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Dysregulation of Nova-mediated alternative pre-mRNA splicing in

the orbital frontal cortex of schizophrenia and bipolar disorders

Taylor Brown

 

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ABSTRACT

Schizophrenia (SCZ) and bipolar disorder (BPD) are two devastating mental disorders,

garnering substantial costs to the affected individuals and society. The underlying etiology of

SCZ and BPD remains largely unknown, but SCZ and BPD are commonly viewed as a complex

disorder of brain development and plasticity involving alterations of multiple genes and various

neurobiological processes contributed by both genetic and environmental factors. Neuro-

oncological ventral antigens (Nova1 and Nova2) are neuron-specific RNA binding proteins or

splicing factors that regulate alternative pre-mRNA splicing, transcription, mRNA stability, and

trafficking of many neuronal genes, most of which are expressed in synapse and associated with

SCZ and BPD. This study is one of the first to describe the dysregulation of Nova proteins and

Nova-mediated splicing regulation in the postmortem orbital frontal cortex (OFC) of SCZ and

BPD patients. Particularly, Nova-regulated splicing in a NMDA receptor subunit (GRIN1), a

GABAA receptor subunits (GABRG2) and a calcium channel (CACNA1B) was found to be

significantly altered in the OFC of SCZ and BPD patients, most likely due to an increased

expression of Nova proteins. In addition, steady-state mRNA levels in several Nova targets

including ARHGAP21, CACNA1B, CAMK2G, EPHA5 and PLCB4, were upregulated in the

OFC of BPD and/or SCZ patients, suggesting potential Nova-mediated regulation at steady-state

mRNA level. These findings strongly endorse a novel hypothesis that dysregulation of Nova-

mediated splicing regulation in various brain regions plays an important role in development and

pathogenesis of SCZ and BPD. Most importantly, it may provide a new perspective on the

underlying etiology of SCZ and BPD, and may potentially lead to the development of novel

therapeutics for the treatment and/or prevention of SCZ and BPD. The current study may also

have an impact on studies of other psychiatric disorders such as depression and autism, some of

which have clinical and pathophysiological features similar to those seen in SCZ and BPD.

 

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INTRODUCTION

Schizophrenia (SCZ) and bipolar disorders (BPD) are chronic, severe and prevalent

mental illnesses, together affecting approximately 1.5% of the population1, 2, and characterized

by affective and cognitive symptoms. SCZ patients often suffer from positive symptoms such as

hallucinations and delusions, and negative symptoms such as alogia, avolition and flat affect, and

depression. BPD patients usually experience abnormally intense emotional states or mood

episodes that can be manic, or depressive, or mixed. When psychosis is exhibited, schizophrenia

and bipolar disorders are at times mistaken for each other due to their many similarities. Both

SCZ and BPD patients have a higher suicidal rate than those nit affected with mental illness.

There are no medications that can cure SCZ and BPD, largely because the causes of SCZ and

BPD are still unknown, and most treatments using antipsychotic drugs simply focus on relieving

the symptoms by blocking receptors or transporters, and are often prescribed for both SCZ and

BPD patients. Although some of the anti-psychotic medications, such as Abilfy and Risperdol,

are prescribed to SCZ patients, they are also used as off-label medications for psychosis and with

mania. Therefore, these devastating neuropsychiatric disorders create substantial financial and

emotional burdens for affected individuals, their families and our society.

The underlying etiology of SCZ and BPD remains largely unknown despite decades of

intense research efforts. Nevertheless, SCZ and BPD should be considered as complex disorders

of brain development and plasticity involving alterations of multiple genes and various

neurobiological processes contributed by both genetic and environmental factors3-­‐11. The

heritability estimated from twin/family studies is quite high (0.81 for SCZ and 0.75 for BPD)10,

which is consistent with estimates of single nucleotide polymorphism (SNP) heritabilities for

SCZ and BPD from recent genome-wide association (GWA) studies12. Linkage and genome-

 

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wide association studies have suggested associations of several chromosomal regions and

multiple genes with SCZ and BPD11, 13. Several hypotheses involving neurotransmitter

dysregulation, such as the dopamine, serotonin and glutamine, calcium channel dysfunction,

immune system and neurodevelopmental dysfunction have been proposed to define the

underlying mechanisms of developing SCZ and BPD14-­‐21. For example, the dopamine hypothesis

explains dysfunction of dopaminergic system in SCZ and BPD14, whereas the glutamate

hypothesis emphasizes disturbed glutamate pathways mainly involved the N-methyl-D-aspartate

(NMDA) receptor15. Neurodevelopmental hypothesis associates neruodevelopmental defects

during the prenatal and early postnatal period with SCZ and BPD22. Using neuroimaging such as

magnetic resonance imaging (MRI), neuromorphological abnormalities have been reported in a

number of brain regions of SCZ and BPD, such as the frontal lobe including the orbital frontal

cortex, temporal lobe, somatosensory and motor cortices, thalamus and hippocampus23-­‐43, some

areas that were investigated in this study.

Neuro-oncological ventral antigens (Nova) including Nova1 and Nova2 were initially

identified in paraneoplastic opsoclonus myoclonus ataxia (POMA), an autoimmune neurologic

disease, as neuron-specific RNA-binding proteins44-47. Nova family proteins include Nova1 with

a molecular weight about 55 kDa on SDS PAGE, and Nova2 expressed as two forms, one with ~

55 kDa and the other, ~72 kDa (Supernova). Both Nova1 and Nova2 proteins are primarily

expressed in the central nervous system, particular in neocortical regions47. Nova proteins bind to

YCAY-rich elements of their target transcripts to regulate their alternative splicing, stability,

trafficking and transport46-48. Nova-mediated splicing regulation acts through YCAY-rich

elements that are commonly located at exon and its adjacent introns, and either enhances or

represses exon splicing depending upon where they bind and how they interact with other

 

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splicing factors (Fig. 1)49-51.

Nova proteins also regulate

steady-state mRNA levels of

their target transcripts through

YCAY-rich elements located

at 5’-untranslated region

(UTR), coding regions and 3’-

UTR, as well as adjacent

introns. Importantly, a large

number of Nova-regulated

targets are proteins critical for

synaptic function and plasticity, such as NMDA receptors, calcium channels, GABAA receptors,

neuregulin-1, and neurexin-1 (Fig. 2)52, which have all been associated with SCZ and BPD. For

example, 6 of 32 proteins located at 22q11 whose deletion was found in 30% of SCZ patients are

the Nova targets: UFD1L, TXNRD7, COMT, ARVCF, RANBP1 and LZTR153.   Moreover,

Changes in alternative splicing of several individual genes have been reported in SCZ and

BPD54-57. Many of these splicing events are regulated by Nova. For example, 5 of 8 genes

(ADAR, BIRC6, MAPK14, STXBP2 and ZDHHC3) showing significant alternative splicing

changes in SCZ patients belong to Nova-regulated targets54. All these observations provide the

rationales for a novel hypothesis that dysregulation of Nova proteins and Nova-mediated

alternative splicing plays an important role in development and pathogenesis of SCZ and BPD.

The present study examines this hypothesis by analyzing RNA and protein samples from the

 

Fig.1. Nova-mediated splicing regulation (Adapted from literature49-51). Shaded light orange and blue areas are the distribution of Nova binding sites (YCAY clusters45,49) positively and negatively regulated by Nova proteins for exon splicing, respectively. Fox, SR proteins, hnRNP and PTBP are also shown to influence Nova-mediated splicing74-81.

 

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postmortem orbital frontal cortex of SCZ and BPD patients using RT-PCR, RT-qPCR and

Western blot analysis.

MATERIALS AND METHODS

RNAs and proteins.

Total RNAs and proteins

extracted from the postmortem

orbital frontal cortex of normal

control subjects (n = 35),

schizophrenia (n = 35) and

bipolar disorder patients (n = 35)

were obtained from Stanley

Medical Research Institute

(SMRI). The demographic

information is listed on Table 1.

Reverse-transcription

(RT)-SYBR green quantitative

polymerase chain reaction

(qPCR) and regular PCR. Total

RNA was treated with Turbo

DNase I to remove potential

contaminated genomic DNA and

reverse transcribed with random

Fig. 2. The synaptic module of 41 proteins encoded by validated Nova-regulated RNA  (Adapted from literature35). Proteins pointed by dashed green arrows are those examined in this study and shown significant (Oval frame) and trended (rectangle frame) changes, respectively (see Table 4). Cav2.2: CACNA1B; GABAARγ2: GABRG2; NDMAR1: GRIN1; EphrinA5R: EPHA5; PLCβ4: PLCB4; Neogenin: NEO1.  

 

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primers and Superscript III reverse

transcriptase (Life Technologies).

The first-strand cDNA was then

used as template in qPCRs with

HotStart SYBR green master mix

(Affymetrix) to amplify Nova1 and

Nova2 mRNAs, as well as 8 Nova-

regulated transcripts with

appropriate primers designed from

constitutively expressed exons

using AB7500 Machine (Applied

Biosystems). The human succinate

Table 1. Demographic summary of samples from SMRI  

  PMI: postmortem interval; RIN: RNA integrity number.

Table 2. Primers and PCR conditions for amplifying steady-state mRNAs

 

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dehydrogenase subunit A (hSDHA), humann beta-2 microglobulin (hB2M) and humen

glyceraldehyde 3-phosphate dehydrogenase (hG3PDH) were used to determine normalization

factor with (C(t)hSDHA x C(t)hB2M x C(t)hG3PDH)1/3 format. All variant C(t) values were normalized

with the normalization factor to obtain ∆C(t). (∆C(t) = C(t)variant – normalization factor). To

analyze Nova-mediated splicing exons, the same first-strand cDNA was used as template in

regular PCRs with appropriate primers designed from Nova-regulated splicing exons, which

generated two PCR fragments, the longer one as exon inclusion and the shorter one as exon

exclusion. Regular PCR conditions including annealing temperature and number of cycles were

optimized. PCR products were then analyzed on 3% of MetaPhor agarose gel, stained with

ethidium bromide, imaged and quantified on ImageLab 4.1, ChemiDoc MP System (Bio-Rad).

All PCR primers and conditions are listed in Tables 2 & 3. Exon inclusion (ψ) was calculated by

dividing the percentage of the bands representing exon inclusion by the total percentage of bands

representing exon inclusion and exclusion. Splicing changes (∆ψ) was determined by subtraction

between SCZ or BPD and Control. A positive ∆ψ value indicates enhanced exon inclusion by

Nova, and negative one suppressed by Nova.

Table 3. Primers and PCR conditions for amplifying Nova-regulated exons

*: The primer sequences were adapted from Tollervey et al82.

 

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Western blot analysis. Protein samples were denatured in SDS sample buffer (25mM

Tris, 0.19M glycine, 2% SDS, 0.1M DTT, 0.25% β-mecaptoethanol) by heating at 100°C for 10

min., separated on pre-casted Any kD stain-free SDS polyacrylamide gel electrophoresis (PAGE)

(Bio-Rad), and transferred onto PVDF membranes using Trans-Blot Turbo (Bio-Rad). Gels were

UV-crosslinked before transfer to conjugate florescent substrate to any proteins on the gel for the

purpose of quantifying the amount of total proteins on transferred PVDF membranes that were

used in normalization for protein loading control. This stain-free technology is designed for

assessment of protein loading with clear advantages in accuracy and simplicity over traditional

method using an antibody against individual house-keeping protein such as Actin and GAPDH.

The PVDF membranes were then blocked in a block solution containing TTBS (10mM

Tris–HCl, pH 7.4, 150mM NaCl, and 0.05% Tween 20), 5% nonfat dried milk at room

temperature for 1 h and incubated with anti-Nova1 antibody (Santa Cruz, 1:1000 dilution) or

anti-Nova2 antibody (ProteinTech, 1:800 dilution) or anti-Nova antibody (a gift from Dr. Robert

Darnell, Rockefeller University (1:3000 dilution) in the block solution at 4°C overnight. After

washing with TTBS buffer, the membrane was incubated with appropriate peroxidase-

conjugated secondary antibodies in TTBS buffer at room temperature for 1 h. After washing with

TTBS buffer, the signals were determined by using Luminata Forte Western HRP substrate

(Millipore), imaged and analyzed on ImageLab 4.1, ChemiDoc MP System. Expression level of

Nova1 and Nova2 bands was determined by dividing band intensity of Nova proteins by

intensity of total protein bands shown on PVDF membranes by stain-free technology.

Statistical analysis. Student t-test was used to analyze statistical significance.

RESULTS

 

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Nova mRNA levels are upregulated in the orbital frontal cortex of BPD, but not SCZ patients.

To determine steady-state mRNA expression level of Nova1 and Nova2 in the orbital

frontal cortex of SCZ and BPD patients and unaffected control subjects, I performed RT-SYBR

green qPCRs using primers designed from constitutively expressed exons. I observed that the

expression levels of both Nova1 and Nova2 mRNAs were significantly higher in BPD patients

than in control subjects (Fig. 3). There was 1.5-fold and 1.7-fold increase of Nova1 and Nova2

mRNAs in BPD as compared to control subjects, respectively. However, the expression levels of

Nova1 and Nova2 mRNAs in SCZ patients seemed lower than those in control subjects, but there

was no statistical significance (Fig. 3). Also, the expression level of Nova1 mRNA was slightly

higher than that of Nova2 mRNA in all three groups with no statistical difference. The mRNA

expression determined by RT-SYBR green qPCR is an indicator of steady-state mRNA level that

is contributed by transcription and

degradation. The results suggested

increased transcription and/or decrease

degradation of Nova1 and Nova2

mRNAs in the orbital frontal cortex of

BPD patients.

Nova proteins are upregulated in the orbital frontal cortex of both SCZ and BPD patients.

 

Fig. 3. Nova mRNAs are upregulated in the oribital frontal cortex of BPD, but not SCZ patients. RT-SYBR qPCR was performed as described in Materials & Methods. Expression level of Nova1 and Nova2 mRNAs in Control (n=34), SCZ (n=34) and BPD (n=33) is shown by 2-

∆C(t) (∆C(t) = C(t)Nova – normalization factor determined from C(t) values of hSDHA, hB2M and hD3PDH). Fold change determined by division between two indicated groups is indicated beside the line. Statistical significance was analyzed by Student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.  

 

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Next, I performed Western blot analyses to examine expression of Nova proteins in the orbital

frontal cortex of the same SCZ, BPD and control subjects. I observed that the expression levels

of Nova1 and Nova2 proteins including the low molecular weight band (~55kDa) and high

molecular weight band (Super Nova2, ~72kDa) were significantly increased in SCZ and BPD

patients as compared to control subjects

(Fig. 4). Similar results were obtained

using an anti-Nova antibody against both

Nova1 and Nova2 proteins (a gift from

Dr. Darnell) (data not shown). The

protein level measured by Western blot

analysis is an indicator of steady-state

protein level that is mainly determined

by translation and degradation. These

 

   

Fig. 4. Nova proteins are upregulated in the orbital frontal cortex of both SCZ and BPD patients. A. Western blots. Western blot analysis using protein samples from Control (n=32), SCZ (n=32) and BPD (n=32) was performed as described in Materials & Methods. The results from 3 representative samples of each group (C1-C3, S1-S3 and B1-B3) using anti-Nova1 (Santa Cruz) and anti-Nova2 (ProteinTech) antibodies are shown. B, C & D. Quantification of Western blots. Expression level of Nova proteins were determined by dividing band intensity of Nova by those of total proteins transferred on PVDF membranes shown by stain-free technology. Fold changes between two groups were indicated by numbers beside lines. Statistical significance was analyzed by Student t-test. *: p < 0.05; **: p < 0.01.  

 

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results suggested that in the orbital frontal cortex of both SCZ and BPD patients Nova proteins

are more efficiently translated and/or less degradated. The increased Nova proteins in BPD

patients were consistent with their upregulated levels of Nova mRNAs (Fig. 3), suggesting that

increased Nova mRNAs may contribute to the enhanced translation of Nova proteins in BPD

patients. On the other hand, the increased Nova proteins in SCZ patients were clearly not

contributed by the changes of their steady-state Nova mRNA levels (Fig. 3), suggesting

regulatory mechanisms at the post-transcriptional level.

Nova-regulated splicing events are altered in the orbital frontal cortex of SCZ and BPD patients.

Nova proteins are neuron-specific splicing factors that regulate alternative splicing of

hundreds of genes, particularly neuronal genes richly expressed in synapse (Fig. 2)52. Increased

Nova proteins in SCZ and BPD patients raise questions as to whether or not these Nova-

regulated splicing events are consequently changed. I then performed RT-PCR to analyze eight

selected splicing events regulated by Nova proteins that were validated in mouse and human

exons52,82 (Table 4). I found that three Nova-regulated exons in CACNA1b, GABRG2 and

GRIN1 genes were significantly upregulated in the OFC of SCZ patients, and three Nova-

regulated exons in EPHA5, NEO1 and PLCB4 genes trended to be changed in SCZ and BPD

patients with no statistical difference, all of which were correlated with the increased Nova

proteins in these patients (Table 4 and Fig. 5). There were no changes in two Nova-regluated

exons in ARHGAP21 and CAMK2G genes. The results suggested that in SCZ and BPD patients

increased Nova proteins led to altered expression of some, but not all, Nova-regulated splicing

events or isoforms, and these alterations may contribute to development of SCZ and BPD. In the

 

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near future, it will be interesting to investigate if any of the validated or non-validated Nova-

regulated splicing events are altered in the

orbital frontal cortex of SCZ and BPD

patients.

Huntsman et al. previously

reported58 that the ratio of γ2L (E11

inclusion) and γ2S (E11 exclusion) of

GABRG2 was increased in the prefrontal

cortex of SCZ, an observation consistent

with the results from the present study. The

γ2L form has extra 8 amino acids bearing a

potential phosphorylation site for protein

kinase C. The functional relevance of the

γ2L/γ2S ratio was suggested by a

 

Fig. 5. Nova-regulated splicing events are altered in the orbital frontal cortex of SCZ and BPD patients. A. RT-PCR products. RT-PCR using RNA samples from Control (n=32), SCZ (n=32) and BPD (n=32) was performed as described in Materials & Methods. The results from three Nova-regulated splicing (GABRG2, CACNA1 and GRIN1) of 3 representative samples of each group (C1-C3, S1-S3 and B1-B3) are shown. Inc: Inclusion; Exc: Exculsion. B, C & D. Quantification of RT-PCR products. Intensities of top and bottom bands from RT-PCR products were quantified as exon inclusion and exclusion, respectively, using ImageLab (Bio-Rad). Changes of exon inclusion (∆ᴪ) were indicated by numbers beside lines. Statistical significance was analyzed by Student t-test. *: p < 0.05; **: p < 0.01; ***: p < 0.001.  

 

 

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difference in GABA-mediated chloride currents59 and GABAA receptor trafficking60, 61 mainly

contributed by the phosphorylation site. The GABAA receptors with more γ2L subunits had

reduced GABA-mediated chloride currents when compared to those with more γ2S subunits.

Steady-state mRNA levels of Nova-regulated targets are upregulated in the orbital frontal

cortex of SCZ and BPD patients.

Nova proteins can regulate steady-state mRNA levels of their targets through binding to

YCAY-rich elements located at 5’-UTR, coding region and 3’-UTR. These YCAY elements are

functionally independent from those modulating splicing. For example, 17% of Nova high-

throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP) tags

were mapped to 3’-UTR17, a region important for mRNA stability, translation and transport. I

found that in the 5’-UTR, coding region and 3’-UTR of the 8 selected genes there are a number

Table 4. RT-PCR analysis of Nov-regulated exons

Note: Data of Nova KO (knockout) mice was obtained from Dr. Darnell’s lab (http://splicing.rockefeller.edu/). Exon inclusions highlighted by light orange had significant changes, and those by light green trended to have changes. Statistical significance was analyzed by Student t-test as compared to Control group. *: p < 0.05; **: p < 0.01; ***: p < 0.001.

 

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of YCAY-rich clusters (data not shown), suggesting that they are potentially subject to be

regulated by Nova at steady-state mRNA level. To examine whether steady-state mRNA levels

of these 8 Nova targets are altered, I performed SYBR green qPCRs using the same RT products

in determining expression of Nova mRNAs and Nova-regulated splicing exons with primers

derived from constitutively expressed exons of these genes (Table 2). I observed that the steady-

state mRNA levels of 4 genes (ARHGAP21, CACCNA1B, CAMK2G and NEO1) in the OFC of

BPD patients were significantly upregulated, while the mRNA levels of 3 genes (EPHA5, NEO1

and PLCB4) were increased in the OFC of SCZ patients (Fig. 6). Interestingly, most of these

increased expressions were not correlated with the changes in Nova-regulated splicing (Table 3

and Fig. 4), except for CACNA1B in BPD. For example, both Nova-regulated splicing exons in

ARHGAP21 and CAMK2G were not altered in BPD, but the steady-state mRNA levels of these

2 genes were upregulated. Nova-regulated splicing exons in NEO1 and PLCB4 were trended to

be downregulated in the OFC of both SCZ and BPD. However, the steady-state mRNA levels of

Fig. 6. Steady-state mRNA levels of Nova targets. RT-SYBR qPCR was performed as described in Materials & Methods. % change of 8 validated Nova-regulated target mRNAs in Control (n=34), SCZ (n=34) and BPD (n=33) is calculated by 2-∆∆C(t) * 100 (∆C(t) = C(t)target – normalization factor determined from C(t) values of hSDHA, hB2M and hD3PDH; ∆∆C(t) = ∆C(t)SCZ or BPD - ∆C(t)Control). *: p < 0.05;

 

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these 2 genes were significantly increased in the OFC of SCZ and/or BPD. These results

suggested that Nova-mediated splicing regulation is independent from potential Nova-mediated

modulation at steady-state mRNA level, although it needs to be experimentally verified.

DISCUSSION

For the first time, the present study demonstrates alterations of Nova1 and Nova2

proteins, and their regulated splicing events in the orbital frontal cortex of SCZ and BPD patients.

This provides strong support for the hypothesis that dysregulation of Nova-mediated alternative

splicing in various brain regions plays an important role in development and pathogenesis of

SCZ and BPD. What makes the hypothesis more attractive is that Nova proteins regulate

expression of many protein isoforms that are expressed in synapses (Fig. 2), some of which have

already been linked to the pathogenesis of these disorders. For example, altered expression and

function of NMDA receptors and GABAA receptors have been extensively reported in various

brain regions of SCZ and BPD patients. Genetic variations in voltage-dependent calcium

channels, NMDA and GABAA receptors have been linked to SCZ and BPD19. As illustrated by

the present study, Nova-mediated alternative splicing of a NMDA receptor subunit (GRIN1), a

GABAA receptor subunit (GABRG2) and a voltage-dependent calcium channel subunit

(CACNA1) is dysregulated in the orbital frontal cortex of SCZ and BPD, as a result of the

increased Nova proteins. These findings raise a suggestive hypothesis that Nova-mediated

splicing regulation is a key player in development and pathogenesis of SCZ and BPD.

It should be noted that the magnitude of the dysregulation was not dramatic for all three

Nova-regulated exons, and the changes in the % of exon inclusion (∆ψ) ranged from 6.3 (GRIN1

in SCZ) to 9.5 (CACNA1B in SCZ) (Table 3 & Fig. 5). This may be correlated with similar

degree of changes in Nova proteins seen in SCZ and BPD patients, ranging from 1.20-fold

 

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increase of Nova1 in BPD to 1.59-fold increase of Nova2(~72kDa) in SCZ (Fig. 4). However,

summation of these small changes in each of these three Nova-regulated events could be

dramatic. Nova proteins regulate alternative splicing of at least 48 validated splicing targets

including neurotransmitter receptors, cation channels, adhesion and scaffold proteins, all of

which are highly expressed in synapse52, in addition to others not yet validated. The present

study reveals that aproximately 37.5% of Nova-regulated splicing events (three of eight) were

dysregulated. Based on this rate, it will account for ~ 18 synaptic isoforms (37.5% of 48). One

can imagine that not only are there cumulative effects, but also synergistic effects among these

dysregulated proteins, which contribute greatly to alterations in synaptic functions and plasticity,

a pathological consequence commonly seen in SCZ and BPD patients. Although the functional

relevance of each Nova-regulated splicing isoform and the functional relationship among these

isoforms in SCZ and BPD needs to be further investigated, the current findings highlight the

potential influence of Nova-mediated splicing regulation in development and pathogenesis of

SCZ and BPD.

The present findings also raise questions regarding how Nova proteins are upregulated in

the orbital frontal cortex of SCZ and BPD patients. As demonstrated here, the underlying

mechanisms of Nova protein regulation are different between SCZ and BPD. In SCZ, the

increase of Nova proteins with no alteration of their mRNAs suggests regulatory mechanisms

mainly at the post-transcriptional level. However, upregulation of both Nova mRNAs and

proteins in BPD implicates the mechanisms at both transcription and/or post-transcription levels.

microRNAs (miRNAs) are a class of non-coding RNAs that regulate gene expression at both

transcription and translation levels, and play important roles in many biological processes such

as neuronal development and differentiation, synapse formation and neuronal plasticity.

 

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Dysregulation of miRNAs has been linked to the cause of several brain disorders including SCZ,

BPS, major depression, Parkinson’s disease, as well as drug addiction62-69. It is most likely that

Nova mRNAs are regulated by miRNAs. Multiple miRNAs have been shown to target Nova

mRNAs based upon several computer models including miRBase, Targetscan, RNAhybrid,

RNA22 and miRWalk. Interestingly, Nova2 is a potential target of mir-185 located within 22q11

whose deletion was found in 30% of SCZ patients53, 70. In the PFC of Df(16)A+/- mice, a mouse

model mimicking human 22q11.2 microdeletion, 10 of 59 downregulated miRNAs potentially

targets Nova171, 72. Downregulation of these miRNAs may explain the upregulation of Nova

proteins in the OFC of SCZ and BPD patients, although speculated. Additionally, alterations of

several miRNAs previously reported in association with SCZ and BPD are also on the list of

miRNAs predicted to target Nova mRNAs, such as miR-13263 and miR-2273, suggesting that

these miRNAs also play an important role in regulating expression of Nova proteins and Nova-

mediated splicing isoforms. If confirmed, it will provide a functional link between miRNA and

Nova-mediated splicing regulation.

Several brain regions including the cingulate cortex, frontal cortex, temporal pole,

thalamus, and hippocampus have been indicated to be critically involved in pathogenesis of SCZ

and BPD. Although the present study only examined the orbital frontal cortex, I hope to extend

our study on expression of Nova proteins and Nova-mediated splicing isoforms in SCZ and BPD

to the other brain regions in the near future. It will be very interesting to see if the other brain

regions have the same or different changes as those observed in the orbital frontal cortex.

Alternative splicing involves coordination of multiple components including cis-acting

elements located within the exons and proximal or distal introns, trans-acting factors, including

the serine/arginine-rich (SR) family proteins, hnRNP proteins, the CELF protein family and

 

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Nova proteins, that interact with the cis-acting elements, and their interactions with the basal

spliceosome. It has been shown that Nova-mediated splicing is greatly influenced by other

splicing factors such as Fox2, PTBP2, hnRNPA1, hnRNPH, hnRNPL and SR proteins (Fig. 1)50,

74-81. Now, it will be interesting to investigate whether these splicing factors are also dysregulated

in SCZ and BPD.

The present study used RNA and protein samples from dissected regions. One caveat of

using these samples is heterogeneity of various cell types in the samples that may differentially

express Nova mRNAs and proteins. It will be essential to examine the expression of Nova

mRNAs and proteins at an anatomic level by using in situ hybridization for Nova mRNAs and

immunohistochemistry for Nova proteins, if such brain tissues are available. The results from

these studies can further direct studies using laser capture microdissection approach that allows

collecting single cell or very small region for examining expression of Nova-regulated splicing

events.

Alterations of steady-state mRNAs in a number of genes bearing YCAY-rich elements at

5’-UTR, coding region and 3’-UTR in the OFC of SCZ and/or BPD patients raises questions

whether or not Nova proteins indeed regulate these targets, and if so, what the underlying

mechanisms are. Steady-state mRNA level is determined by many other factors such as

transcription factors that can also be altered in the OFC of SCZ and/or BPD patients. I believe

that alteration of Nova proteins can coordinate with changes of other factors, and such

coordination at least partly contributed to modulation of these steady-state mRNA levels.

 

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CONCLUSION AND FUTURE WORK

The present study shows that Nova proteins and Nova-regulated splicing isoforms were

dysregulated in the postmortem orbital frontal cortex of SCZ and BPD, providing a strong

support for a new hypothesis that dysregulation of Nova-mediated splicing in brain plays an

important role in development and pathogenesis of SCZ and BPD. The significances of the

present findings are three-fold. First, majority of Nova-regulated genes are critical in

maintaining normal functions of synapse, particularly in the cortex regions. Summation of small

changes in splicing of these genes can potentially alter synaptic formation, development and

plasticity, leading to the development of SCZ and BPD. Alterations of just three Nova-regulated

splicing isoforms in GRIN1, GABA and CACNA1 genes discovered in the present study bridge

together of three systems, the NMDA receptor, the GABAA receptor and calcium channels,

whose alterations are commonly used for explaining the pathogenesis of SCZ and BPD.

Involvement of multiple Nova-regulated targets is also supporting the notion that SCZ and BPD

are complex brain disorders contributed by dysfunction of multiple genes. Second, further

exploring mechanisms underlying regulation of Nova mRNAs and proteins and their targets will

possibly link to regulation of miRNA and/or epigenetics, as well as other splicing factors,

providing even broad and deep understanding of the roles of Nova proteins in SCZ and BPD.

Third, if we can establish the functional relevance of Nova-regulated isoforms, it will lead to the

development of new drugs for the treatment and/or prevention of SCZ and BPD. For example,

we can use siRNA targeting Nova mRNAs to downregulate Nova proteins in the orbital frontal

cortex and therefore correct aberrant Nova-mediated splicing. Alternatively, we can deliver Nova

proteins into brain regions where Nova proteins are downregulated by using an appropriate viral

 

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vector. Also by using morpholino antisense oligonucleotide approach, we can correct altered

splicing events.

The present study raises many intriguing questions that need to be addressed in the near

future regarding the role of Nova-mediated splicing regulation in development of SCZ and BPD.

1) How are the other 40 synaptic protein isoforms regulated by Nova proteins expressed in the

orbital frontal cortex of SCZ and BPD? 2) How are Nova proteins and Nova-regulated isoforms

expressed in other brain regions of SCZ and BPD? 3) What are the molecular mechanisms

underlying regulation of Nova mRNAs and proteins? 4) What is the functional relevance of

Nova-regulated isoforms, particularly for the genes already shown to be important in SCZ and

BPD, such as NMDA receptor (GRIN1), GABAA receptor (GABRG1) and calcium channel

(CACNA1) that are reported here? 5) Can we develop an animal model that mimics SCZ or BPD

in which Nova proteins and Nova-mediated splicing are dysregulated in a similar way to those in

the human postmortem brains? 6) Are other splicing factors, particularly for those related to

Nova-mediated splicing regulation, dysregulated in brain regions of SCZ and BPD? 7) What is

the impact of current antipsychotic drugs used for treating SCZ and BPD on expression of Nova

proteins and Nova-regulated isoforms? 8) What are the potentials for developing new

therapeutics to treat and/or prevent SCZ and BPD based upon the hypothesis raised by the

present study.

 

22    

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