AAV-FMRP transduction in
discrete brain regions
(a) Diagrammatic representation of FMRP transgene expression in Fmr1 KO mice treated
with AAV-FMRP. The levels of transduction were graded as indicated. (b) Representative low
magnification (top row) and higher magnification (bottom row) sagittal images showing FMRP
expression in PBS-injected WT, AAV-FMRP-injected Fmr1 KO mouse, and PBS-injected Fmr1
KO mouse brain. (c) Photomicrographs depicting neuronal specificity of the AAV-FMRP
63
transduction in the cingulate cortex of mice collected at PND 61. Brain sections were double
labeled using anti-FMRP antibody and either the neuronal marker NeuN or the glial specific
marker S100β. Scale bars = 20 µm (top row), 50 µm (bottom row). (d) Higher magnification
photomicrographs illustrating similar cytosolic distributions of FMRP in the cortex of WT and
AAV-FMRP-injected KO mice. Scale bars = 20 µm in all panels.
5.4. Phase 1 - Behavioral analyses
Behavioral experiments were carried out to determine if AAV-induced restoration of FMRP
in the brains of Fmr1 mice could rescue or ameliorate pathological behaviors typically seen in
Fmr1 KO mice (and observed here in PBS-injected Fmr1 KO mice). In the behavioral tests, if a
mouse displayed an obvious abnormality that could influence the test, such as weight loss
following malocclusion, it was excluded from the analysis. Overall, this happened infrequently
and did not appear to be restricted to any particular treatment group. Additional contributions to
the variable number of mice analyzed in each test include the assessment of only male mice in
the ultrasonic vocalization analysis, and in tube test, the N values represent the total number of
pairings; this depended on the availability of age-matched groups at the time of testing.
In the short arm of the study, the tests included measurement of locomotor activity, marble
burying (as a measure of repetitive behavior), and audiogenic seizure susceptibility. Separate
groups of mice were examined in the long arm of the study (i.e. no mice studied in the short arm
were again studied in the long arm). In the long arm, motor activity and marble burying were
measured along with two additional tests: ultrasonic vocalizations to examine mouse-to-mouse
communication during courtship behavior, and the tube test which measures social dominance.
In both the short and long arms, preliminary statistical analyses were conducted using two-way
ANOVA on males and females (i.e. gender and treatment as the main effects). These results
indicated no significant interaction effect between males vs. females; therefore further statistical
analyses were only carried out on pooled data from males and females combined.
64
In the motor activity test, a significant increase in the total distance travelled was seen in both
PBS-injected and AAV-FMRP injected Fmr1 KO mice compared with age and gender-matched
PBS-injected WT mice in the shorts and long arms of the study (Figs. 21a and 20). Thus, AAV-
FMRP vector administration did not rescue the motor hyperactivity in Fmr1 KO mice.
In the marble burying test, used as a measure of repetitive behavior, a significant increase
was observed in the number of marbles buried by PBS-injected Fmr1 KO mice compared to
PBS-injected WT mice (Fig. 21b; F = 29.79, p < 0.001), similar to previous observations made
in some strains of the Fmr1 KO mouse (Spencer et al., 2011). There was a significant decrease
in the number of buried marbles in Fmr1 mice injected with AAV-FMRP compared to PBS-
injected Fmr1 KO mice (Fig. 21b), indicating that the introduction of FMRP rescued
stereotypical behavior. In the short arm of the study this trend was also observed but did not
reach statistical significance due to the lower number of marbles buried by mice in all groups at
PND 32 (Fig. 20; F = 4.01, p > 0.05). The variable level of FMRP transgene expression in
AAV–FMRP-injected animals provided us an opportunity to examine potential correlations
between FMRP expression and behavioral rescue. A preliminary analysis based on a small set
of data points (n = 10), suggested a possible inverse correlation between the number of marbles
buried and cortical levels of FMRP protein expression (Fig. 22; r2
= 0.37, p = 0.06). To
summarize, the trend towards reversal of elevated repetitive behavior seen in the short arm was
more robust and statistically significant in the long arm of the study.
In summary, FMRP transgene expression in the phase 1 study resulted in full reversal of the
elevated repetitive behavior and the deficit in social dominance behavior seen in
PBS-injected Fmr1 KO mice. In other behavioral tests such as audiogenic seizure, motor
activity and ultrasonic vocalizations, there was no statistically significant difference between the
Fmr1 KO mice treated with PBS or AAV-FMRP.
65
Fig. 18. FMRP transgene expression in the striatum, hippocampus, cingulate
cortex, and retrosplenial cortex 61 days after i.c.v. administration of AAV-
FMRP on PND 5.
FMRP transgene expression in the striatum, hippocampus, cingulate cortex, and retrosplenial
cortex 61 days after i.c.v. administration of AAV-FMRP on PND 5. Brain sections were double
labeled using anti-FMRP (green) and anti-NeuN (red). Scale bars = 50 µm.
66
Fig. 19. Quantitative analysis of AAV-FMRP transduction in cingulate
cortex.
The cells were counted in 3 consecutive coronal 25 μm thick sections (100 μm apart) of the
cingulate cortex from each brain (total of 9 sections from 3 mice) and reported as the average
number of cells per square millimeter ± S.E.M.
For the analysis of audiogenic seizure susceptibility, as expected from previous observations
in our laboratory WT C57/BL6J mice were not susceptible to audiogenic seizures (Pacey et al.,
2009). In PBS-injected Fmr1 KO mice, 36% of the females and 31% of the males had seizures.
In the combined male plus female data, the incidence of audiogenic seizures in both PBS-
injected Fmr1 KO mice and AAV-FMRP-injected mice was significantly elevated compared to
PBS-injected WT mice (Fig. 20c; p < 0.05). However, the AAV-FMRP group was not different
than the PBS-injected Fmr1 group.
Animal # FMRP+ cells/mm2
NeuN+/FMRP+
cells/mm2
% of neuronal
transduction
1 1714 ± 470 1588 ± 403 94 ± 2
2 2154 ± 734 2097 ± 731 96 ± 2
3 2042 ± 666 2003 ± 656 98 ± 1
67
Fig. 20. Summary of behavioral analyses of the short arm of the study.
Summary of the behavioral analyses in the short arm of the phase 1 of the study. PBS-
injected Fmr1 KO mice exhibited (a) Elevated motor activity (b) Increased repetitive behavior
and (c) Higher susceptibility to audiogenic seizures compared to PBS-injected WT mice. There
was a trend towards rescue in the marble burying test, but not in motor activity or seizure
susceptibility. Each bar represents the average ± SEM. For panel c, Fisher’s exact test was
performed; each bar represents the percentage of mice that had seizures (* p < 0.05; *** p <
0.001).
68
Fig. 21. Summary of the behavioral results from the long arm of the study.
Summary of the behavioral results from the long arm of the study at postnatal days 55 to 61.
PBS-injected Fmr1 KO mice exhibited hyperactivity, increased repetitive behavior, decreased
ultrasonic vocalizations, and a reduction in social dominant behavior compared to PBS-injected
WT mice. (a) Total horizontal activity over 20 min. (b) Rescue of stereotypical behavior as
seen by total number of marbles buried during 30 min. (c) Total number of ultrasonic
vocalization during a 4 min encounter in naive (left) and treated mice (right). (a-c) Each bar
represents the average ± SEM (*** p < 0.001). (d) Rescue of impaired social dominance in the
tube test in AAV-FMRP Fmr1 KO mice compared to PBS-injected Fmr1 KO mice. Columns
indicate total % of wins for each group. The tube test was administered to PBS-treated WT
paired with PBS-Fmr1 KO mice (top), PBS-treated Fmr1 KO mice paired with AAV-FMRP-
treated Fmr1 KO mice (middle), and WT PBS paired with AAV-FMRP mice (bottom). The
number of wins for each group was tallied and a chi-square analysis was used to determine
whether the scores were significantly different from the 50:50 win/loss outcome expected by
chance. n = the number of pairings. ** p < 0.01, *** p < 0.001.
Ultrasonic vocalizations were recorded from PND 59 male mice. In a preliminary
experiment, we tested adult naïve uninjected WT and Fmr1 KO and observed a significant
69
decrease in the number of ultrasonic vocalized calls in Fmr1 KO mice compared to WT mice
(Fig. 21c left panel; F = 2.53, p < 0.01). This result confirmed a previous finding showing
reduced vocalizations in Fmr1 mice (Rotschafer et al., 2012). We then conducted a second
study comparing male PBS-injected WT mice with male PBS-injected and AAV-FMRP-
injected Fmr1 mice. Although the number of ultrasonic vocalizations was lower in the PBS-
Fmr1 KO group compared to AAV-FMRP-Fmr1 KOs and PBS-treated WT mice, there was no
statistically significant difference among the 3 groups (Fig. 21c, right panel; F = 0.32, p > 0.05).
Impaired social dominance is an established characteristic of Fmr1 KO mice (Spencer et al.,
2005; Pacey et al., 2011). The tube test measures social dominance and aggressive tendencies
without allowing mice to injure one another. Dominant versus submissive behaviors were
scored for two age and sex-matched mice during a brief pairing. For each trial the more
dominant mouse pushes the other out of the tube and is deemed the winner while the second
(subordinate) mouse is the loser. All combinations of WT-PBS, Fmr1-PBS and Fmr1-AAV-
FMRP were evaluated and tallied as the percent of wins by each group against their opponents.
As expected based on previous studies (Spencer et al., 2005; Pacey et al., 2011), Fmr1 KO mice
injected with PBS won significantly fewer matches than expected by chance against WT mice
injected with PBS (Fig. 21d; p < 0.001). Male and female Fmr1 animals injected with AAV-
FMRP won significantly more matches against gender-matched PBS-injected Fmr1 KO mice
(Fig. 21d, p < 0.01). Thus, the deficit in social dominance in Fmr1 KO mice was rescued in the
AAV-FMRP- treated mice.
70
Fig. 22. Correlation between the number of marbles buried and FMRP
transgene expression levels in the cerebral cortex.
The location of each point on the graph depicts the level of FMRP transgene expression as
determined by western blotting from an individual mouse mapped to the marble burying
behavior of that mouse. Linear regression of the data points showed a noticeable, albeit non-
significant, correlation of r2
= 0.37, p = 0.06, F = 4.61, n = 10.
71
Chapter 6. FXS gene therapy-Phase 2:
Fmr1 transgene delivery in postnatal day 0 Fmr1 mice
Acknowledgements: Vector injections, Western blotting and behavioral experiments were
performed with assistance from Dr. Jason Arsenault (Post-doctoral fellow).
6.1. Specific hypotheses, objectives and rationale
We hypothesized that i.c.v. delivery of viral vectors at PND 0-1 may facilitate AAV
distribution in the brain, and therefore provide a more comprehensive behavioral rescue,
compared to PND 5 injections, as conducted in phase 1 of this work. Our rationale for
delivering the AAV viral vectors at PND 0-1 is based on (a) our own observations from pilot
experiments (see below), and (b) a previous study by Kim, et al (2013) both indicating the most
widespread diffusion of AAV vectors after i.c.v. delivery at PND 0-1 and diminished spread of
the virus with age (PND 2-5), particularly in distant structures from the site of injection,
including the brainstem and cerebellum. These authors also demonstrated that the widespread
expression of virally-encoded transgene is apparent within one day post-injection, during early,
critical stages of neuronal development, and it is also stable for up to 12 months post-injection
(Kim et al., 2013b). Here, we re-introduced the FMRP protein at an earlier time point in order
to elevate the transduction levels as well as heighten the overall brain distribution of the
transgene, while avoiding potentially pathological over-expression. Since the ependymal cell
lining surrounding the ventricles of the brain is still immature during the first 24 hours after
birth, the AAV vectors can more easily transfer from the CSF to the brain parenchyma (Passini
and Wolfe, 2001; Kim et al., 2014; Yamazaki et al., 2014). Moreover, while taking advantage
of the innate mouse-to-mouse variability of transduction levels seen in previous studies
(Gholizadeh et al., 2014), we sought to investigate what range of FMRP transduction and
expression levels would be most adequate for long-term behavioral correction in Fmr1 KO
mice.
72
Another objective in this project was to evaluate the potential effects of FMRP over-
expression in the brain on mouse behavior. Therefore, WT C57/BL6 mice were also injected
with a range of AAV-FMRP vector particles (0.75 to 1 µl of 1010
genome/µl) to investigate the
effects of over-expression. These variable levels of transgene expression could give us better
insight into the proper range of FMRP needed in different regions of the brain to achieve a
proper rescue. The effects of FMRP over-expression in humans and mice are presently unclear.
To date, few patients with duplication of the entire FMR1 gene have been reported (Rio et al.,
2010; Nagamani et al., 2012; Vengoechea et al., 2012; Hickey et al., 2013) . FMRP duplication
in these case reports has been associated with severe developmental delay, fine motor and
speech delay, and progressive seizures, which support the notion that regulated Fmr1 gene
dosage is critical for normal neurocognitive function. To our knowledge, only one study
investigated the effects of FMRP over-expression on mouse behavior, suggesting that over-
expression may be pathological in some but not all of behavioral phenotypes associated with
Fmr1 mice (Peier et al., 2000). These conclusions were based on the finding that while KO
mice demonstrate hyperactivity and reduced anxiety, transgenic mice that over-express Fmr1,
demonstrate hypo-activity and elevated anxiety. However, these conclusions might have been
premature since the transgenic mouse used in these experiments expressed the human FMRP
protein at levels 10 times greater than average FMRP levels in healthy individuals. Although
the mouse and human FMRP share 97% homology, there are important differences in their
mRNA binding properties (Denman and Sung, 2002), and over-expression of the human FMRP
isoform at immensely high levels in the brains of transgenic mice, might have obvious
detrimental effects on their behavior.
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Table 9. Summary of the main differences in the experimental design between
Phase 1 and Phase 2 Main differences Phase 1 Phase 2
Age of mice receiving
viral injections
PND 5 PND 0-1
Control injections PBS AAV-null
Injection groups 1. Fmr1 KO : PBS
2. Fmr1 KO: AAV-FMRP
3. WT : PBS
1. Fmr1 KO: AAV-null
2. Fmr1 KO: AAV-FMRP
3. WT: AAV-null
4. WT: AAV-FMRP
Behavioral analysis Short arm (starting on PND 27) and
Long arm (starting on PND 55)
Only long arm (starting on PND 55)
The two phases of the gene therapy study differed in the age of injection, control injections,
and behavioral analysis.
Fig. 23. Fmr1 gene delivery (Phase 2): Overview of experimental plan for
injections, and behavioral analyses
Overview of tests following AAV injections; The behavioral tests started on PND 55, which
is 54-55 days after bilateral i.c.v. injections of viral vectors on PND 0-1. The behavioral tests
were performed at least 2 days apart and all animals were tested only once in each behavioral
paradigm.
6.2. Quantification of FMRP expression levels following neonatal i.c.v.
injection of AAV-FMRP in postnatal day 0 mice
A total of 206 neonatal WT and Fmr1 mice were included in the phase 2 study. All mice
were injected under blinded condition with either AAV-null or AAV-FMRP. Among the 206
injected mice, 27 displayed hydrocephalus and were immediately euthanized or, following post
mortem analysis, were seen to have had brain damage from the procedure and were excluded
from the study, on the assumption that their behavior would be adversely affected. Brain
damage was not restricted to any one treatment group or genotype, but was evident across all
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groups and both genotypes. The absence in elevated glial fibrillary acidic protein (GFAP)
staining following AAV administrations into the CNS suggested a lack of astrocyte activation
and neuroinflammation in response to AAV injections (Fig. 24).
Fig. 24. Analysis of GFAP following AAV-FMRP injections in WT animals.
GFAP was probed to investigate astrocyte activation. A-B) Western immunoblotting and
protein quantification of GFAP in 2-month-old naive WT (n = 4) or WT-AAV-FMRP (n = 5)
mice, shows no elevation in GFAP levels in the striatum (A) and hippocampus (B).
Western immunoblotting was used to determine the relative expression rates (compared to
WT-null) found in the mouse brain. Five brain regions were assessed for FMRP transgene
quantification. Fig. 25A shows a topographical view of the brain regions examined. The cortex
(C) comprises the retrosplenial area, the anterior cingulate area, the visual area, and the posterior
somatomotor area. The frontal cortex (FC) comprises the anterior somatomotor area, the pre-
limbic area, the orbital area and the inferior limbic area. The cerebellums, hippocampus and
striatum were also collected separately for analysis. Fig. 25B shows these same brain regions as
seen by a sagittal view. Fig. 25C shows exemplified western blotting of the cortex (top left),
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frontal cortex (top right) and hippocampus (bottom left) of Fmr1 KO mice injected with AAV-
FMRP (KO + FMRP). AAV-null injected WT (WT-null) was used to normalize protein
expression while AAV-null injected Fmr1 KO was used as a negative control to confirm
antibody specificity. The cortex of WT mice injected with AAV-FMRP (WT+FMRP) can also
be seen (bottom right). Fig. 25D shows the bar charts representing the average values of each of
these brain regions. We observed a 54 ± 7% expression rate in the cortex of Fmr1 KO mice
injected with AAV-FMRP, 115 ± 18% in the frontal cortex, 80 ± 15% in the hippocampus,
34±8% in the striatum and 2±1% in the cerebellum. The variable range of FMRP transgene
expression levels in forebrain regions was likely the result of mouse-to-mouse variation in the
ability of the vector to diffuse from the site of the injection in the lateral ventricles. FMRP was
detected at very low levels in the cerebellum, which likely reflects the longer distance from the
site of injection in the lateral ventricles compared to forebrain regions.
In the WT mice injected with AAV-FMRP, we observed an average expression level of 235
± 47% in the cortex, 182 ± 30% in the frontal cortex, 209 ± 45% in the hippocampus, 113 ±
24% in the striatum and 91 ± 4% in the cerebellum, compared to WT-null-injected mice. The
quantitative western immunoblotting results showed that in addition to achieving a wider
distribution of the FMRP transgene compared to that seen in phase 1, the overall levels of CNS
expression were higher in the mice analyzed in phase 2.
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Fig. 25. Western blots of FMRP expression in AAV-FMRP-treated KO and
WT mice.
AAV-FMRP transgene, and PSD-95 protein expression levels. A. Schematic representation
of the brain regions investigated (top view). The regions comprise the cortex (C; see text for
anatomical description), the frontal cortex (FC), the cerebellum (CB), the striatum (S) and the
hippocampus (H). B. Sagittal representation of the brain regions investigated. C.
Representative western blots of FMRP expression in the mouse brain. The FMRP transgene
expression level in Fmr1 KO mice injected with AAV-FMRP shown in the cortex (top left), the
frontal cortex (top right) and the hippocampus (lower left). The lower right panel shows the
FMRP expression levels in the cortex of WT mice injected with AAV-FMRP. D. Summary of
quantification of FMRP levels in different brain regions of Fmr1 KO mice (left) and WT
(middle) injected with AAV-FMRP. E. Quantification of PSD-95 in the cortex. All protein
levels were normalized to WT-null expression rates and corrected with Gapdh protein levels.
Results are shown as the average ± SEM. *p<0.05.
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6.2.1. Levels of FMRP substrates in AAV-FMRP-treated mice
Expression of the FMRP transgene, at or near WT levels in the KO mouse brain, is expected
to restore the natural functions of the WT protein, and normalize the translation of its substrate
mRNAs. To investigate this, we analyzed the protein expression levels of two mRNA substrates
of FMRP, namely PSD-95 and MeCP2 in the brains of AAV-FMRP-injected KO mice.
PSD-95 (post-synaptic density-95) is a synaptic adapter and transducer protein that is highly
expressed throughout the CNS, and also functions as a scaffold protein to link downstream
signaling components to mGluRs. PSD-95 mRNA is a well-established target for FMRP, and
previous studies have reported reduced PSD-95 protein levels in the Fmr1 KO mouse brain
(Zalfa et al., 2007). This down-regulation is at least partly explained by the well-established
role of FMRP in maintaining PSD-95 mRNA stability (Zalfa et al., 2007). PSD-95 protein in
the cerebral cortex of 2-month-old KO-null mice was reduced by 28±7% compared to that
observed in the WT-null group (Fig. 25E). In the KO+FMRP-treated group, PSD-95 levels
were restored to the level seen in the WT-null mice. A small elevation of PSD-95 was observed
in the WT+FMRP group compared to the WT-null group, indicating that FMRP over-expression
over native WT levels had relatively little impact on PSD-95.
MeCP2 is a transcription factor that is severely down-regulated due to gene mutation in Rett
syndrome, another monogenic autism spectrum disorder. MeCP2 was listed as a substrate for
FMRP in a previous study (Ascano et al., 2012). To confirm MeCP2 as an mRNA substrate for
FMRP, we initially examined cellular co-localization of FMRP and MeCP2. FMRP, which is
principally localized in the cytosol, and MeCP2, a nuclear protein, are co-expressed within the
same cells in the cortex (Fig. 26A), the striatum (Fig. 26B) and the hippocampus (Fig. 26C).
Despite the different compartmentalization of the two proteins, almost all MeCP2 positive cells
were also positive for FMRP. Subsequently, an mRNA pull down followed by quantitative RT-
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PCR was performed to determine if MeCP2 mRNA would be enriched in the anti-FMRP
immunoprecipitate. There was a modest enrichment of MeCP2 mRNA in whole forebrain
isolates from WT C57/BL6 mice, where PSD-95 mRNA served as a positive control (Fig. 26D).
A significant MeCP2 enrichment was also seen in FMRP immunoprecipitate in the human
embryonic kidney 293 cell line (Fig. 26E). These results indicate that both homo sapiens and
mus musculus MeCP2 mRNA interacts with FMRP.
To ascertain the functional relevance of this finding, we quantified MeCP2 protein
expression levels in the cortex (Fig. 26F). In contrast to reduced PSD-95 protein levels in Fmr1
KO mice, MeCP2 expression levels were elevated by 32 ± 8% in the cortex compared to WT-
null levels (*p<0.05). The introduction of FMRP significantly reduced the MeCP2 protein
levels compared to KO-null (Fig. 26F, **p<0.01). WT+FMRP group also had a significant
reduction in MeCP2 protein levels compared to KO-null (Fig. 26F, ***p<0.001). Since micro
gene duplications of MeCP2 was previously reported to cause severe intellectual disabilities in
humans (Fieremans et al., 2014), it is plausible to assume that high levels of MeCP2 in Fmr1
KO mice might be a contributing factor to at least some of the pathological mechanisms of this
disorder. Overall, these results showed that the AAV-FMRP was able to correct the up-
regulation in MeCP2 protein levels and partially ameliorate the down-regulation of PSD-95
levels seen in Fmr1 KO animals. The FMRP transgene thus recapitulated the native
translational modulation of endogenous FMRP.
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Fig. 26. Characterization of MeCP2 as an mRNA substrate for FMRP and
quantification of MeCP2 levels in AAV-treated WT and KO mice.
A-C: High magnification of WT coronal sections labeled with anti-FMRP 5c2 (green) and
anti-MeCP2 antibodies (red) of the A. cortex, B. Striatum and C. Hippocampus. D-E: FMRP
immunoprecipitation and mRNA quantification. D. Total mRNA enrichment of Gapdh, PSD-
95, and MeCP2 from whole mouse forebrain. E. Total mRNA enrichment of Gapdh and
MeCP2 from Hek 293 cell lysates. Results were normalized to Gapdh mRNA levels. Results
are shown ± S.E.M. *p<0.05. F. Quantification of MeCP2 levels in the cortex of WT-null,
WT+FMRP, KO-null, and KO+FMRP. MeCP2 protein levels were normalized to WT-null
expression rates and corrected for Gapdh protein levels. Results are shown as the average ±
SEM. *p<0.05, **p<0.01, *** p<0.001.
6.3. Distribution and cellular selectivity of transgene expression
Immunocytochemical analysis of AAV-injected KO mice sacrificed on PND 65 revealed
expression of the FMRP transgene in the striatum, hippocampus, retrosplenial cortex, cingulate
cortex, piriform cortex, prefrontal cortex, amygdala, thalamus, and at lower levels in dispersed
cells in superior colliculus, inferior colliculus and cerebellum (Fig. 27). A representative
sagittal image of FMRP expression from AAV-FMRP-injected KO mice is shown in Fig. 27A
emphasizing the brain regions with higher levels of FMRP transgene expression. The highest
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transduction efficiencies were observed in the hippocampus and most cortical regions. Unlike
phase 1, FMRP transgene expression was detected in the anterior forebrain such as the
orbitofrontal cortex and the anterior olfactory nucleus, which were not transduced in PND 5-
treated animals (Gholizadeh et al., 2014). Fig. 27B shows higher magnification coronal images
at different brain regions of 2-month-old Fmr1 KO animals injected with AAV-FMRP.
Compared to the vector tropism in phase 1, here FMRP transgene is expressed more extensively
in regions more distal from the site of i.c.v. injection including the amygdala, inferior colliculus,
thalamus and some cells in the cerebellum.
Fig. 27. Immunocytochemical analysis of AAV-FMRP transduction in
discrete brain regions of AAV-treated KO mice.
A
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Immunocytochemical analysis of AAV-FMRP transduction in discrete brain regions. (A)
Representative sagittal image showing FMRP expression in AAV-FMRP-injected Fmr1 KO
mouse brain on PND 65. (B) FMRP transgene expression in different brain regions 65 days
after i.c.v. administration of AAV-FMRP on PND 0-1. Brain sections were double labeled
using anti-FMRP (green) and anti-NeuN (red).
As we previously reported, the synapsin promoter endows a neuronal specific tropism of the
FMRP protein in the CNS (Gholizadeh et al., 2013; Gholizadeh et al., 2014). This was again
confirmed in phase 2 study where double-labeling with the neuron-specific marker NeuN
showed that FMRP was predominantly observed in NeuN-positive cells, in all of the brain
regions analyzed, indicating strong preferential expression in neuronal populations (Fig. 27B).
Quantitative analysis of FMRP positive cells co-expressing NeuN in the cingulate cortex
revealed over 90% neuronal transduction (Table 10).
B
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Table 10. Quantitative analysis of AAV-FMRP transduction in cingulate
cortex.
FMRP+ cells were counted in 3 consecutive coronal 25 μm thick sections (100 μm apart) of
the cingulate cortex from each brain (total of 9 sections from 3 mice) and reported as the
average number of cells per visual field. Percent of FMRP+ cells that expressed co-expressed
NeuN, total DAPI+ cells and NeuN+ neurons that expressed FMRP are calculated separately.
Analysis of FMRP over-expression in brains of WT mice injected with AAV-FMRP and
sacrificed on PND 65 revealed obvious FMRP over-expression in hippocampus, striatum,
retrosplenial cortex, cingulate cortex and motor cortex but no obvious over-expression in
cerebellum, compared to matched brain sections from AAV-null injected WT mice (Fig. 28).
These results suggest similar transduction efficiencies of AAV-FMRP in WT and KO neonatal
brain. We also quantified FMRP expression in the cingulate cortex and striatum of AAV-null or
AAV-FMRP-treated WT mice by measuring the total number of FMRP+ cells, average cell size,
and the mean intensity of the FMRP signal in each cell. As expected from the high abundance
and ubiquitous expression of FMRP protein in the brain, there was no difference in the number
of FMRP+ cells and size of the cells in AAV-null compared to AAV-FMRP-treated brains in
cingulate cortex or striatum. However, AAV-FMRP injections resulted in a 67% increase in
mean FMRP expression level in each cell (FMRP signal intensity/cell) in cingulate cortex and
Animal #
FMRP+ cells/mm2
% FMRP+/NeuN+
% DAPI+/FMRP+
% NeuN+/FMRP+
1 173 96 87 98
2 184 99 92 97
3 171 92 92 98
4 168 99 89 97
5 172 92 91 96
Average 173.6 95.6 90.2 97.2
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52% increase in striatum, compared to AAV-null injected mice (Fig. 29). There was a
remarkable variability in FMRP levels in different neurons both in the striatum and cingulate
cortex of AAV-treated WT brain, which possibly stems from a. the natural variability of FMRP
expression levels in different neurons in the WT brain, and b. the fact that more than one virion
can transduce a given neuron resulting in various FMRP levels in different cells.
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Fig. 28. Immunocytochemical analysis of FMRP over-expression in different
brain regions of AAV-SYN-Fmr1-treated WT mice.
NeuN NeuN
FMRP FMRP
DAPI DAPI
Merge Merge
NeuN NeuN
FMRP FMRP
DAPI DAPI
Merge Merge
NeuN NeuN
FMRP FMRP
DAPI DAPI
Merge Merge
WT AAV-null WT AAV-syn
Cingulate cortex Retrosplenial cortex Motor cortex
WT AAV-null WT AAV-syn WT AAV-null WT AAV-syn
Matched brain sections of AAV-null or AAV-SYN-Fmr1 treated WT mice were
double labeled using anti-FMRP (green) and anti-NeuN (red).
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Fig. 29. Quantitative analysis of FMRP over-expression in AAV-SYN- Fmr1-
treated WT mice.
Total number of FMRP+ cells per visual field, the area encompassing each cell (µm2) and
the mean intensity of FMRP+ signal per cell per µm2 is shown in the cingulate cortex or
striatum of AAV-FMRP or AAV-null-treated WT mice.
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6.4. Phase 2 - Behavioral Analyses
The first behavioral analysis was assessment of the reversibility of the well-established
locomotor hyperactivity in Fmr1 KO mice (Kazdoba et al., 2014; Wrenn et al., 2015) after
AAV-FMRP administration. As expected, KO-null compared to WT-null mice displayed a
significant elevation in the horizontal activity (Fig. 30A) and ambulatory activity (Fig. 30B),
while showing a reduction in the number of rest episodes (Fig. 30C). There was also a moderate
but not significant decrease in the total rest time (Fig. 30D). The total distance travelled
between groups was also not significantly different (data not shown). The KO+FMRP group
showed reduced horizontal activity, ambulatory activity and an elevation of the number of rest
episodes, all characteristic of a reduction in hyperactive behavior. Thus, the locomotor
hyperactivity was partially rescued in KO+FMRP mice, with no statistically significant
difference compared to the WT-null group. FMRP over-expression (WT+FMRP) did not
induce significant changes in the motor activity parameters.
Based on the high mouse-to-mouse variability of FMRP transduction rates seen in AAV-
FMRP injected mice, a potential correlation may exist between the total distance travelled and
FMRP or MeCP2 protein levels. There was an inverse trend observed between motor activity
and FMRP levels (Fig. 30E), indicating that FMRP levels might be inversely correlated with the
severity of the hyperactive phenotype. Considering the high number of data points, the innate
variability of the mice, and the high variability of FMRP transduction levels from the vector
delivery, this trend did not reach statistical significance (n = 56, r2= 0.055; p<0.0806).
However, a significant positive correlation was found between MeCP2 levels, and total distance
travelled. (n = 57, r2=0.1231, p<0.0074, Fig. 30F).
In summary, we conclude that (a) FMRP and MeCP2 are co-expressed in many if not most
neurons in the CNS, (b) MeCP2 mRNA is a substrate for FMRP, (c) MeCP2 is intrinsically
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over-expressed in Fmr1 KO mice (d) the combined analysis of all four treatments groups
demonstrated that cortical MeCP2 expression correlated with the severity of the hyperactive
phenotype, and (e) that FMRP transgene expression eliminated the pathological over-expression
of brain MeCP2 levels observed in Fmr1 KO-null-treated mice and concomitantly rectified
motor hyperactivity.
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Fig. 30. Analysis of locomotor activity.
A. Horizontal activity (beam breaks) for the WT-null, WT+FMRP, KO-null, and KO+FMRP
groups during locomotor testing. B. Ambulatory activity shown as total number of events; C.
Rest episodes as measured by the number of pauses (absence of beam breaks for ≥1s); and D.
Total rest time. Results are presented as an average ± SEM. *p<0.05. E. Linear correlation
between the FMRP protein levels as a function of the total distance travelled for the WT-null,
WT+FMRP and KO+FMRP groups combined. F. Correlation between the MeCP2 protein
levels and total distance travelled for WT-null, WT+FMRP, KO-null, and KO+FMRP groups.
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Impaired sensorimotor gating and auditory hyper-excitability are also among the robust,
well-established behavioral impairments in Fmr1 KO mice (Paylor et al., 2008; Rotschafer et
al., 2012), therefore pre-pulse inhibition and the base startle response were investigated. The
KO-null group had a significantly higher base startle response than the WT-null group (Fig.
31A). Furthermore, the KO+FMRP group showed a full rescue with a significant reduction in
this parameter compared KO-null and rendered no different than the WT-null group. The
WT+FMRP group, having the same trend as the KO+FMRP, showed a slight but not significant
reduction in the startle response compared to the WT-null group. The pre-pulse inhibition at 72
dB was not significantly different between null groups (Fig. 31B). Pre-pulse inhibition at 78 dB
(Fig. 31C) and 82 dB (Fig. 31D) also did not show any difference between AAV-null injected
groups, but interestingly, this difference was accentuated between AAV-FMRP injected groups
showing a statistical elevation of pre-pulse inhibition in the KO+FMRP group compared to the
reduced inhibition seen in WT+FMRP. Linear regression did not show any statistical
significance when compared to FMRP expression levels in the quantified mice (data not shown).
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Fig. 31. Analysis of sensorimotor gating.
A. Basal startle reaction corresponding to the pulse alone for the WT-null, WT+FMRP, KO -
null, and KO+FMRP groups. B. Percentage of inhibition to the startle response by submitting
the mice to a 74 dB pre-pulse; C. Percentage of inhibition to a 78 dB pre-pulse, and D.
Percentage of inhibition to a 82 dB pre-pulse. Results are presented as an average ± SEM.
*p<0.05, *** p<0.001.
In the elevated plus maze, which measures anxiety, the Fmr1 KO mice spent significantly
more time in the open arms (Fig. 32A), which is consistent with a previous report (Qin et al.,
2011). Intriguingly, this behavioral abnormality was fully reversed in the KO+FMRP treatment
group (Fig. 32A). Furthermore, the KO-null mice showed a significantly higher number of open
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arm entries compared to WT-null, which was also reversed in KO+FMRP group (Fig. 32C).
FMRP over-expression in the WT+FMRP group showed a significant increase in the number of
open arm entries, compared to WT-null (Fig. 32C), but no difference in the time spent in open
arms (Fig. 32A). The total number of entries was not different among the four groups (Fig.
32D). No significant correlation was detected in the elevated plus maze parameters when
compared to FMRP transgene levels (data not shown).
In the marble burying test, which measures stereotypic behavior in mice, our phase 1 results
(Gholizadeh et al., 2014), together with a previous report (Spencer et al., 2011), showed a
significant elevation in the number of marbles buried in Fmr1 KO mice. In the second phase of
this study, the KO-null and KO+FMRP groups displayed a modest but non-significant increase
in marbles buried compared to the WT-null group (Fig. 32E). The WT+FMRP over-expression
group, however, buried significantly less marbles compared to both KO groups, indicating that
higher levels of FMRP contribute to lowering the total number of marbles buried, as was
previously observed (Gholizadeh et al., 2014).
The number of ultrasonic vocalizations during courtship behavior was found to be reduced in
Fmr1 KO mice in phase 1 (Gholizadeh et al., 2014), as well as a previous study (Rotschafer et
al., 2012). Here, the KO-null mice and the WT+FMRP showed non-significant reductions in
the number of vocalizations (Fig. 32F). The number of vocalizations in the KO+FMRP group
was significantly lower than the number in the WT-null group, indicating no reversal of the
trend towards reduced vocalization in the KO-null mice.
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Fig. 32. Analysis of anxiety, stereotypical behavior, and courtship behavior.
.
A. Elevated plus maze trial showing the total time spent in the open arm for the WT-null,
WT+FMRP, KO-null, and KO+FMRP groups. B. Elevated plus maze trial showing the number
of closed arm entries, C. the number of open arm entries, and D. the total number of entries. E.
Marble burying assay for repetitive, stereotyped behavior showing the total number of marbles
buried during a 30 min. interval. F. Measurement of the total number of ultrasonic vocalizations
of male mice during courtship behavior. Results are presented as an average ± SEM. *p<0.05,
**p<0.01, *** p<0.001.
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Chapter 7. Discussion
7.1. FMRP expression in the developing and adult brain
The purpose of this part of my research was to fully document the cell type expression of
FMRP in neurons and glia in selected brain regions of the developing and mature WT mouse
CNS. Our results demonstrated a predominantly neuronal expression of FMRP in all of the
brain regions analyzed, except the corpus callosum, where only a small number of neurons were
present and FMRP expression was mainly observed in astrocytes. Our data also revealed the
presence of FMRP in cells expressing markers for astrocytes (S100β), microglia (Iba-1) and
oligodendrocyte precursor cells (NG2) in the developing brain. In general, glial expression of
FMRP diminished to low or undetectable levels in the adult brain following different trajectories
in different brain regions.
FMRP expression was abundant in astrocytes at PND 0 in all of the brain regions analyzed;
thereafter, expression in astrocytes showed a gradual decline in striatum and hippocampus,
reaching negligible numbers of FMRP+/S100β+ cells in the adult mouse brain. Pacey and
Doering (2007) reported FMRP expression in cells expressing the astrocytic marker GFAP in
the hippocampus and the ependymal cells surrounding the third ventricle at PND 1 and PND 7,
but no FMRP/GFAP co-localization in the brains of adult (2-month-old) mice. Our results
confirmed the findings of Pacey and Doering, (2007) in the hippocampus using a different
astrocytic marker (S100β), and extended these findings to other brain regions including the
cingulate cortex, corpus callosum, striatum, and cerebellum. Quantitative analysis of cell-type
expression of FMRP indicated that more than 20% of FMRP+ cells in the corpus callosum co-
expressed S100β and this co-localization level remained relatively constant from PND 0 to adult
mice. In contrast, there were few NeuN+ cells detected in the corpus callosum.
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Expression of FMRP in astrocytes could be of crucial importance in regulating the
established functions of astrocytes in synapse formation and neural plasticity. More
specifically, during the early postnatal weeks in rodents when astrocytes play a critical role in
regulation of neuronal growth and synapse formation, the lack of FMRP could contribute to the
abnormal dendritic morphology and synapse development seen in FXS (Jacobs and Doering,
2010). Astrocytes dynamically regulate synaptic transmission, partly by releasing soluble
synaptogenic molecules such as the astrocyte-secreted matricellular proteins, including Hevin,
and thrombospondin-1 (Jones and Bouvier, 2014), or neurotrophic factors such as Neurotrophin-
3 (Yang et al., 2012), which are highly expressed within first few weeks of postnatal
development and decrease as the brain matures. Interestingly, Hevin, also known as SPARC-
like 1, and Neurotrophin-3 have been identified as mRNA substrates for FMRP (Darnell et al.,
2011) and excessive neurotrophin-3, secreted from Fmr1 KO astrocytes, has been suggested to
contribute to abnormal neuronal dendritic development in Fmr1 KO mouse model (Yang et al.,
2012). Furthermore, thrombospondin-1 protein is reduced in astrocytes of Fmr1 KO mice and
synaptic deficits are corrected by its protein replacement in Fmr1 KO mouse hippocampal
neurons (Cheng et al., 2014). These findings highlight the important role for FMRP expression
in astrocytes during early postnatal weeks, which coincides with the peak of synaptogenesis.
FMRP expression has been reported in oligodendrocyte precursors in vitro (Wang et al.,
2004) and in the early postnatal cerebellum (Pacey et al., 2013). Our data extend these findings
by reporting a more comprehensive analysis of FMRP/NG2 co-localization in different regions
of the mouse brain at multiple time points during postnatal CNS development. FMRP
expression is also present in mature MBP+ oligodendrocytes of rodents as well as human
oligodendrocytes (Giampetruzzi et al., 2013). FMRP was previously shown to bind to MBP
mRNA and down-regulate its translation in vitro (Wang et al., 2004). Moreover, the lack of
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FMRP is associated with delayed myelination in the Fmr1 KO mouse cerebellum (Pacey et al.,
2013). In the present study, we observed a gradual decline in the number of FMRP+ cells co-
expressing NG2 from PND 0 to adult mice; this pattern was observed in all of the brain regions
examined, but was most prominent in the corpus callosum and striatum. A decline in FMRP
levels during oligodendrocyte differentiation was previously reported in primary cultures of
oligodendrocytes derived from neonatal rat brain (Wang et al., 2004). This gradual decline
indicates that FMRP likely plays a more important role in myelin formation during CNS
maturation, rather than in mature CNS.
FMRP has been previously reported in cultures of microglial cells (Yuskaitis and Jope, 2009;
Yuskaitis et al., 2010a). However, the findings reported here provide the first direct evidence
for FMRP expression in microglia in brain tissue. Post mortem studies have identified
neuroinflammation and glial activation in the brains of individuals with idiopathic autism
(Tetreault et al., 2012), and other studies suggest a similar pathology may be present in FXS
(Jacobs et al., 2012). FMRP expression was reported in the BV-2 microglial cell line (Yuskaitis
and Jope, 2009). However, despite microglial expression of FMRP, there was no difference in
the production of pro-inflammatory cytokines IL-6 and TNFα after acute lipopolysaccharide-
induced activation of microglia in WT or Fmr1 KO mice (Yuskaitis and Jope, 2009). Recent
results from our laboratory demonstrated that a persistent activation of astrocytes exists in the
adult Fmr1 KO mouse brain; however, no evidence was observed for activated microglia in
Fmr1 mice (Pacey et al., 2015).
In the current study, FMRP expression in microglia in WT mice was detected most
prominently during the first 10 postnatal days and declined thereafter. Interestingly, the number
of microglial cells in mice is immensely increased during the first two postnatal weeks when the
vast majority (>95%) of microglia are born (Alliot et al., 1999). These findings suggest a
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potential regulatory role for FMRP in the cell lineage and maturation of microglia during early
development. However, the exact molecular function of FMRP in these cells during brain
development and its contribution to behavioral abnormalities in Fmr1 KO mice remains to be
elucidated.
Quantitative analysis of FMRP+ cells in the cingulate cortex and corpus callosum of the WT
mouse revealed the highest number of FMRP+ cells at PND 0 which diminished steadily
throughout brain development. The abundant FMRP expression at birth and during the first 1-2
postnatal weeks indicates that the functional requirement of FMRP in glia is highest during this
critical period of early brain development. In the drosophila nervous system, FMRP appears to
be present sequentially, first in neuroblasts and then in glia, and glial expression was required
for neuroblast reactivation (Callan et al., 2012). Similar to the mouse model, the expression of
the drosophila FMRP homolog is developmentally regulated (Tessier and Broadie, 2008) and its
re-introduction during brain development rescues the dendritic impairment in the drosophila
model (Gatto and Broadie, 2009), indicating a causal relationship between FMRP expression
during a distinct developmental time-window and normal synapse formation. In the mouse
CNS, the most intense period of synaptogenesis (Zito and Svoboda, 2002) and the most severe
abnormalities in dendritic spine morphology converge at the end of the first postnatal week,
during which FMRP is abundantly expressed (Nimchinsky et al., 2001).
7.2. Gene therapy as a potential therapeutic avenue for the treatment of
Fragile X syndrome
There is no pharmacological cure for FXS and the currently prescribed medications, such
as anti-psychotics, anti-depressants, and stimulants, only partially alleviate selected symptoms
and are associated with deleterious side effects. Recently failed clinical trials of metabotropic
glutamate receptor antagonists and a GABAB receptor agonist to treat FXS relied on single
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molecular targets that were expected to alleviate certain symptoms, but did not address the core
underlying issue—the pathological absence or reduction of FMRP in the brain (Hampson et al.,
2012). Given that FMRP regulates the translation and stability of hundreds of mRNAs in the
CNS (Darnell et al., 2011; Ascano et al., 2012), it is not surprising that manipulation of single
molecular targets, such as metabotropic glutamate receptors, p21-activated kinase, and
membrane metalloproteinase-9, failed to produce long-term benefits (Choi et al., 2011; Dolan et
al., 2013; Gkogkas et al., 2014). In fact, to date none of the phase 3 clinical trials of small
molecules against single molecular targets have been successful, in spite of promising results in
pre-clinical or early clinical stage testing (Paribello et al., 2010; Scharf et al., 2014). However,
considering the plethora of genes whose expression is regulated by FMRP, a priori, it may be
expected that restoring FMRP expression in the CNS could provide a more comprehensive
reversal of the disorder compared to targeting single molecules.
7.3. Major factors affecting cellular tropism, efficiency and distribution
of AAV-induced transduction
Our initial results demonstrated that i.c.v. administration of AAV9- eGFP on PND 5,
compared to PND 21, resulted in broader distribution of the transgene throughout the brain.
Transgene delivery at early postnatal days is also favourable for the development of gene
therapy strategies for neurodevelopmental disorders, such as FXS, as earlier re-introduction of
the missing protein very early in postnatal period at the peak of neurogenesis is expected to
translate into a higher level of phenotypic rescue.
A goal of this study was to compare cell type specificity of virus transduction after treatment
of AAV9-eGFP as directed by the CMV vs. the synapsin-1 promoter (Gholizadeh et al., 2013).
AAV9-CMV-eGFP vector preferentially targeted astrocytes in neonatal mice and neurons in
juvenile mice, whereas treatment with the same AAV9 vector construct, but swapping the CMV
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promoter for the synapsin 1 promoter (AAV9-SYN-eGFP), resulted in extensive neuron-specific
transduction, regardless of the time of administration. Differences in cell-type-specific
transduction profiles at different stages in development are important considerations when
planning gene therapy studies for monogenic neurological disorders, where the aim of the study
is to introduce the missing protein specifically to either neurons or glia.
Our results demonstrated that i.c.v. treatment with AAV9 vectors displayed very good
diffusion properties in the mouse brain, especially when administered during the early postnatal
period. Another advantage of delivering AAV vectors into the CSF is the possibility of
targeting ependymal cells that line the inside perimeter of the ventricles and form the interface
between the ventricles and brain parenchyma. For transgenes that produce soluble proteins,
uptake into ependymal cells, which are susceptible to a wide range of viral vectors, provides the
potential to secrete the transgene into the cerebrospinal fluid, where it can diffuse continuously
into the brain; this strategy has been successfully employed to treat mouse models of lysosomal
storage disorders (Ghodsi et al., 1999; Liu et al., 2005; Fu et al., 2007; Haurigot et al., 2013;
Yamazaki et al., 2014).
Based on our initial results, we expected that administration of the AAV9 vector driven by
the SYN promoter at early postnatal days would result in wide diffusion of the vectors, and
consequently predominant expression of the therapeutic protein in neurons, where it is most
critically required in many neurological disorders. Early postnatal administration of AAV-based
therapeutics would be applicable not only to FXS but may also be a useful strategy in other
neurodevelopmental disorders such as Angelman Syndrome and Rett Syndrome where the
disease pathology is caused primarily by the absence or near absence of a missing protein in
neurons (Hampson et al., 2012). Together, these findings highlight the important effects of
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AAV serotype, the promoter, and the age of administration on the intensity, distribution and cell
type specificity of AAV transduction.
7.4. FMRP transgene expression in the brains of Fmr1 KO mice
As outlined in Chapter 3, FMRP is widely distributed in most neurons throughout the adult
mammalian CNS. This wide distribution throughout the CNS presents a challenge from the
perspective of virally mediated gene therapy in terms of achieving sufficiently wide distribution
of the viral vector. Other groups investigating approaches for treating CNS disorders in animal
models had previously reported success after direct CNS injections (Daily et al., 2011; Gadalla
et al., 2012), and after intra-venous injection of AAV vectors in the mouse (Garg et al., 2013)
and monkey (Samaranch et al., 2012). Of the various AAV vectors currently available, AAV9
was used in the current study because of its documented propensity to disseminate within the
brain parenchyma and its neuronal tropism (Nonnenmacher and Weber, 2012; Rothermel et al.,
2013).
We chose i.c.v. administration into the lateral ventricles for several reasons including (a) the
desire to avoid possible off-target tissue effects such as the liver, a known AAV-targeted organ,
(b) to minimize the amount of vector given to the animal, and (c) to specifically scrutinize the
effects of FMRP restoration in the brain. The results of our early work indicated substantial
diffusion of a vector coding for eGFP from the ventricles and transgene expression in multiple
forebrain regions of the mouse CNS (Gholizadeh et al., 2013). Relevant conclusions gleaned
from that AAV9-eGFP study were that the age of injection and the promoter employed were
critical factors in determining the distribution and cell type transduction specificity of the
transgene. We speculate that one parameter that may promote the spread of the viral vector in
the brain after i.c.v. administration is the recently characterized “glymphatic system”. Similar in
function to the peripheral lymphatic system, in brain parenchyma, para-vascular exchange
100
between cerebrospinal fluid and interstitial fluid has been shown to be facilitated by astroglial
water transport mediated in part by the aquaporin-4 water channel (Iliff et al., 2013).
In the adult mammalian CNS, FMRP is highly expressed in neurons (Cruz-Martín et al.,
2010; Harlow et al., 2010). In the present study, double labeling immunocytochemical analysis
demonstrated that AAV-FMRP transduction occurred primarily in neurons rather than glia, as
expected from the incorporation of the neuron-selective synapsin promoter in the AAV-FMRP
vector construct. In the first phase of the study where mice were treated at PND5, analysis of
the anatomical distribution of the recombinant FMRP transgene in AAV-FMRP-treated KO
mice indicated that it was present in several forebrain regions including the retrosplenial and
cingulate cortices, the hippocampus, and the striatum. FMRP was not detected in brain regions
located more distal from the lateral ventricles such as the piriform cortex, inferior colliculus,
cerebellum, and brainstem. However, in the second phase of the study where mice received
AAV-FMRP on PND 0 or 1, immunocytochemical analyses revealed increased distribution of
the vectors in brain regions devoid of FMRP expression in phase 1, such as amygdala, thalamus,
inferior colliculus, superior colliculus, and the cerebellum. Quantitative WB analysis further
confirmed more efficient FMRP transgene expression after PND 0-1 vector injections, with
higher FMRP levels in the cerebral cortex, frontal cortex and hippocampus, detected
approximately two months after PND 0-1 injections compared to PND 5 AAV delivery at phase
1.
Because severely low or elevated levels of FMRP expression have both been associated with
pathological outcomes, the correct level of expression of FMRP was previously suggested to be
of critical concern (Nagamani et al., 2012). In phase 2 we examined four different treatment
groups with disparate levels of FMRP in the forebrain. The KO-null group with none, the
KO+FMRP group had a midpoint average expression level compared to WT levels, the WT-null
101
had normal expression levels, while the WT+FMRP showed an almost two fold increase in the
expression of largest isoform of FMRP. In addition, the innate mouse-to-mouse variability in
the degree of vector diffusion and cellular transduction also facilitated the delineation of the
effects of a wide spectrum of FMRP expression levels on behavior.
7.5. Behavioral effects of FMRP restoration in the brains of Fmr1 KO
mice
In the first phase of the research, we assumed one potential reason that might explain the
absence of reversal of abnormal behavior on some tests might be the limited FMRP expression
in brain regions located distally from the injection site in the lateral ventricles. This may be
particularly pertinent to more caudal regions such as the cerebellum and brainstem, where very
limited FMRP transgene expression was detected. The presence of the FMRP transgene in the
observed brain regions (cerebral cortex, hippocampus, and striatum), and its absence or very low
expression in other regions provided an opportunity to link the restoration of FMRP expression
in specific brain regions to corrected pathological behaviors observed in Fmr1 KO mice. Our
data from phase 1 suggested that the presence of FMRP in the cortex and/or the striatum of the
AAV-FMRP-treated mice could have been a contributing factor for the reduction in repetitive
behavior, as assessed by the marble burying test. Several lines of evidence have shown that
lesions of the dorsal or ventral striatum in rodents (Aliane et al., 2011) and non-human primates
(Saka et al., 2004) block stereotyped behavior. Moreover, in a longitudinal study of brain
development in children, it was shown that an abnormally increased growth rate of the striatum
was correlated with more severe repetitive behavior in autistic children compared to control
subjects (Langen et al., 2013).
It could also be speculated that FMRP transgene expression in neurons of the cingulate
cortex might have contributed to the rescue of impaired social dominance (as assessed using the
102
“tube test”). The medial prefrontal cortex, which includes the cingulate cortex, sub-callosal
cortex, and the medial frontal gyrus, has been shown to be linked with social dominance in
mice. Based on recordings from pyramidal neurons of the medial prefrontal cortex, Wang et al,
2011 reported that social rank in mice directly correlates with the synaptic strength of pyramidal
neurons in the medial prefrontal cortex (Wang et al., 2011). It has also been reported that levels
of key proteins involved in regulating synaptic function, including the NMDA receptor subunits
NR1, NR2A, and NR2B, are decreased in the medial prefrontal cortex of Fmr1 KO mice
(Krueger et al., 2011).
Our findings in the elevated plus maze test (phase 2) demonstrated that AAV-null injected
Fmr1 KO mice exhibited lower non-social anxiety (more time in the open arms). Abnormally
low anxiety has been reported in previous studies of FXS KO mice (Liu et al., 2011; Heulens et
al., 2012), as well as another mouse model of autism lacking the mu-opioid receptor (Ide et al.,
2010; Becker et al., 2014). In the present work, we speculate that FMRP transgene expression
in the amygdala of the AAV-FMRP-treated mice could have been a contributing factor for
normalizing the reduction in anxiety levels as assessed by the elevated plus maze test. Several
lines of evidence have shown the pivotal role of amygdala in regulating the emotional symptoms
associated with FXS such as anxiety and unstable mood (for a review see Suvrathan and
Chattarji, 2011). Whole-cell recordings in brain slices from adult Fmr1 KO mice revealed a
decrease in mGluR-dependent long-term potentiation at thalamic inputs to principal neurons in
the lateral amygdala (Suvrathan et al., 2010).
Analysis of pre-pulse inhibition test in phase 2 showed a higher basal startle response in
Fmr1 KO mice compared to WT mice which was rescued in the AAV-FMRP-treated mice.
Fmr1 KO AAV-null-treated mice displayed abnormal responses to auditory stimuli, as seen in
increased incidence of audiogenic seizure, and auditory startle response. Abnormal pre-pulse
103
inhibition responses to auditory stimuli has also been reported in FXS patients (Frankland et al.,
2004; Hessl et al., 2009; Yuhas et al., 2011; Kohl et al., 2013). In our study, FMRP transgene
expression in medial prefrontal cortex of AAV-FMRP-treated KO mice might have contributed
to the rescue of the elevated startle response. Lesions in the medial prefrontal cortex of rodents
result in reduced pre-pulse inhibition responses (Bubser and Koch, 1994) and pharmacological
blockade of dopaminergic D1 receptors in the medial prefrontal cortex reduces pre-pulse
inhibition in a rat model of schizophrenia (Swerdlow et al., 2006).
In contrast to the reversal of behavioral abnormalities described above, there was no rescue
of other pathological behaviors including motor hyperactivity, reduced ultrasonic vocalizations
and susceptibility to audiogenic seizures. There are several possible explanations for this
outcome. One possibility is that undetectable or very low levels of FMRP transgene expression
in the cerebellum of AAV-treated KO mice, may be a factor contributing to the inability to
achieve full behavioral recovery of motor hyperactivity. FMRP is expressed extensively in all
neuronal populations of the cerebellum (Pacey et al., 2013), and the cerebellum has been
consistently associated with neuropathology in FXS (Koekkoek et al., 2005; Ellegood et al.,
2010; Fatemi et al., 2012), and autism (Whitney et al., 2008). The cerebellum has also been
linked with motor hyperactivity in Fmr1 KO mice (Rogers et al., 2013). Similarly, the absence
of FMRP in the inferior colliculus and the brainstem of the AAV-FMRP-treated Fmr1 KO mice
might explain the lack of reversal of audiogenic seizures in phase 1. These brain regions have
been linked to auditory processing and are thought to be important for propagation of auditory
seizures (Garcia-Cairasco, 2002; Byrns et al., 2014).
Perplexingly, increased repetitive behavior was rescued in phase 1 but not in phase 2 of the
gene therapy study. There are several potential explanations for the lack of statistically
significant reversal of this phenotype in phase 2. The number of animals tested in each group in
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phase 2 and the use of AAV-null injections instead of PBS as control groups can both
potentially contribute to differences in statistical analyses of behavioral results. Also,
differences in FMRP transgene expression levels, delivered at PND 0-1 instead of PND 5 in the
striatum, a brain region closely associated to modulating repetitive behavior in mice, might
attribute to differences in number of marbles buried in the two phases of the study. For
instance, in the long arm of phase 1 project, FMRP was detected at18 ±5% of WT expression in
the striatum; however in phase 2, FMRP transgene expression levels reached 33% of WT
expression, which is almost two times higher than FMRP levels in the AAV-FMRP group in
phase 1. Based on a preliminary analysis from a small set of data points in the phase 1 (n = 10),
a possible inverse correlation exists between the number of marbles buried and FMRP
expression level (r2 = 0.37 p = 0.06), which may explain differences in repetitive behavior in
AAV-FMRP-treated KO mice in the different phases of the study.
7.6. Possible contributions of aberrant MeCP2 expression to
pathological abnormalities in FXS
We established that MeCP2 mRNA is a substrate for FMRP and that MeCP2 protein levels
are elevated (about 1.5 to 2-fold over-expression) in the brains of adult naive Fmr1 KO mice
compared to WT mice. Moreover, in the WT mouse brain, most neurons co-expressed MeCP2
and FMRP in the brain regions examined. Finally, elevated MeCP2 levels in the cortex of Fmr1
KO mice were normalized after FMRP transgene expression. Intriguingly, we detected a
correlation between elevated MeCP2 expression in the forebrain and increased motor activity.
Evidence supporting a relationship between MeCP2 and motor activity comes from transgenic
mouse lines over-expressing MeCP2 at levels comparable to what we observed in Fmr1 mice
(about 1.5 to 2-fold over-expression), where increased vertical activity (rearing) and increased
aggressive behavior have been reported (Collins et al., 2004; Bodda et al., 2013). Moreover,
105
similar to our results with male Fmr1 mice, brain MeCP2 expression in female MeCP2
heterozygous mice was also reported to be correlated with motor activity (Wither et al., 2013).
Further ancillary evidence of a link between MeCP2 and FMRP derives from the demonstration
that MeCP2-repressed genes overlap extensively with genes that are FMRP mRNA substrates
(Gabel et al., 2015). The relationship observed between increased MeCP2 levels and motor
hyperactivity in the Fmr1 mouse, together with the phenotypic overlap between Fmr1 KO mice
and mouse models over-expressing MeCP2, raises the question as to whether elevated brain
MeCP2 might also contribute to other features of the fragile X phenotype. Mice over-
expressing MeCP2 also display impairments in anxiety, hippocampal long term potentiation,
deficits in learning and memory (Shang et al., 2009; Na et al., 2012; Neuhofer et al., 2015), and
increased seizures and stereotypic behavior (Collins et al., 2004). Moreover, patients with
fragile X syndrome, duplication of the FMR1 gene, or MeCP2 gene duplication syndrome all
display several over-lapping phenotypes (Fig. 33). Gene duplication in either FMR1 or MeCP2
is associated with developmental delay, mental retardation, and seizures (Smyk et al., 2008).
Furthermore, cerebellar abnormalities such as Purkinje cell loss has been reported in MeCP2
duplication patients (Reardon et al., 2010), as well as elderly males with FXS (Greco et al.,
2011). The relationship observed between increased MeCP2 levels and motor hyperactivity in
the Fmr1 mouse, together with the phenotypic overlap between Fmr1 KO mice and mouse
models over-expressing MeCP2, raises the intriguing possibility that elevated brain MeCP2
might also contribute to other features of the fragile X phenotype and may provide a common
mechanistic link across some populations of autism spectrum disorders.
106
Fig. 33. Overlapping characteristics of FXS, Rett syndrome, FMR1
duplication syndrome, and MeCP2 duplication syndrome.
107
7.7. Concluding remarks and future directions
Our results demonstrated a predominantly neuronal expression profile of native FMRP in all
of the brain regions analyzed of the adult WT mouse, except in the corpus callosum, where only
a small number of neurons were present and FMRP expression was mainly observed in
astrocytes. Our data also revealed the presence of FMRP in cells expressing markers for
astrocytes, microglia and oligodendrocyte precursor cells in the developing brain. In general,
glial expression of FMRP diminished to low or undetectable levels in the adult brain following
different trajectories in different brain regions, with the exception of the corpus callosum in
which predominant FMRP expression in astrocytes remained almost unaffected from birth to
adulthood.
The findings gleaned from our viral vector gene therapy work provide a proof-of-principle
demonstrating that expression of recombinant FMRP in specific regions of the CNS can reverse
or alleviate selected neurological abnormalities in the Fmr1 KO mouse. The exceptionally
protracted expression of AAV-mediated transgenes as shown here (at least 7 months post-
transfection), and in other studies using AAV transduction in the brain (up to 1 year post-
injection), makes this approach especially attractive for treating neurodevelopmental disorders
where the presence of the missing protein is likely required over an extended time frame.
Conceivably, the introduction of FMRP into Fmr1 KO mouse neurons could indirectly result
in correction of disrupted neuron-to-astrocyte signaling in FXS at a critical time during early
developmental stages during which astrocyte support of neuronal growth and synaptic formation
is critical (Cheng et al., 2012). Since glia play indispensable roles in synaptic development,
developmentally regulated FMRP expression in glia might regulate synaptic development of
contiguous neurons. Future efforts could be directed towards determining whether FMRP re-
introduction in astrocytes alone, or concomitantly in both astrocytes and neurons, may provide
108
enhanced benefit (correction of abnormal behaviors) over that seen when expressed only in
neurons, as done in the current research.
One potential reason for limited transgene expression in cerebellum might be low expression
levels of the synapsin gene within the first two postnatal weeks in the mouse cerebellum
(Melloni et al., 2004), which could have contributed to low levels of promoter-driven FMRP
transgene expression in our study. This issue might be resolved by using other promoters in the
AAV construct such as the Fmr1 promoter (which has not yet been fully mapped out), or by
using one of the more established ubiquitous promoters such as cytomegalovirus, or the chicken
beta-acting promoter in our future attempts. A potential problem from the use of ubiquitous
promoters could be ectopic expression in non-neuronal cells such as glia in adults, where FMRP
is almost exclusively expressed in neurons. Therefore another alternative is the use of combined
cytomegalovirus mini-promoter and synapsin, as used in a recent study to provide widespread
neuronal transduction (Huda et al., 2014). A better understanding of the brain regions FMRP
needs to be delivered to and levels of FMRP needed to cause significant changes on
pathological behavior will facilitate development of more efficacious gene therapies for FXS.
Another potential parameter to consider is delivery of AAV vectors containing other Fmr1
isoforms. First, it must be established whether expression of isoform 1, which encodes for the
longest isoform of FMRP in the CNS, is sufficient to restore normal function. Once the most
functionally essential FMRP isoforms are fully characterized, co-administration of multiple
AAV vectors, each containing a different isoform, might potentially lead to more favorable
outcomes compared to using vectors expressing isoform 1 alone. It has been shown that only
two FMRP isoforms (isoforms 1 and 7) have the major phosphorylation site at Ser-499, which is
believed to affect the ability of the protein to bind to stalled ribosomes and inhibit translation
(Brackett et al., 2013). Therefore, in the next phase of the study, we might consider delivering
109
isoform 7 using viral vectors on its own and in combination with isoform 1. Expression of each
isoform individually would enable the characterization of the functional impact of each isoform
in modulating behavior, independent from the contribution of other isoforms.
Based on our AAV-FMRP experiments in the WT mouse, forebrain over-expression was
associated with a reduction in ultrasonic vocalizations, stereotyped behavior, and pre-pulse
inhibition. Of course, how these abnormalities in mice might relate to human FMR1 gene
duplication syndrome remains unknown. FMR1 gene duplication in humans is characterized
with symptoms including mental retardation, developmental delay and seizures (Rio et al., 2010;
Nagamani et al., 2012; Vengoechea et al., 2012; Hickey et al., 2013). The present work
provides the first evidence supporting the notion that modest (1.5- 2-fold) postnatal over-
expression of the dominant mouse isoform of FMRP in the brain results in behavioral
abnormalities. Overall our findings indicate that establishing the therapeutic window for Fmr1
gene delivery and careful regulation of the level of transgene expression, are critical for
achieving successful rescue of this disorder.
In conclusion, this work has established a proof-of-principle by demonstrating that abnormal
behaviors in Fmr1 KO mice can be reversed after treatment with AAV-FMRP and transduction
of forebrain neurons. PND 0-1 bilateral i.c.v. injection of AAV-FMRP resulted in wider
distribution compared to AAV treatment at PND 5. We believe that the neurodevelopmental
effects of early FMRP introduction may also promote normal neurogenesis and neuronal
maturation in the Fmr1 brain and that this might be a contributing factor in the behavioral rescue
seen in the adult mice. In addition, brain levels of PSD-95 and MeCP2 proteins (two key FMRP
substrates) were normalized in the cortex and likely contributed to establishing proper synaptic
integrity. It remains to be seen whether FMRP expression in more distal brain regions, such as
the cerebellum, would further correct additional pathological and behavioral abnormalities in
110
Fmr1 KO mice. The findings of this study contribute useful new information in the quest for
eventually translating our work here in the mouse model of FXS to human gene therapy.
111
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