Modeling cognitive dysfunction inneurofibromatosis-1Kelly A. Diggs-Andrews and David H. Gutmann

Department of Neurology, Washington University School of Medicine, Box 8111, 660 South Euclid Avenue, St. Louis, MO 63110,

USA

Review

Cognitive dysfunction, including significant impair-ments in learning, behavior, and attention, is found inover 10% of children in the general population. However,in the common inherited cancer predisposition syn-drome, neurofibromatosis type 1 (NF1), the prevalenceof these cognitive deficits approaches 70%. As a mono-genic disorder, NF1 provides a unique genetic tool toidentify and dissect mechanistically the molecular andcellular bases underlying cognitive dysfunction. In thisreview, we discuss Nf1 fly and mouse systems thatmimic many of the cognitive abnormalities seen in chil-dren with NF1. Further, we describe discoveries fromthese models that have uncovered defects in the regula-tion of Ras activity, cAMP generation, and dopaminehomeostasis as key mechanisms important for cognitivedysfunction in children with NF1.

IntroductionNF1 is one of the most common neurogenetic disordersaffecting the nervous system. The hallmark of NF1 is thedevelopment of tumors involving the central and periph-eral nervous systems. In this condition, affected individu-als are prone to the formation of peripheral nerve sheathtumors (i.e., neurofibromas, plexiform neurofibromas, andmalignant peripheral nerve sheath tumors) and braintumors (optic pathway gliomas and malignant gliomas).Moreover, 50–70% of children with NF1 manifest specificcognitive impairments, including difficulties with atten-tion, executive function, language, visual perception, andlearning [1–4]. Although most children exhibit some formof cognitive deficit that negatively impacts upon theirscholastic performance, the specific cognitive abnormalitypresent (i.e., attention deficit, spatial memory impairment,fine motor delay) and the severity of the deficit variesgreatly from child to child.

In concert with clinical studies characterizing the spec-trum of learning, behavioral, and motor delays in childrenwith NF1, laboratory investigations have begun to definethe molecular and cellular etiologies for these commonproblems. Using Nf1 genetically engineered strains of miceand flies, investigators have successfully modeled many ofthe cognitive and behavioral deficits seen in children withNF1, and employed these model systems to better definethe role of the NF1 protein (neurofibromin) in normalcentral nervous system (CNS) neuronal function. This

Corresponding author: Gutmann, D.H. (gutmannd@neuro.wustl.edu)Keywords: neurofibromin; cyclic AMP; dopamine; NF1; learning; attention deficits.

0166-2236/$ – see front matter � 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.101

review will highlight the basic neurobiological insightsthat have derived from the use of these robust preclinicalstrains as well as their importance for the identificationand validation of new therapeutic drug targets relevant tothe treatment of children with NF1.

Clinical features of NF1NF1 is a common nervous system disorder, affecting 1 in3500 people globally [5]. NF1 is inherited in an autosomaldominant manner, although �50% of individuals presentwith de novo mutations, and represent the first member oftheir family with NF1 [6]. Although NF1 genetic testing isavailable for select individuals, the diagnosis of NF1 ismost often established on clinical grounds (Table 1). To begiven the diagnosis of NF1, individuals must have at leasttwo features of the condition, including greater than fivecafe-au-lait macules (birthmarks), skinfold (underarm orgroin) freckling, Lisch nodules (iris hamartomas), neurofi-bromas, an optic pathway glioma, a distinctive bone ab-normality (i.e., tibial dysplasia), or a first degree relativewith NF1 [7]. In addition to these features, individualswith NF1 may also manifest learning/behavioral problems,malignant gliomas, T2 hyperintensities on neuroimaging(i.e., magnetic resonance imaging) studies, enlarged heads(macrocephaly), gross and fine motor delays, short stature,and other less common cancers [8–13].

Cognitive and behavioral deficits in children with NF1

Cognitive problems are the most frequently observed neu-rological impairments in children with NF1. The majorityof children display some degree of cognitive deficits [1],which limit their full academic achievement and overallquality of life. Clinical studies examining cognitive pro-blems in NF1 have revealed a left shift in average IQ,ranging from low to normal IQs, with specific learningdeficits being observed in 30–70% of children [1,14,15].Additionally, children with NF1 exhibit poor performanceon tasks of reading, spelling, and mathematics, impairedexpressive and receptive language skills, deficits in visuo-spatial and visuoperceptual skills, and defects in executivefunction (planning and concept formation) [1,16,17]. Al-though less common, there is also an increased incidence ofautistic spectrum disorder in children with NF1 [18].Problems with attention and behavior in children withNF1 can also negatively affect school performance andsocial interactions [19–21]. Nearly 70% of children withNF1 report deficits in one or more of the attention system

6/j.tins.2012.12.002 Trends in Neurosciences, April 2013, Vol. 36, No. 4 237

Table 1. NF1 clinical manifestations

Clinical Feature Diagnostic criteria

(fulfillment of �2)aPrevalence

(% of NF1

population)

[114,115]

Family history 50%

Neurocutaneous

Cafe -au-lait spots 6 or more 99% in adults

Axillary/inguinal freckling 2 or more 85%

Lisch nodules 2 or more 95% in adults

Neurofibromas 2 or more 99% in adults

Plexiform neurofibromas 1 or more 20–45%

Skeletal

Scoliosis ND 5–10%

Bone dysplasia 1–5%

Pseudarthrosis 2%

Short stature ND 30%

Macrocephaly ND 45%

Tumors

Optic pathway glioma 15%

Malignant peripheral

nerve sheath tumor

ND 2–5%

Low-grade glioma

Malignant glioma ND 2–3%

Optic pathway glioma ND 1–2%

aND, non-diagnostic; denotes NF1 clinical features associated with disease, but

are not assessed for diagnosis

Ras Ras*

MEK

AC

MAPKP

PI3K

GTP

(

Flie

hsNNf

Nf

Nf

Mi

Nf(12hy

NfGF

NfSyn

NfDlx

9a 23a

(a)

(b)

Neurofibromin

GDP

RTK

CSRD GRD LRD

Inac�ve Ac�ve

Akt

mTOR

ATP

cAMP

Neurofibromin

Figure 1. Neurofibromin structure and function. (a) Neurofibromin is a 2818 amino acid p

red bars) and encodes several distinct functional domains, including a cysteine-rich dom

(b) Neurofibromin serves as a negative regulator of Ras by accelerating the hydrolysi

receptor tyrosine kinase (RTK) activation, Ras guanosine exchange (GDP to GTP) pro

including MEK/MAPK and PI3K/Akt/mTOR. Ras can also positively regulate adenylate c

associated learning and attention abnormalities. Abbreviations: Dlx5/6, distal-less hom

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domains (sustained, selective, divided, and shifting atten-tion) [1,22,23], and one-third to one-half of children arediagnosed with attention-deficit hyperactivity disorder(ADHD) [1,2]. Moreover, children with NF1 tend to beimpulsive, and often have difficulty detecting and respond-ing to social cues.

Neurofibromin structure and functionThe NF1 gene resides on chromosome 17 [5,24] andencodes a large cytoplasmic protein (neurofibromin),encompassing 2818 amino acids and over 60 exons. Neu-rofibromin contains several domains, including a cysteine-rich domain (CSRD), a leucine repeat domain (LRD), and aRas-GAP domain (GRD) (Figure 1a). The NF1 gene alsocontains at least 3 alternatively spliced exons, 9a, 23a, and48a, each with unique properties. Exon 9a-containing neu-rofibromin is a neuron-specific isoform [25,26], whereasexon 48a-containing neurofibromin is expressed in muscle[27]. Exon 23a neurofibromin interrupts the normal func-tion of the GAP domain, but has a more widespread tissuedistribution [28–30]. Interestingly, mice engineered to lackexon 23a do not have an increased tumor predisposition,but manifest specific learning impairments [28]. Currentstudies are focused on defining the functional conse-quences of NF1 alternative splicing, especially exon 9a,on neurofibromin signaling and NF1-associated tumor andcognitive deficits.

c)Modeling NF1-associated cogni�ve deficits

Nf1 muta�on Cogni�veabnormality

Refs

s

f1/+;1P2

Heterozygote Olfactory associa�ve learning

[67]

1P1and P2 Null mutant Olfactory learningmemory

[67,68]

1c00617 Null mutant Memory acquisi�on [68]

ce

1+/−

9/C57brid)

Heterozygote Spa�al learningmemorya�en�on

[45, 49,51]

1flox/−

AP–CreHeterozygosity inneurons

Spa�al learningmemorya�en�on

[80,87]

1flox/+

1–CreHeterozygosity in neuronal subset

Spa�al learningmemory

[51]

1flox/+

5/6–CreHeterozygosity in interneurons

Spa�al learningmemory

[51]

48a

TRENDS in Neurosciences

rotein that contains multiple alternatively spliced exons (9a, 23a, and 48a shown as

ain (CSRD), a Ras-GAP domain (GRD), and a leucine-repeat domain (LRD) [5,24–30].

s of the GTP-bound active Ras, producing inactive GDP-bound Ras [31–34]. Upon

motes Ras activity. Activated Ras, in turn, stimulates its downstream effectors,

yclase (AC) activity in some cell types. (c) Current fly and mouse models of NF1-

eobox 5 and 6; GFAP, glial fibrillary acidic protein; Syn1, synapsin 1.

Review Trends in Neurosciences April 2013, Vol. 36, No. 4

The neurofibromin GRD functions in a similar fashion toother GTPase-activating proteins (GAPs), which negative-ly regulate the activity of the p21-Ras proto-oncogene(Figure 1b). Ras is recruited to the plasma membrane byadaptor proteins and is activated by receptor tyrosinekinases (RTKs) following growth factor binding. At themembrane, guanine nucleotide exchange factors (GEFs)enable Ras to bind GTP to become active and transmitgrowth-promoting signals. In its active state, Ras signals toseveral downstream effectors, including the mitogen-acti-vated protein kinase (MAPK) and phosphoinositide 3 ki-nase (PI3K)/mammalian target of rapamycin (mTOR)signaling pathways. Neurofibromin acts as an inhibitorof Ras activity by catalyzing the hydrolysis of active GTP-bound Ras to inactive GDP-bound Ras, such that in cellslacking neurofibromin, there are high levels of Ras path-way (i.e., MEK/MAPK and PI3K/mTOR) activity and in-creased cell growth [31–34].

In addition to Ras regulation, neurofibromin is also apositive regulator of intracellular cAMP levels. Studiesinitially performed in Nf1 mutant flies revealed that re-duced Drosophila larval size did not result fromderegulated Ras signaling, but rather was dependent oncAMP-mediated protein kinase A (PKA) activity [35,36].Subsequent studies in mice likewise demonstrated thatboth Nf1-deficient embryonic brains and postnatal astro-cyte cultures had reduced cAMP levels [37,38]. Althoughthe precise mechanism(s) underlying neurofibromin con-trol of cAMP homeostasis has yet to be fully elucidated,there appear to be both Ras-dependent and Ras-indepen-dent modes of regulation [36,37,39].

Nf1 models of learning, memory, and attention deficitsInspection of the predicted protein sequence of the Dro-sophila and murine Nf1 homologs reveals 60% and greaterthan 98% identity to the human NF1 gene product, respec-tively, supporting the use of Nf1 mutant mouse and flymodels to study many of the cognitive and behavioralfeatures seen in the human condition [35,40,41]. In thisregard, robust mouse and fly models have been developedand employed for preclinical discovery and validationinitiatives aimed at improving clinical outcomes for chil-dren and adults with NF1 (Figure 1c).

The role of Ras activation in learning and memory

Studies pioneered in the late 1990s by Alcino Silva andcolleagues employed mice harboring a germline inactivatingmutation in one Nf1 allele. These Nf1 heterozygous (Nf1+/�)mice are genetically similar to children with NF1 who startlife with one functional and one non-functional copy of theNF1 gene in every cell. Nf1+/�mice exhibit impaired spatiallearning in the Morris watermaze, a behavioral task used toassess hippocampal-based learning [42,43]. These learningdefects improve with training time, supporting clinicalobservations that children with NF1 can learn with addi-tional focused training and resources [3]. Concomitant withdefects in spatial learning, Nf1+/�mice also have decreasedCA1 hippocampal long-term potentiation (LTP) followingtheta-burst stimulation [43]. Further characterization ofthis LTP deficit revealed abnormalities in both early-phase and long-term LTP maintenance [43,44]. Whereas

early-phase LTP is thought to underlie immediate learning,late-phase LTP regulates long-term memory formation [45].In this manner, abnormalities in both early-phase and late-phase LTP could contribute to the learning and memorydeficits in Nf1+/� mice.

To define better the connection between reduced neu-rofibromin and impaired hippocampal function, Silva andcolleagues explored the possibility that increased Rasactivity was responsible for the observed cognitive defi-cits [43]. Their demonstration of increased Ras and MEKactivity in the brains of Nf1+/� mice prompted geneticrescue experiments, where Nf1+/�mice were crossed withmice to reduce Ras (K-Ras and N-Ras) expression (Nf1+/�;KRas+/� or Nf1+/�;NRas+/� mice). In these studies, per-formance of Nf1+/� mice with either reduced K-Ras or N-Ras expression was comparable to that found in wild typecontrol mice [43]. Similarly, pharmacological inhibition ofRas using a drug that blocks the post-translational far-nesylation of Ras (Lovastatin) improved performance ofNf1+/� mice on hippocampal-based spatial learning tests[43,46]. Collectively, these findings provide strong evi-dence for a functional link between reduced brain neu-rofibromin expression, elevated Ras activity, andcognitive deficits, and prompted clinical trials using Lov-astatin-like treatments for children with NF1 [47].

The cellular and neurochemical bases for impaired LTPand learning in Nf1+/� mice have also been investigated.Using Nf1 conditional knockout (CKO) mice in which onecopy of the Nf1 gene was selectively inactivated in neuronsor astrocytes, the consequence of reduced neurofibrominexpression in distinct cell populations on mouse learningand memory was evaluated [48]. Although reduced Nf1expression in neurons recapitulated the learning andmemory deficits observed in Nf1+/� mice, reduced Nf1expression in glial fibrillary acidic protein (GFAP)-positiveastrocytes had no effect on learning or memory. Neuronsubpopulation-specific Cre driver lines have also beenemployed to address learning deficits after selective Nf1inactivation. Specifically, reduced Nf1 expression in excit-atory and inhibitory neurons (synapsin-I–Cre mice) orGABA-containing neurons [distal-less homeobox 5/6(Dlx5/6)–Cre mice], but not forebrain pyramidal neurons[i.e., Ca2+/calmodulin-dependent protein kinase II (CAM-KII)–Cre mice], resulted in defects in learning and memorysimilar to those observed in Nf1+/� mice. These observa-tions demonstrate a primary neuronal defect as a respon-sible cellular etiology underlying murine Nf1 learning andmemory dysfunction.

Based on the finding that inhibitory neurons were sen-sitive to the effects of reduced Nf1 gene expression, it wassubsequently shown that Nf1+/� mice had increased hip-pocampal GABA inhibitory tone. In these studies, elevatedMAPK-mediated synapsin-I phosphorylation and in-creased GABA release in hippocampal inhibitory inter-neurons were shown to cause impaired Nf1+/� mouseLTP and learning [43,48]. Consistent with this model,treating Nf1 mutant mice with a GABAA receptor antago-nist (i.e., picrotoxin) restored normal LTP in the hippocam-pus [43]. These findings established a direct relationshipbetween Ras regulation and GABAergic signaling relevantto the Nf1 learning deficits.

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The notion that deregulated Ras activation can causecognition dysfunction is further underscored by severalhuman disorders caused by genetic mutations that leadto increased Ras signaling. For example, patients withLegius syndrome, a NF1-like disorder that results frominactivating mutations in the SPRED1 Ras-MEK/MAPKregulator, exhibit mild neurocognitive deficits [49–51].Similarly to Nf1+/�mice, defective spred1 function in miceresulted in increased MAPK signaling [49,52,53], in addi-tion to deficits in learning and memory [54]. Costellosyndrome is a Ras disorder (‘Ras-opathy’) caused by anactivating mutation in the HRAS gene, leading to consti-tutive Ras signaling [55]. Children with Costello syndromeoften have cognitive impairments, low IQs, and increasedanxiety [56]. Similarly to their human counterparts,HRasG12V knock-in mice exhibit increased anxiety beha-viors and cognitive impairments [57]. Lastly, in additionalmodels of aberrant Ras activity, pharmacological inhibi-tion of PI3K or the MAPK kinase MEK was effective atrestoring normal synaptic plasticity and learning inrodents [58–61]. Taken together, these studies demon-strate that inappropriate regulation of Ras activity candisrupt cognitive function, providing a preclinical rationalefor Ras pharmacological treatments for NF1-associatedcognitive deficits.

The role of cAMP in learning and memory

In Drosophila, neurofibromin modulates cAMP by activat-ing the adenylyl cyclase 1 (AC1) homolog, rutabaga (rut)[35,36]. Although the exact mechanism remains to beelucidated, activation of rut-AC is initiated by an interac-tion with the C terminus of neurofibromin [39]. Further,this interaction is essential for learning and memory inflies [62,63]. Using a classic Pavlovian olfactory behavioralparadigm, flies lacking Nf1 or rut-AC show reduced per-formance, indicative of learning and memory impairments[62–66]. Restoration of neurofibromin expression usingdNf1 heat-shock transgenes, or correction of the cAMPdefect by ectopic protein kinase A (PKA) expression, re-stored normal learning and memory in Nf1 mutant flies[62]. Recent studies have revealed that distinct domains ofDrosophila neurofibromin can modulate different compo-nents of learning and memory. Whereas mutations in theGAP domain cause defects in long-term memory, thecAMP-activating C-terminal region of neurofibromin med-iates immediate memory or learning [45]. A direct linkbetween cAMP homeostasis and cognitive defects has notbeen firmly established in Nf1 mouse models, but supportfor a role of cAMP in learning and memory derives fromstudies in other rodent models. In these reports, pharma-cological and genetic manipulation of cAMP levels en-hanced LTP, learning, memory, and hippocampalneurogenesis [67–71].

In Nf1 mutant mice, some of the defects observed inCNS neurons were found to be dependent on cAMP signal-ing. Following complete Nf1 loss in neural stem cells(NSCs), reduced secondary somatosensory cortex thick-ness was observed [72]. Further examination of theseNf1 CKO (i.e., Nf1BLBPCKO) mice revealed that this re-duction in cortical thickness resulted from decreased neu-ronal arborization, rather than from reductions in the total

240

number of neurons. Using in vitro methods, it was demon-strated that neurons differentiating from Nf1-deficientNSCs also had shortened neuronal processes [72]. Thebiochemical basis for this defect in neuronal morphologywas shown to result from impaired cAMP generation be-cause both Nf1BLBPCKO mouse brains and Nf1�/� primaryneurons had lower cAMP levels in vitro and in vivo [72–74].Re-expression of the Ras-regulatory NF1-GAP domain inNf1BLBPCKO mice did not correct the cortical thicknessdeficit. Similarly, expression of an activated K-Ras allele inNSCs both in vitro and in vivo did not alter neurite length.Instead, treatment of Nf1BLBPCKO mice with pharmaco-logical agents that increase cAMP levels restored normalcortical thickness in vivo and neurite lengths in primaryNf1�/� neurons in vitro [72–74].

Because the brains of children with NF1 are composed ofneurons with reduced, not absent, NF1 gene expression,subsequent studies have employed Nf1+/� CNS neurons asa relevant model (Figure 2a). Similar to Nf1-deficientneurons, Nf1+/� CNS neurons exhibited 25% and 40%reductions in neurite (axonal) length and growth-conearea, respectively [72,73]. These abnormalities resultedfrom impaired cAMP generation, and were corrected bytreatments that elevated intracellular cAMP levels, in-cluding Rolipram (a phosphodiesterase-4 inhibitor), non-hydrolyzable cAMP analogs, and adenylyl cyclase (AC)activators (e.g., forskolin). Conversely, blocking cAMP pro-duction, using 2,3 dideoxyadenosine (DDA) to inhibit ade-nylyl cyclase function, was sufficient to attenuate neuriteextension in wild type neurons [73,74]. Lastly, neurofibro-min regulation of actin cytoskeletal dynamics was found tobe dependent on cAMP-mediated PKA activation of Rho/ROCK/MLC signaling [74], such that correcting the defec-tive signaling conferred by any component along this path-way normalized Nf1+/� axon lengths and growth cone areasin vitro (Figure 2b).

Further examination of these abnormal Nf1+/� CNSneurons also revealed small increases in programmed celldeath. In studies examining the impact of reduced neuro-fibromin expression upon retinal ganglion cells (RGCs),optic glioma formation in Nf1 mutant strains was associ-ated with increased RGC apoptosis [73,75]. As observed invitro, treatment of Nf1 optic glioma-bearing mice withRolipram (to raise intracellular cAMP levels) almostcompletely ameliorated the increased RGC programmedcell death in vivo [73], underscoring the importance ofneurofibromin-mediated cAMP homeostasis in the main-tenance of normal CNS neuronal function. Interestingly,CNS neurons with reduced Nf1 expression had similarlevels of cAMP to neurons completely lacking neurofibro-min expression, emphasizing the profound effect of Nf1heterozygosity on CNS neuronal morphology and function[74]. Further studies using hippocampal neurons demon-strated a requirement of neurofibromin for dendritic spineformation, such that Nf1 knockdown reduced spine forma-tion in mature neurons in vitro [76]. Correspondingly,dendritic spine densities were also reduced in the brainsof Nf1+/� mice [77]. Together, altered dendritic spinedensity, axonal morphological defects, and reduced neuro-nal survival may lead to impaired synaptic efficacy andreduced cognitive function in children with NF1.

GPCR

AC

GTP

Gα*

ATP

PKAROCK

RhoA P

Neurofibromin

MLC

Ac�ncytoskeleton

cAMP

Gα β

γ

(b)(a) Nf1+/+

Nf1+/+

+ FOR

Nf1+/+

+ DDANf1+/−

+ DDA

Nf1+/−

+ FOR

Nf1+/−

TRENDS in Neurosciences

Figure 2. Neurofibromin regulation of neuronal morphology is cAMP-mediated. (a) Neuronal morphology (growth cone areas and neurite length) is attenuated by reduced

neurofibromin expression and cAMP levels. Top panel: primary cultured Nf1+/� hippocampal neurons have decreased neurite lengths (not shown) and growth cone areas

as a result of reduced cAMP generation [73]. Middle panel: stimulating adenylyl cyclase (AC) with forskolin (FOR) elevates cAMP levels and rescues growth cone areas in

Nf1+/� hippocampal neurons. Bottom panel: decreasing cAMP levels in wild type neurons by blocking AC activity with the AC inhibitor 2,3-dideoxyadenosine (DDA) blunts

growth cone areas, comparably to Nf1+/� neurons. Scale bar, 20 mm. Adapted, with permission, from [73]. (b) Schematic diagram illustrating mechanism of cAMP-mediated

morphological changes. In neurons, low cAMP generation due to reduced Nf1 expression, in turn, leads to decreased PKA activation, RhoA/ROCK activity, and MLC

phosphorylation. These changes contribute to impaired actin cytoskeletal dynamics, resulting in smaller growth cones and shortened axons [74]. Abbreviations: GPCR, G

protein-coupled receptor; MLC, myosin light chain; PKA, protein kinase A; ROCK, Rho-associated protein kinase.

Review Trends in Neurosciences April 2013, Vol. 36, No. 4

The role of dopamine homeostasis in learning, memory,

and attention

Owing to the high prevalence of attention problems inchildren with NF1, stimulant medications, such as meth-ylphenidate (MPH), have proved to be effective treat-ments in this affected population [2]. MPH restoresdopamine homeostasis by blocking dopamine reuptakethrough the DA transporter (DAT), allowing increasedextracellular dopamine availability in dopaminergic neu-rons [78,79]. Although brain dopaminergic defects haveyet to be reported in children with NF1, impaired dopa-mine homeostasis has been shown in a Nf1 geneticallyengineered mouse strain [80]. In these studies, Nf1+/�

mice with total Nf1 loss in GFAP+ astroglial cells (Nf1+/�GFAPCKO mice) were used. Similarly to Nf1+/� mice,Nf1+/�GFAPCKO mice had reduced exploratory behaviorsand performed poorly on tests of selective and non-selec-tive attention. Moreover, there were reduced dopaminelevels and reduced postsynaptic dopamine signaling inthe striatum, as measured by decreased dopamine- andcAMP-regulated phosphoprotein-32 (DARPP32) phos-phorylation. In addition, cell-autonomous decreases inthe growth cone areas and neurite lengths of dopaminer-gic neurons in vitro were observed, resulting in attenu-ated cell projections to the striatum in vivo. Althoughpost-synaptic dopamine receptor expression was normal,presynaptic dopamine transporters [i.e., DAT and vesic-ular monoamine transporter (VMAT)] and tyrosine hy-droxylase (TH) expression were significantly reducedin Nf1+/�GFAPCKO mice [80] (Figure 3). To provide apreclinical measure of reduced striatal dopaminelevels, positron emission tomography (PET) imaging

with 11C-raclopride demonstrated increased binding inthe striata of Nf1+/�GFAPCKO mice, indicative of lowendogenous dopamine [81]. Consistent with a dopamineavailability deficit, and similar to children with NF1,treatment with dopamine-elevating drugs, such asMPH and L-DOPA, increased striatal dopamine levelsand ameliorated attention defects in these mice [80,81].

Dopamine system function is also critical for learningand memory in rodents, such that disruption of hippocam-pal dopaminergic innervations and loss of hippocampal D1/D5 dopamine receptor function result in spatial learningdefects [82–84]. A recent study has explored the relation-ship between dopamine homeostasis and learning in Nf1+/�GFAPCKO mice further [85]. This study found Nf1+/�GFAPCKO mice to have reduced dopamine levels in thehippocampus, similar to the striatum. Moreover, L-DOPAadministration rescued the post-acquisition Morris water-maze probe trial deficits in vivo, and dopamine D1 receptoragonist (i.e., SKF-38393) treatment corrected the LTPabnormalities in hippocampal slice preparations in vitro.These findings, coupled with previous observations, estab-lish defective dopaminergic function as a contributingfactor important for both spatial learning/memory andattention system dysfunction in Nf1 mutant mice.

Although the precise mechanism by which neurofibro-min modulates dopamine signaling has yet to be identified,these findings suggest that dopamine-targeted therapiesmay be useful treatments for some children with NF1-associated cognitive abnormalities. Furthermore, thesestudies highlight the potential utility of raclopride PETimaging to stratify children with NF1 into subgroups mostlikely to respond to dopamine-elevating treatments.

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TRENDS in Neurosciences

AC*

ATP

PKA

DARPP 32P

Dopamine

HO

OH

O

NH2

TH

L-DOPA

AC*

ATP

PKA

DARPP 32P

Dopamine

TH

L-DOPA

(a) (b)

AC*

ATP

PKA

DARPP 32P

Dopamine

TH

L-DOPA

(c)

* GTPcAMPDopami neMPHDA transp orter

Key:

GPCR

Reduced dopa minesignaling in Nf1 mutant mice

Dopamine si gna lingpathway in wildtype mice

Drug targets of dopa minepathway restore si gna ling andbehavior in Nf1 mutant mice

α

α

βγ

α

α

βγ

α

α

βγ

HO

OH

O

NH2 HO

OH

O

NH2

Figure 3. Role of dopamine in NF1-associated behavior. (a) Tyrosine hydroxylase (TH)-positive neurons in the substantia nigra project to the striatum and modulate

dopamine signaling. Presynaptically, TH converts tyrosine to L-DOPA. Upon release, dopamine returns to the presynaptic dopamine pool by binding dopamine transporters

or it activates adenylyl cyclase (AC) by binding to postsynaptic dopamine receptors. AC activation stimulates cAMP generation and dopamine- and cAMP-regulated

phosphoprotein-32 (DARPP32) phosphorylation in the striatum. (b) In Nf1 mutant mice, TH expression is reduced, leading to lower striatal dopamine levels and lower

effector signaling, as indicated by reduced DARPP32 phosphorylation [80]. Abnormal dopamine homeostasis also results in attention system dysfunction in the Nf1 mutant

model [80,81]. (c) Treatment with drugs that increase dopamine synthesis (L-DOPA), exogenous dopamine levels (dopamine), or block dopamine reuptake [e.g.,

methylphenidate (MPH)] restores striatal dopamine levels and reverses the attention phenotype in Nf1 mutant mice [80,81]. Abbreviation: GPCR, G protein-coupled

receptor.

Review Trends in Neurosciences April 2013, Vol. 36, No. 4

Clinical heterogeneity and implicationsAlthough it is convenient to regard monogenetic disorders ashomogenous medical conditions, the marked clinical vari-ability between members of the same family with the samegermline NF1 genetic mutation argues to the contrary.Instead, it is more likely that NF1 is composed of numerousdistinct diseases, each defined by factors including patientage, patient sex, the timing of NF1 inactivation, the specificcell type, genomic modifiers, and microenvironmental influ-ences. These factors have begun to be elucidated in cancersarising in Nf1 genetically engineered mice where the timingof bi-allelic Nf1 gene inactivation [86–88], the cell type (cellof origin) [89–96], the genetic (strain) background [97–99],and the non-neoplastic cell microenvironment [93,100–102]play critical deterministic roles.

Based on these observations, we propose a model ofNF1-associated cognitive abnormalities in which the widerange of clinical features represents an admixture of dis-tinct cellular and molecular etiologies. As such, there is nosingle molecular abnormality that underlies all of thecognitive dysfunction observed in children with NF1. Rath-er, the specific collection of cognitive and behavioral def-icits observed in any given child with NF1 reflects therelative contributions of multiple cellular and molecular

242

defects (Figure 4). Emerging evidence implicates specificneuronal populations, several molecular defects, and othercontributing factors; as such, we envisage a model ofcognitive dysfunction in which the resulting clinical phe-notype reflects the interplay of all of these potential ab-normalities.

First, genomic factors likely influence the specific con-stellation of cognitive and behavioral deficits observed inchildren with NF1. In this regard, attention deficits aremore common in boys in the general population, but areequally represented in both sexes in children with NF1 [1].This finding suggests that sexually dimorphic genomicfactors may contribute to the phenotypes observed inprepubescent children, as have been reported for otherCNS abnormalities in mice [103–105]. Second, not allCNS neuronal populations appear to be equally affectedby reduced Nf1 gene expression. The use of conditionalknockout mouse strains has already revealed that Nf1+/�

forebrain pyramidal neurons do not contribute to abnormallearning and memory [48]. It is possible that distinctneuronal populations differentially respond to reducedneurofibromin expression, and contribute in unique fash-ions to the learning, memory, and attention deficits thatcharacterize mice and people with a germline inactivating

Non-neuronalmicroenvironment

Neuronalheterogeneity

Molecularsignaling

Gene�cfactors

Alleleexpression

MEK PI3K

Excitatory(glutamatergic)

Inhibitory(GABAergic)

NF1cogni�ve

phenotype

Oligodendrocytes

AstrocytesMicroglia

Genomic modifiers(e.g. methyla�on)

CpG CpG

Germline muta�onNF1

cAMP

Ras*

*

Dopaminergic InterneuronNF1

Gender

Age

MRI T2 hyperintensi�es

Otherfactors

Dopamine

AC

A

R

a

TRENDS in Neurosciences

Figure 4. Factors influencing the NF1 cognitive phenotype. The specific cognitive phenotype observed in any given individual with NF1 reflects the confluence of genomic,

molecular, cellular, and environmental factors. The specific germline NF1 gene mutation, allele expression, and genomic modifying events (including methylation), may

influence clinical heterogeneity and create variations in neurofibromin expression in different cell types. Heterogeneity in neuronal subpopulations may also factor into the

overall cognitive phenotype. In this manner, the relative contributions of defects in different populations of CNS neurons (e.g., GABAergic, dopaminergic) may lead to a

distinct spectrum of cognitive and behavioral abnormalities. Similarly, less well-studied characteristics, such as patient sex, age, and NF1-associated brain abnormalities

(e.g., T2 hyperintensities), may also contribute to the observed patient cognitive profile. In addition, the effects of abnormal signaling through specific molecular pathways

(i.e., signaling heterogeneity) could likewise differentially affect distinct neuronal populations and lead to unique cognitive phenotypes. Despite being understudied to date,

it is also possible that NF1+/� oligodendrocytes, microglia, and/or astrocytes contribute to abnormal neuronal function as a result of disrupted axonal signal propagation,

impaired synaptic pruning, and/or changes in glutamate or other neurotransmitter availability.

Review Trends in Neurosciences April 2013, Vol. 36, No. 4

Nf1 gene mutation. Third, the specific germline NF1 genemutation may create different abnormalities in CNS neu-rons that reflect the degree of neurofibromin reduction or aspecific impairment in cAMP or Ras signaling. Althoughthere are few studies examining the effect of specific NF1gene mutations on neurofibromin expression and function,Nf1+/� mice maintained on different genetic backgroundsexhibit a wide spectrum of reduced Nf1 mRNA expression[99]. Fourth, it is possible that some of the more severeneurocognitive abnormalities occasionally seen in childrenwith NF1 may reflect total loss of neurofibromin expressionin select populations of CNS neurons. Recent evidencedemonstrates gross brain abnormalities following Nf1 bial-lelic inactivation in developing neural stem cells [106].Further study will be required to formally evaluate thisintriguing possibility. Fifth, Nf1 mutant mouse strainshave been instructive in elucidating the major neurochem-ical defects responsible for learning, memory, and atten-tion abnormalities associated with NF1. Intelligent use of

these preclinical mouse strains may provide insights intopotential treatments that reflect the predominant signal-ing defect (e.g., cAMP, Ras, dopamine) in individuals withNF1.

Finally, it is possible that non-neuronal cell types,including NF1+/� oligodendrocytes, astrocytes, and micro-glia, may contribute to neuronal dysfunction in the brainsof children and adults with NF1. In this regard, astrocytesare important for glutamate homeostasis in one tuberoussclerosis complex (TSC) mouse model of CNS disease, suchthat impaired astrocyte glutamate transport is partlyresponsible for the seizures and synaptic plasticity defectsin Tsc1 conditional knockout mice [107,108]. Similarly,oligodendrocytes can maintain neuronal integrity and pro-mote axonal signal propagation. Previous studies revealedthat spinal cord oligodendrocyte precursor differentiationis regulated by neurofibromin [109], such that twofoldmore NG2+ progenitor cells reside in the spinal cords ofNf1+/� mice. Lastly, microglia can regulate synaptic

243

Child A Child B Child C

Cogni�ve profile:

Personalized therapy:

Children with neurofibromatosis 1

Unaffected

Ras DA Ras DARas DARas DA

Ras ac�vity-loweringdrug (e.g., Lovasta�n)

Dopamine-eleva�ng drug(e.g., Methylphenidate)

Severe a�en�onand mild learning

deficits, impulsivity

Severe spa�allearning and

memory deficits

Moderate spa�allearning deficits,

distrac�bility

Combina�ontreatment

TRENDS in Neurosciences

Figure 5. Model of clinical heterogeneity and implications for treatment. The cognitive profile of children with NF1 likely includes a diverse set of learning, memory,

attention, and motor deficits, each linked to a distinct molecular abnormality (i.e., increased Ras, reduced cAMP, or low dopamine). In this regard, each child with NF1 has a

different level of Ras, dopamine, and cAMP (not shown) signaling that collectively contribute to the overall cognitive phenotype. Identifying the primary set of cellular and

molecular abnormalities may lead to individualized treatments with a higher likelihood of improving the neurocognitive deficits specific to that child or adult with NF1.

Review Trends in Neurosciences April 2013, Vol. 36, No. 4

plasticity and pruning [110,111]. Increased numbers ofmicroglia have been observed in Nf1+/� mouse brains[100], where they represent critical modulators of opticglioma growth [101,102]. Their roles in NF1-associatedlearning, memory, and attention deficits have not beenexplored.

Concluding remarksIn this review, we have highlighted the roles of Ras, cyclicAMP, and dopamine as key molecular targets that modu-late NF1-associated cognitive dysfunction. Each of thesefactors represents a potential therapeutic target; however,the efficacy of any particular treatment regimen will relyheavily on the specific constellation of molecular and

Box 1. Outstanding questions

� How does neurofibromin regulate cAMP homeostasis in CNS

neurons?

� How does neurofibromin regulate dopamine homeostasis in the

brain?

� What is the relationship between neurofibromin cAMP, Ras, and

dopamine regulation in CNS neurons?

� Does reduced neurofibromin function in distinct CNS neuronal

populations differentially contribute to the cognitive and beha-

vioral abnormalities observed in children with NF1?

� How do non-neuronal cells (such as microglia, oligodendrocytes,

and astrocytes) influence the cognitive and behavioral abnormal-

ities in children with NF1?

� How do combinations of molecular and cellular abnormalities

result in specific cognitive profiles in children with NF1?

� What underlies the clinical heterogeneity in children with NF1-

associated cognitive and behavioral abnormalities (genomic

modifiers, environmental factors, patient sex, etc.)? What are the

implications for treatment?

� How can we stratify children with NF1-associated cognitive and

behavioral abnormalities based on their unique molecular and

cellular defects? What are the implications for personalized therapy?

244

cellular abnormalities present in a given child with NF1(Figure 5). This idea is underscored by the outcomes ofseveral recent NF1 clinical trials. Although treatment withthe Lovastatin analog, Simvastatin, did not improve cog-nitive function in the group as a whole, it did enhanceperformance on some features of learning and memory in asubgroup of patients [47]. A more recent Phase I Lovastat-in trial demonstrated some improvement in verbal andnonverbal memory, without any effect on attention systemdysfunction [112]. Similarly, some reports have shown thatMPH can improve working memory in children withADHD [113] as well as learning in NF1 patients [2]. Byconsidering the interplay between each of these cellularand molecular abnormalities, future approaches to man-aging the learning, memory, and attention system deficitsin children with NF1 may consider integrating targetedtherapies specific to the biochemical and neurochemicalabnormalities unique to each child (Box 1).

AcknowledgmentsThis work was supported by grants from the Department of Defense(W81XWH-10-1-0884) and National Cancer Institute (U01-CA141549) toD.H.G.. K.D.A. is supported by a Diversity Supplement from the NationalCancer Institute (U01-CA141549).

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