Paul G. Matteson

Autism-Relevant Social Abnormalities and Cognitive Deficits in Engrailed-2 Knockout Mice

ENGRAILED 2 (En2), a homeobox transcription factor, functions as a patterning gene in the early development and connectivity of rodent hindbrain and cerebellum, and regulates neurogenesis and development of monoaminergic pathways. To further understand the neurobiological functions of En2, we conducted neuroanatomical expression profiling of En2 wildtype mice. RTQPCR assays demonstrated that En2 is expressed in adult brain structures including the somatosensory cortex, hippocampus, striatum, thalamus, hypothalamus and brainstem. Human genetic studies indicate that EN2 is associated with autism. To determine the consequences of En2 mutations on mouse behaviors, including outcomes potentially relevant to autism, we conducted comprehensive phenotyping of social, communication, repetitive, and cognitive behaviors. En2 null mutants exhibited robust deficits in reciprocal social interactions as juveniles and adults, and absence of sociability in adults, replicated in two independent cohorts. Fear conditioning and water maze learning were impaired in En2 null mutants. High immobility in the forced swim test, reduced prepulse inhibition, mild motor coordination impairments and reduced grip strength were detected in En2 null mutants. No genotype differences were found on measures of ultrasonic vocalizations in social contexts, and no stereotyped or repetitive behaviors were observed. Developmental milestones, general health, olfactory abilities, exploratory locomotor activity, anxiety-like behaviors and pain responses did not differ across genotypes, indicating that the behavioral abnormalities detected in En2 null mutants were not attributable to physical or procedural confounds. Our findings provide new insight into the role of En2 in complex behaviors and suggest that disturbances in En2 signaling may contribute to neuropsychiatric disorders marked by social and cognitive deficits, including autism spectrum disorders.

Identification of a Schizophrenia-Associated Functional Noncoding Variant in NOS1AP

OBJECTIVE The authors previously demonstrated significant association between markers within NOS1AP and schizophrenia in a set of Canadian families of European descent, as well as significantly increased expression in schizophrenia of NOS1AP in unrelated postmortem samples from the dorsolateral prefrontal cortex. In this study the authors sought to apply novel statistical methods and conduct additional biological experiments to isolate at least one risk allele within NOS1AP. METHOD Using the posterior probability of linkage disequilibrium (PPLD) to measure the probability that a single nucleotide polymorphism (SNP) is in linkage disequilibrium with schizophrenia, the authors evaluated 60 SNPs from NOS1AP in 24 Canadian families demonstrating linkage and association to this region. SNPs exhibiting strong evidence of linkage disequilibrium were tested for regulatory function by luciferase reporter assay. Two human neural cell lines (SK-N-MC and PFSK-1) were transfected with a vector containing each allelic variant of the SNP, the NOS1AP promoter, and a luciferase gene. Alleles altering expression were further assessed for binding of nuclear proteins by electrophoretic mobility shift assay. RESULTS Three SNPs produced PPLDs >40%. One of them, rs12742393, demonstrated significant allelic expression differences in both cell lines tested. The allelic variation at this SNP altered the affinity of nuclear protein binding to this region of DNA. CONCLUSIONS The A allele of rs12742393 appears to be a risk allele associated with schizophrenia that acts by enhancing transcription factor binding and increasing gene expression.

Autism Associated Haplotype Affects the Regulation of the Homeobox Gene, ENGRAILED 2

BACKGROUND Association analysis identified the homeobox transcription factor, ENGRAILED 2 (EN2), as a possible autism spectrum disorder (ASD) susceptibility gene (ASD [MIM 608636]; EN2 [MIM 131310]). The common alleles (underlined) of two intronic single nucleotide polymorphisms (SNPs), rs1861972 (A/G) and rs1861973 (C/T), are over-transmitted to affected individuals both singly and as a haplotype in three separate datasets (518 families total, haplotype p = .00000035). METHODS Further support that EN2 is a possible ASD susceptibility gene requires the identification of a risk allele, a DNA variant that is consistently associated with ASD but is also functional. To identify possible risk alleles, additional association analysis and linkage disequilibrium (LD) mapping were performed. Candidate polymorphisms were then tested for functional differences by luciferase (Luc) reporter transfections and electrophoretic mobility shift assays (EMSAs). RESULTS Association analysis of additional EN2 polymorphisms and LD mapping with Hapmap SNPs identified the rs1861972-rs1861973 haplotype as the most appropriate candidate to test for functional differences. Luciferase reporters for the two common rs1861972-rs1861973 haplotypes (A-C and G-T) were then transfected into human and rat cell lines as well as primary mouse neuronal cultures. In all cases the A-C haplotype resulted in a significant increase in Luc levels (p < .005). The EMSAs were then performed, and nuclear factors were bound specifically to the A and C alleles of both SNPs. CONCLUSIONS These data indicate that the A-C haplotype is functional and, together with the association and LD mapping results, supports EN2 as a likely ASD susceptibility gene and the A-C haplotype as a possible risk allele.

The orphan G protein-coupled receptor, Gpr161, encodes the vacuolated lens locus and controls neurulation and lens development

The vacuolated lens (vl) mouse mutant causes congenital cataracts and neural tube defects (NTDs), with the NTDs being caused by abnormal neural fold apposition and fusion. Our positional cloning of vl indicates these phenotypes result from a deletion mutation in an uncharacterized orphan G protein-coupled receptor (GPCR), Gpr161. Gpr161 displays restricted expression to the lateral neural folds, developing lens, retina, limb, and CNS. Characterization of the vl mutation indicates that C-terminal tail of Gpr161 is truncated, leading to multiple effects on the protein, including reduced receptor-mediated endocytosis. We have also mapped three modifier quantitative trait loci (QTL) that affect the incidence of either the vl cataract or NTD phenotypes. Bioinformatic, sequence, genetic, and functional data have determined that Foxe3, a key regulator of lens development, is a gene responsible for the vl cataract-modifying phenotype. These studies have extended our understanding of the vl locus in three significant ways. One, the cloning of the vl locus has identified a previously uncharacterized GPCR-ligand pathway necessary for neural fold fusion and lens development, providing insight into the molecular regulation of these developmental processes. Two, our QTL analysis has established vl as a mouse model for studying the multigenic basis of NTDs and cataracts. Three, we have identified Foxe3 as a genetic modifier that interacts with Gpr161 to regulate lens development.

Engrailed-2 (En2) deletion produces multiple neurodevelopmental defects in monoamine systems, forebrain structures and neurogenesis, and behavior

Many genes involved in brain development have been associated with human neurodevelopmental disorders, but underlying pathophysiological mechanisms remain undefined. Human genetic and mouse behavioral analyses suggest that ENGRAILED-2 (EN2) contributes to neurodevelopmental disorders, especially autism spectrum disorder. In mouse, En2 exhibits dynamic spatiotemporal expression in embryonic mid-hindbrain regions where monoamine neurons emerge. Considering their importance in neuropsychiatric disorders, we characterized monoamine systems in relation to forebrain neurogenesis in En2-knockout (En2-KO) mice. Transmitter levels of serotonin, dopamine and norepinephrine (NE) were dysregulated from Postnatal day 7 (P7) to P21 in En2-KO, though NE exhibited the greatest abnormalities. While NE levels were reduced ∼35% in forebrain, they were increased 40 -: 75% in hindbrain and cerebellum, and these patterns paralleled changes in locus coeruleus (LC) fiber innervation, respectively. Although En2 promoter was active in Embryonic day 14.5 -: 15.5 LC neurons, expression diminished thereafter and gene deletion did not alter brainstem NE neuron numbers. Significantly, in parallel with reduced NE levels, En2-KO forebrain regions exhibited reduced growth, particularly hippocampus, where P21 dentate gyrus granule neurons were decreased 16%, suggesting abnormal neurogenesis. Indeed, hippocampal neurogenic regions showed increased cell death (+77%) and unexpectedly, increased proliferation. Excess proliferation was restricted to early Sox2/Tbr2 progenitors whereas increased apoptosis occurred in differentiating (Dcx) neuroblasts, accompanied by reduced newborn neuron survival. Abnormal neurogenesis may reflect NE deficits because intra-hippocampal injections of β-adrenergic agonists reversed cell death. These studies suggest that disruption of hindbrain patterning genes can alter monoamine system development and thereby produce forebrain defects that are relevant to human neurodevelopmental disorders.

Autism Associated Gene, ENGRAILED2, and Flanking Gene Levels Are Altered in Post-Mortem Cerebellum

Background Previous genetic studies demonstrated association between the transcription factor ENGRAILED2 (EN2) and Autism Spectrum Disorder (ASD). Subsequent molecular analysis determined that the EN2 ASD-associated haplotype (rs1861972-rs1861973 A-C) functions as a transcriptional activator to increase gene expression. EN2 is flanked by 5 genes, SEROTONIN RECEPTOR5A (HTR5A), INSULIN INDUCED GENE1 (INSIG1), CANOPY1 HOMOLOG (CNPY1), RNA BINDING MOTIF PROTEIN33 (RBM33), and SONIC HEDGEHOG (SHH). These flanking genes are co-expressed with EN2 during development and coordinate similar developmental processes. To investigate if mRNA levels for these genes are altered in individuals with autism, post-mortem analysis was performed. Methods qRT-PCR quantified mRNA levels for EN2 and the 5 flanking genes in 78 post-mortem cerebellar samples. mRNA levels were correlated with both affection status and rs1861972-rs1861973 genotype. Molecular analysis investigated whether EN2 regulates flanking gene expression. Results EN2 levels are increased in affected A-C/G-T individuals (p = .0077). Affected individuals also display a significant increase in SHH and a decrease in INSIG1 levels. Rs1861972-rs1861973 genotype is correlated with significant increases for SHH (A-C/G-T) and CNPY1 (G-T/G-T) levels. Human cell line over-expression and knock-down as well as mouse knock-out analysis are consistent with EN2 and SHH being co-regulated, which provides a possible mechanism for increased SHH post-mortem levels. Conclusions EN2 levels are increased in affected individuals with an A-C/G-T genotype, supporting EN2 as an ASD susceptibility gene. SHH, CNPY1, and INSIG1 levels are also significantly altered depending upon affection status or rs1861972-rs1861973 genotype. Increased EN2 levels likely contribute to elevated SHH expression observed in the post-mortem samples

Chronic desipramine treatment rescues depression-related, social and cognitive deficits in Engrailed-2 knockout mice

Engrailed‐2 (En2) is a homeobox transcription factor that regulates neurodevelopmental processes including neuronal connectivity and elaboration of monoaminergic neurons in the ventral hindbrain. We previously reported abnormalities in brain noradrenergic concentrations in En2 null mutant mice that were accompanied by increased immobility in the forced swim test, relevant to depression. An EN2 genetic polymorphism has been associated with autism spectrum disorders, and mice with a deletion in En2 display social abnormalities and cognitive deficits that may be relevant to multiple neuropsychiatric conditions. This study evaluated the ability of chronic treatment with desipramine (DMI), a selective norepinephrine (NE) reuptake inhibitor and classical antidepressant, to reverse behavioral abnormalities in En2−/− mice. Desipramine treatment significantly reduced immobility in the tail suspension and forced swim tests, restored sociability in the three‐chambered social approach task and reversed impairments in contextual fear conditioning in En2−/− mice. Our findings indicate that modulation of brain noradrenergic systems rescues the depression‐related phenotype in En2−/− mice and suggest new roles for NE in the pathophysiology of the social and cognitive deficits seen in neuropsychiatric disorders such as autism or schizophrenia.

Cut-like homeobox 1 and nuclear factor I/B mediate ENGRAILED2 autism spectrum disorder-associated haplotype function

Both common and rare variants contribute to autism spectrum disorder (ASD) risk, but few variants have been established as functional. Previously we demonstrated that an intronic haplotype (rs1861972-rs1861973 A-C) in the homeobox transcription factor ENGRAILED2 (EN2) is significantly associated with ASD. Positive association has also been reported in six additional data sets, suggesting EN2 is an ASD susceptibility gene. Additional support for this possibility requires identification of functional variants that affect EN2 regulation or activity. In this study, we demonstrate that the A-C haplotype is a transcriptional activator. Luciferase (luc) assays in mouse neuronal cultures determined that the A-C haplotype increases expression levels (50%, P < 0.01, 24 h; 250%, P < 0.0001, 72 h). Mutational analysis indicates that the A-C haplotype activator function requires both associated A and C alleles. A minimal 202-bp element is sufficient for function and also specifically binds a protein complex. Mass spectrometry identified these proteins as the transcription factors, Cut-like homeobox 1 (Cux1) and nuclear factor I/B (Nfib). Subsequent antibody supershifts and chromatin immunoprecipitations demonstrated that human CUX1 and NFIB bind the A-C haplotype. Co-transfection and knock-down experiments determined that both CUX1 and NFIB are required for the A-C haplotype activator function. These data demonstrate that the ASD-associated A-C haplotype is a transcriptional activator, and both CUX1 and NFIB mediate this activity. These results provide biochemical evidence that the ASD-associated A-C haplotype is functional, further supporting EN2 as an ASD susceptibility gene.

NOS1AP protein levels are altered in BA46 and cerebellum of patients with schizophrenia.

Dear Editors, Brzustowicz and colleagues (2004) identified significant linkage disequilibrium between schizophrenia and markers within the gene encoding nitric oxide synthase 1 (neuronal; NOS1) adaptor protein (NOS1AP; also termed carboxyl-terminal PDZ ligand of nNOS or CAPON). Quantitative real-time PCR (qRT-PCR) analysis of mRNA from human postmortem dorsolateral prefrontal cortex further revealed that expression of the short isoform of the NOS1AP gene (NOS1AP-S) is significantly increased in patients with schizophrenia (Xu, et al., 2005). More recently, the group also identified a functional risk allele within NOS1AP and showed that this change increased NOS1AP mRNA expression in a cell culture system (Wratten, et al., 2009). Despite these recent reports establishing linkage between NOS1AP and schizophrenia, little is known about NOS1AP protein expression in the brains of affected patients. Previous reports described two distinct NOS1AP isoforms: full-length NOS1AP-L (10 exons, ~75kD) and NOS1AP-S, a C-terminal specific transcript that encodes only the PDZ domain (Jaffrey, et al., 1998; Xu, et al., 2005). We have now identified a novel isoform, NOS1AP-S’ (Figure 1A), in mouse and human tissue using qRT-PCR (data not shown). To evaluate the expression levels of these three NOS1AP isoforms in human brain tissue, postmortem samples from Brodmann’s Area (BA) 46, BA11, Medial Temporal Lobe (MTL), Occipital Lobe (OL), and cerebellum of unaffected patients and those with schizophrenia were obtained from the Human Brain and Spinal Fluid Resource Center (Los Angeles, CA) and subjected to Western blotting with normalization to GAPDH or actin, as previously described (Xu, et al., 2005). Investigators were blinded to all subject information until after statistical analysis. The logarithms of the normalized values for subjects with schizophrenia and unaffected control patients within the same brain region were compared using the standard t-test. Correction for testing of multiple expression levels was made using permutations of case/control labels. Secondary examination of linear models with other covariates was based on the AICc model selection criterion (Burnham, 2002). The L (p = 0.0067; reported p-values are nominal), S´ (p = 0.0082) and S (p = 0.0041) isoforms were increased in BA46 of patients with schizophrenia (Figure 1B) at a nominal significance level, although only the increase in the S isoform was significant (p<0.05) under permutation-based multiple testing adjustment. These data are consistent with previous reports strongly implicating this region in the etiology of schizophrenia (Barch, 2005; Bunney and Bunney, 2000; Xu, et al., 2005). The L (p = 0.0031), S (p = 0.0060), and S´ (p = 0.0156) isoforms were decreased in cerebellum of affected individuals (Figure 1B), although only the decrease of the L isoform was significant under adjustment. While some reports have indicated that schizophrenia may affect the cerebellum, the results are not as consistently observed as in other regions, namely the prefrontal cortex (Avila, et al., 2002; Kapoor, et al., 2006). There were no significant differences in NOS1AP expression between control and affected patients in BA11, the MTL, or the OL (Figure 1B). Additional analysis of NOS1AP expression reveals that no significant changes were evident in BA11, the MTL, or the OL of patients with schizophrenia versus those who are unaffected. While some studies have reported a role for BA11 in schizophrenia, others find no changes in expression of NMDA receptor pathway proteins (Toro and Deakin, 2005). Figure 1 (A) NOS1AP isoforms. The intron/exon boundaries and the predicted transcriptional (arrow) and translational (ATG) start sites for the different NOS1AP isoforms are illustrated. The new NOS1AP isoform is characterized by a unique 5’ exon and transcriptional ... Our data show an alteration of three NOS1AP isoforms in specific regions of the brain for patients diagnosed with schizophrenia. Initially identified in rat, NOS1AP plays a role in the inhibition of glutamate neurotransmission via disruption of NOS1 binding to Postsynaptic Density Protein-95 and -93. This results in uncoupling of NOS1 from the NMDA receptor, and ultimately, inhibition of receptor function (Brzustowicz, 2008; Jaffrey, et al., 1998; Xu, et al., 2005). These data suggest a role for NOS1AP in glutamate receptor hypofunction and manifestation of schizophrenia.

Quantitative trait loci affecting phenotypic variation in the vacuolated lens mouse mutant, a multigenic mouse model of neural tube defects

The vacuolated lens (vl) mouse mutant arose spontaneously on the C3H/HeSn background and exhibits neural tube defects (NTDs), congenital cataract, and occasionally a white belly spot. We previously reported that 1) the vl phenotypes are due to a mutation in an orphan G protein-coupled receptor (GPCR), Gpr161; 2) the penetrance of the vl NTD and cataract phenotypes are affected by genetic background, allowing three unlinked quantitative trait loci (QTL) to be mapped (modifiers of vacuolated lens, Modvl1-3); and 3) phenotype-based bioinformatics followed by genetic and molecular analysis identified a lens-specific transcription factor that contributes to the cataract-modifying effect of Modvl3. We now extend this analysis in three ways. First, using the Gpr161 mutation to unequivocally identify mutant adults and embryos, we determined that approximately 50% of vl/vl NTD-affected embryos die during development. Second, the MOLF/Ei genetic background suppresses this embryonic lethality but increases the incidence of the adult belly spot phenotype. Additional QTL analysis was performed, and two novel modifiers were mapped [Modvl4, logarithm of odds ratio (LOD) 4.4; Modvl5, LOD 5.0]. Third, phenotype-based bioinformatics identified candidate genes for these modifiers including two GPCRs that cause NTD or skin/pigmentation defects (Modvl4: Frizzled homolog 6; Modvl5: Melanocortin 5 receptor). Because GPCRs form oligomeric complexes, these genes were resequenced and nonsynonymous coding variants were identified. Bioinformatics and protein modeling suggest that these variants may be functional. Our studies further establish vl as a multigenic mouse model for NTDs and identify additional QTL that interact with Gpr161 to regulate neurulation.