BACKGROUND

Several candidate gene studies support RELN as susceptibility gene for autism. Given the complex inheritance pattern of autism, it is expected that gene-gene interactions will exist. A logical starting point for examining potential gene-gene interactions is to evaluate the joint effects of genes involved in a common biological pathway. RELN shares a common biological pathway with APOE, and Persico et al. have observed transmission distortion of the APOE-2 allele in autism families.

OBJECTIVE

We evaluated RELN and APOE for joint effects in autism susceptibility.

METHODS

A total of 470 Caucasian autism families were analyzed (265 multiplex; 168 trios with no family history; 37 positive family history but only one sampled affected). These families were genotyped for 11 RELN polymorphisms, including the 5' untranslated region repeat previously associated with autism, as well as for the APOE functional allele. We evaluated single locus allelic and genotypic association with the pedigree disequilibrium test and geno-PDT, respectively. Multilocus effects were evaluated using the extended version of the multifactorial dimensionality reduction method.

RESULTS

For the single locus analyses, there was no evidence for an effect of APOE in our data set. Evidence for association with RELN (rs2,073,559; trio subset P=0.07 PDT; P=0.001 geno-PDT; overall geno-PDT P=0.05), however, was found. For multilocus geno-PDT analysis, the joint genotype of APOE and RELN rs2,073,559 was highly significant (trio subset, global P=0.0001), probably driven by the RELN single locus effect. Using the extended version of the multifactorial dimensionality reduction method to detect multilocus effects, there were no statistically significant associations for any of the n-locus combinations involving RELN or APOE in the overall or multiplex subset. In the trio subset, 1-locus and 2-locus models selected only markers in RELN as best models for predicting autism case status.

CONCLUSIONS

Thus, we conclude that there is no main effect of APOE in our autism data set, nor is there any evidence for a joint effect of APOE with RELN. RELN, however, remains a good candidate for autism susceptibility.

Malformations of cortical development (MCD) represent a major cause of developmental disabilities, severe epilepsy, and reproductive disadvantage. Genes that have been associated to MCD are mainly involved in cell proliferation and specification, neuronal migration, and late cortical organization. Lissencephaly-pachygyria-severe band heterotopia are diffuse neuronal migration disorders causing severe global neurological impairment. Abnormalities of the LIS1, DCX, ARX, RELN, VLDLR, ACTB, ACTG1, TUBG1, KIF5C, KIF2A, and CDK5 genes have been associated with these malformations. More recent studies have also established a relationship between lissencephaly, with or without associated microcephaly, corpus callosum dysgenesis as well as cerebellar hypoplasia, and at times, a morphological pattern consistent with polymicrogyria with mutations of several genes (TUBA1A, TUBA8, TUBB, TUBB2B, TUBB3, and DYNC1H1), regulating the synthesis and function of microtubule and centrosome key components and hence defined as tubulinopathies. MCD only affecting subsets of neurons, such as mild subcortical band heterotopia and periventricular heterotopia, have been associated with abnormalities of the DCX, FLN1A, and ARFGEF2 genes and cause neurological and cognitive impairment that vary from severe to mild deficits. Polymicrogyria results from abnormal late cortical organization and is inconstantly associated with abnormal neuronal migration. Localized polymicrogyria has been associated with anatomo-specific deficits, including disorders of language and higher cognition. Polymicrogyria is genetically heterogeneous, and only in a small minority of patients, a definite genetic cause has been identified. Megalencephaly with normal cortex or polymicrogyria by MRI imaging, hemimegalencephaly and focal cortical dysplasia can all result from mutations in genes of the PI3K-AKT-mTOR pathway. Postzygotic mutations have been described for most MCD and can be limited to the dysplastic tissue in the less diffuse forms.

Neuronal migration disorders account for a substantial number of cortical malformations, the most severe forms being represented by lissencephalies. Classical lissencephaly has been shown to result from mutations in LIS1 (PAFAH1B1; MIM#601545), DCX (Doublecortin; MIM#300121), ARX (Aristaless-related homeobox gene; MIM#300382), RELN (Reelin; MIM#600514) and VLDLR (Very low density lipoprotein receptor; MIM#224050). More recently, de novo missense mutations in the alpha-tubulin 1a gene (TUBA1A) located on chromosome 12q13.12, have also been associated with more or less severe defects of cortical development, resulting in complete agyria in the most severe cases of lissencephaly. We report here the cerebral lesions in a 36 weeks' gestation female foetus with a novel de novo missense mutation in the TUBA1A gene, presenting the most severe antenatal phenotype reported so far. Using routine immunohistochemistry and confocal microscopy, we show evidence for defects in axonal transport in addition to defects in neuronal migration and differentiation, giving new insights to the pathophysiology of this form of lissencephaly.

In developing mammalian (mouse) brain, Reelin (Reln) is secreted by the Cajal-Retzius (CR) neurons in the marginal zone, binds apolipoprotein E receptor 2 (ApoER2) and very low density lipoprotein receptor (Vldlr), and induces the phosphorylation of the downstream cytoplasmic molecule disabled-1 (Dab1) in cortical plate neurons. Although this is a well-characterized signaling pathway in mice, it has not been well defined in human brain. In this paper we examined the expression of RELN, APOER2, VLDLR, and DAB1 in the developing human brain by RT-PCR. We further determined the cellular expression of the proteins RELN and DAB1 in 50 human brains ranging in age from 10 gestational weeks (GW) to 62 years using immunochemistry. We found that the pattern of expression of RELN and DAB1 in the human brain isnot identical to that observed in the mouse brain. In particular, we report the novel finding that human DAB1and RELN are coexpressed in CR neurons during cortical development and in cortical pyramidal neurons after neuronal migration is complete. Thus, in the human brain, the whole RELN signaling pathway is present within selected populations of cortical neurons throughout life. We speculate that RELN and DAB1 coexpression in these neurons is necessary for both normal cortical development and mature function.

The Reelin gene (RELN) encodes a secretory glycoprotein critical for brain development and synaptic plasticity. Post-mortem studies have shown lower Reelin protein levels in the brains of patients with schizophrenia and bipolar disorder (BP) compared with controls. In a recent genome-wide association study of schizophrenia, the strongest association was found in a marker within RELN, although this association was seen only in women. In this study, we investigated whether genetic variation in RELN is associated with BP in a large family sample. We genotyped 75 tagSNPs and 6 coding SNPs in 1,188 individuals from 318 nuclear families, including 554 affected offspring. Quality control measures, transmission-disequilibrium tests (TDTs), and empirical simulations were performed in PLINK. We found a significant overtransmission of the C allele of rs362719 to BP offspring (OR = 1.47, P = 5.9 x 10(-4)); this withstood empirical correction for testing of multiple markers (empirical P = 0.048). In a hypothesis-driven secondary analysis, we found that the association with rs362719 was almost entirely accounted for by overtransmission of the putative risk allele to affected females (OR(Female) = 1.79, P = 8.9 x 10(-5) vs. OR(Male) = 1.12, P = 0.63). These results provide preliminary evidence that genetic variation in RELN is associated with susceptibility to BP and, in particular, to BP in females. However, our findings should be interpreted with caution until further replication and functional assays provide convergent support.

Patients with severe deficiency of methylenetetrahydrofolate reductase (MTHFR) suffer from a wide variety of neurological problems, which can begin in the neonatal period. MTHFR is a critical enzyme in folate metabolism; the product of the MTHFR reaction, 5-methyltetrahydrofolate, is required for homocysteine remethylation to methionine and synthesis of S-adenosylmethionine (SAM). To understand the mechanisms by which MTHFR deficiency leads to significant neuropathology, we examined early postnatal brain development in mice with a homozygous knockout of the Mthfr gene. These mice displayed a dramatically reduced size of the cerebellum and cerebral cortex, with enlarged lateral ventricles. Mthfr deficiency affected granule cell maturation, but not neurogenesis. Depletion of external granule cells and disorganization of Purkinje cells were mainly confined to the anterior lobules of mutant cerebella. Decreased cellular proliferation and increased cell death contributed to the granule cell loss. Reduced expression of Engrailed-2 (En2), Reelin (Reln) and inositol 1,4,5-triphosphate receptor type 1 (Itpr1) genes was observed in the cerebellum. Supplementation of Mthfr(+/-) dams with an alternate methyl donor, betaine, reduced cerebellar abnormalities in the Mthfr(-/-) pups. Our findings suggest that MTHFR plays a role in cerebellar patterning, possibly through effects on proliferation or apoptosis.

One of the challenges in mammalian reproduction is to understand the basic physiology of oocyte quality. It is believed that the follicle status is linked to developmental competence of the enclosed oocyte. To explore the link between follicles and competence in cows, previous research at our laboratory has developed an ovarian stimulation protocol that increases and then decreases oocyte quality according to the timing of oocyte recovery post-FSH withdrawal (coasting). Using this protocol, we have obtained the granulosa cells associated with oocytes of different qualities at selected times of coasting. Transcriptome analysis was done with Embryogene microarray slides and validation was performed by real-time PCR. Results show that the major changes in gene expression occurred from 20 to 44 h of coasting, when oocyte quality increases. Secondly, among upregulated genes (20-44 h), 25% were extracellular molecules, highlighting potential granulosa signaling cascades. Principal component analysis identified two patterns: one resembling the competence profile and another associated with follicle growth and atresia. Additionally, three major functional changes were identified: (i) the end of follicle growth (BMPR1B, IGF2, and RELN), involving interactions with the extracellular matrix (TFPI2); angiogenesis (NRP1), including early hypoxia, and potentially oxidative stress (GFPT2, TF, and VNN1) and (ii) apoptosis (KCNJ8) followed by iii) inflammation (ANKRD1). This unique window of analysis indicates a progressive hypoxia during coasting mixed with an increase in apoptosis and inflammation. Potential signaling pathways leading to competence have been identified and will require downstream testing. This preliminary analysis supports the potential role of the follicular differentiation in oocyte quality both during competence increase and decrease phases.

Development of visual system circuitry requires the formation of precise synaptic connections between neurons in the retina and brain. For example, axons from retinal ganglion cells (RGCs) form synapses onto neurons within subnuclei of the lateral geniculate nucleus (LGN) [i.e., the dorsal LGN (dLGN), ventral LGN (vLGN), and intergeniculate leaflet (IGL)]. Distinct classes of RGCs project to these subnuclei: the dLGN is innervated by image-forming RGCs, whereas the vLGN and IGL are innervated by non-image-forming RGCs. To explore potential mechanisms regulating class-specific LGN targeting, we sought to identify differentially expressed targeting molecules in these LGN subnuclei. One candidate targeting molecule enriched in the vLGN and IGL during retinogeniculate circuit formation was the extracellular matrix molecule reelin. Anterograde labeling of RGC axons in mutant mice lacking functional reelin (reln(rl/rl)) revealed reduced patterns of vLGN and IGL innervation and misrouted RGC axons in adjacent non-retino-recipient thalamic nuclei. Using genetic reporter mice, we further demonstrated that mistargeted axons were from non-image-forming, intrinsically photosensitive RGCs (ipRGCs). In contrast to mistargeted ipRGC axons, axons arising from image-forming RGCs and layer VI cortical neurons correctly targeted the dLGN in reln(rl/rl) mutants. Together, these data reveal that reelin is essential for the targeting of LGN subnuclei by functionally distinct classes of RGCs.

BACKGROUND

The challenges in gene identification for psychiatric disorders have awakened interest toward quantitative traits and endophenotypes that are potentially more closely related to the underlying biology and provide more power in the linkage and association analyses. Previously, we successfully replicated schizophrenia linkage on chromosome 7q21-32 in Finnish families and demonstrated that an intragenic short tandem repeat (STR) allele of the regional Reelin (RELN) gene is associated with multiple cognitive traits representing central cognitive functions regarded as valid endophenotypes for schizophrenia.

METHODS

Here, we used an extended sample of 290 Finnish families with schizophrenia and 375 control subjects in an association analysis between 96 SNPs and three STRs in RELN and diagnostic categories, clinical disorder features, as well as central cognitive functions impaired in schizophrenia.

RESULTS

We replicated the original association between RELN intragenic STR allele and working memory in individuals (n = 342) not overlapping with the previous study. This risk allele remained central in the whole study sample by being associated with impaired cognitive functioning and more severe positive and negative symptoms of schizophrenia (p = .0005-.00002). Additionally, multiple SNPs indicated association with the severity of positive symptoms of schizophrenia and together showed potential additive effect on the severity of the symptoms (p = .0000001). However, no significant associations with clinical diagnostic categories emerged.

CONCLUSIONS

The strongest effects on cognitive functions were detected among the affected individuals. We thus propose a particular role for RELN as a modifier gene of the pathogenesis of schizophrenia.

Reelin is the protein defective in reeler mice, an extensively studied model of brain development. The reelin gene (symbol Reln) codes for a protein of the extracellular matrix that contains eight successive repeats of 350 to 390 amino acids. In this work, we describe the genomic structure of the mouse reelin gene and the 5'-flanking genomic DNA sequences. The reelin gene is composed of 65 exons spread over approximately 450 kb of genomic DNA. We identified different reelin transcripts, formed by alternative splicing of a microexon as well as by use of two different polyadenylation sites. All splice sites conform to the GT-AG rule, except for the splice donor site of intron 30, which is GC instead of GT. A processed pseudogene is present in intron 42. Its nucleotide sequence is 86% identical to the sequence of the rat RDJ1 cDNA, which codes for a DnaJ-like protein of the Hsp40 family. Comparison of 8 intron positions in mouse and human reelin genes reveals a highly conserved genomic structure, suggesting a similar structure of the whole gene in both species. We identified two transcription start sites embedded within a CpG. The promoter region contains putative recognition sites for the transcription factors Sp1 and AP2 but lacks TATA and CAAT boxes. The presence of tandemly repeated regions in the Reelin protein suggests that gene duplication events occurred during evolution. By comparison of the amino acid sequences of the eight repeats and the positions of introns, we suggest a model for the evolution of the repeat coding portion of the reelin gene from a putative ancestral minigene.

The reeler gene (Reln(rl), formerly rl) product Reelin controls neuronal migration and positioning and thereby plays a key role in brain development. Mutation of Reln leads to widespread disruption of laminar cortical regions and ectopia in some brainstem nuclei. In the embryonic striatum of normal mice, a substantial expression of reelin mRNA has been documented; however, the anomalous positioning of neurons in the basal ganglia of reeler mice remains to be studied. We provide first evidence for a potential role of Reelin in the developmental formation of the substantia nigra. In reeler mutant mice lacking Reelin, dopaminergic neurons destined for the substantia nigra fail to migrate laterally and become anomalously clustered just lateral to the ventral tegmental area. Their axons appear to project to striatal patches forming "dopamine islands." Results from the normal mice show that, at the midembryonic stage, Reelin identified with CR-50 is highly concentrated in the ventral mesencephalon, where nigral dopaminergic neurons are in progress to migrate laterally to their eventual position of the adult brain. A combination of CR-50 labeling and anterograde axonal tracing provided evidence that embryonic striatal neurons may supply the ventral portion of the mesencephalon with Reelin through their axonal projections. We hypothesize that Reelin plays a role in the positioning of nigral dopaminergic neurons and that it can act as an environmental cue at a remote site far from its birthplace via a transaxonal delivery system.

Dystonia is a genetically heterogenous disease and a prototype disorder where next-generation sequencing has facilitated the identification of new pathogenic genes. This includes the first two genes linked to recessively inherited isolated dystonia, that is, HPCA (hippocalcin) and COL6A3 (collagen VI alpha 3). These genes are proposed to underlie cases of the so-called DYT2-like dystonia, while also reiterating two distinct pathways in dystonia pathogenesis. First, deficiency in HPCA function is thought to alter calcium homeostasis, a mechanism that has previously been forwarded for CACNA1A and ANO3. The novel myoclonus-dystonia genes KCTD17 and CACNA1B also implicate abnormal calcium signaling in dystonia. Second, the phenotype in COL6A3-loss-of-function zebrafish models argues for a neurodevelopmental defect, which has previously been suggested as a possible biological mechanism for THAP1, TOR1A, and TAF1 based on expression data. The newly reported myoclonus-dystonia gene, RELN, plays also a role in the formation of brain structures. Defects in neurodevelopment likewise seem to be a recurrent scheme underpinning mainly complex dystonias, for example those attributable to biallelic mutations in GCH1, TH, SPR, or to heterozygous TUBB4A mutations. To date, it remains unclear whether dystonia is a common phenotypic outcome of diverse underlying disease mechanisms, or whether the different genetic causes converge in a single pathway. Importantly, the relevance of pathways highlighted by novel dystonia genes identified by high-throughput sequencing depends on the confirmation of mutation pathogenicity in subsequent genetic and functional studies. However, independent, careful validation of genetic findings lags behind publications of newly identified genes. We conclude with a discussion on the characteristics of true-positive reports.

The human gene encoding Reelin (RELN), a pivotal protein in neurodevelopment, includes a polymorphic GGC repeat in its 5' untranslated region (UTR). CHO cells transfected with constructs encompassing the RELN 5'UTR with 4-to-13 GGC repeats upstream of the luciferase reporter gene show declining luciferase activity with increasing GGC repeat number (P < 0.005), as predicted by computer-based simulations. Conversely, RELN 5'UTR sequences boost reporter gene expression above control levels in neuronal SN56 and N2A cell lines, but 12- and 13-repeat alleles still yield 50-60% less luciferase activity compared to the more common 8- and 10-repeat alleles (P < 0.0001). RELN "long" GGC alleles significantly blunt gene expression and may, through this effect, confer vulnerability to human disorders, such as schizophrenia and autism.

Schizophrenia (SZ) and bipolar disorder (BPD) patients show a downregulation of GAD67, reelin (RELN), brain-derived neurotrophic factor (BDNF), and other genes expressed in telencephalic GABAergic and glutamatergic neurons. This downregulation is associated with the enrichment of 5-methylcytosine and 5-hydroxymethylcytosine proximally at gene regulatory domains at the respective genes. A pharmacological strategy to reduce promoter hypermethylation and to induce a more permissive chromatin conformation is to administer drugs, such as the histone deacetylase (HDAC) inhibitor valproate (VPA), that facilitate chromatin remodeling. Studies in mouse models of SZ indicate that clozapine induces DNA demethylation at relevant promoters, and that this action is potentiated by VPA. By activating DNA demethylation, clozapine or its derivatives with VPA or other more potent and selective HDAC inhibitors may be a promising treatment strategy to correct the gene expression deficits detected in postmortem brain of SZ and BPD patients.

BACKGROUND

The dyslexia susceptibility 1 candidate 1 (DYX1C1) gene has recently been associated with dyslexia and reading scores in several population samples. The DYX1C1 has also been shown to affect neuronal migration and modulate estrogen receptor signaling.

METHODS

We have analyzed the molecular networks of DYX1C1 by gene expression and protein interaction profiling in a human neuroblastoma cell line.

RESULTS

We find that DYX1C1 can modulate the expression of nervous system development and neuronal migration genes such as RELN and associate with a number of cytoskeletal proteins. We also show by live cell imaging that DYX1C1 regulates cell migration of the human neuroblastoma cell line dependent on its tetratricopeptide repeat and DYX1 protein domains. The DYX1 domain is a novel highly conserved domain identified in this study by multiple sequence alignment of DYX1C1 proteins recovered from a wide range of eukaryotic species.

CONCLUSIONS

Our results contribute to the hypothesis that dyslexia has a developmental neurobiological basis by linking DYX1C1 with many genes involved in neuronal migration disorders.

A recent study by Persico et al. [2001: Mol Psychiatry 6:150-159] suggests alleles of a CGG polymorphism, just 5' of the reelin gene (RELN) initiator codon, confer liability for autism, especially alleles bearing 11 or more CGG repeats (long alleles). The association is consistent across both a case-control and family-based sample. We attempted to replicate their finding using a larger, independent family-based sample from the NIH Collaborative Programs of Excellence in Autism (CPEA) Network. In our data, allele transmissions to individuals with autism versus unaffected individuals are unbiased, both when alleles are classified by repeat length and when they are classified into long/short categories. Because of the apparent linkage of autism to chromosome 7q, particularly related to the development of language, we also evaluate the relationship between Reelin alleles and the age at which autism subjects use their first word or first phrase. Neither is significantly associated with Reelin alleles. Our results are not consistent with a major role for Reelin alleles in liability to autism.

Reelin is a large secreted extracellular matrix glycoprotein playing an important role in early neurodevelopment. Several genetic studies found an association between RELN gene and increased risk of autism suggesting that reelin deficiency may be a vulnerability factor in its etiology. Moreover, a reduced reelin expression has been observed in several brain regions of subjects with Autism Spectrum Disorders. Since a number of reports have documented presence of vocal and neuromotor abnormalities in patients with autism and suggested that these dysfunctions predate the onset of the syndrome, we performed a fine-grain characterization of the neonatal vocal and motor repertoire in reelin mutant mice to explore the developmental precursors of the disorder. Our findings evidence a general delay in motor and vocal development in heterozygous (50% reduced reelin) and reeler (lacking reelin gene) mutant mice. As a whole, an increased number of calls characterized heterozygous pup's emission. Furthermore, the typical ontogenetic peak in the number of calls characterizing wild-type pups on postnatal day 4 appeared slightly delayed in heterozygous pups (to day 6) and was quite absent in reeler littermates, which exhibited a flat profile during development. We also detected a preferential use of a specific call category (two-components) by heterozygous and reeler mice at postnatal days 6 and 8 as compared to their wild-type littermates. With regard to the analysis of spontaneous movements, a differential profile emerged early in development among the three genotypes. While only slight coordination difficulties are exhibited by heterozygous pups, all indices of motor development appear delayed in reeler mice. Overall, our results evidence a genotype-dependent deviation in ultrasonic vocal repertoire and a general delay in motor development in reelin mutant pups.

GABAergic inhibitory interneurons (IN) represent a heterogeneous population with different electrophysiological, morphological, and molecular properties. The correct balance between interneuronal subtypes is important for brain function and is impaired in several neurological and psychiatric disorders. Here we show the data of 123 molecularly and electrophysiologically characterized neurons of juvenile rat barrel cortex acute slices, 48 of which expressed Reelin (Reln). Reln mRNA was exclusively detected in Gad65/67-positive cells but was found in interneuronal subtypes in different proportions: all cells of the adapting-Somatostatin (SST) cluster expressed Reln, whereas 63% of the adapting-neuropeptide Y (NPY, 50% of the fast-spiking Parvalbumin (PVALB), and 27% of the adapting/bursting-Vasoactive Intestinal Peptide (VIP) cluster were Reln-positive. Silhouette analysis revealed a high impact of the parameter Reln on cluster quality. By analyzing the co-localization of RELN immunoreactivity with those of different IN-markers, we found that RELN is produced layer-independently in SST-, NPY-, and NOS1-expressing INs, whereas co-localization of RELN and VIP was mostly absent. Of note, RELN co-localized with PVALB, predominantly in INs of layers IV/V (>30%). Our findings emphasize RELN's role as an important IN-marker protein and provide a basis for the functional characterization of Reln-expressing INs and its role in the regulation of inhibitory IN networks.

We have recently reported that mice born from dams stressed during pregnancy (PRS mice), in adulthood, have behavioral deficits reminiscent of behaviors observed in schizophrenia (SZ) and bipolar (BP) disorder patients. Furthermore, we have shown that the frontal cortex (FC) and hippocampus of adult PRS mice, like that of postmortem chronic SZ patients, are characterized by increases in DNA-methyltransferase 1 (DNMT1), ten-eleven methylcytosine dioxygenase 1 (TET1) and exhibit an enrichment of 5-methylcytosine (5MC) and 5-hydroxymethylcytosine (5HMC) at neocortical GABAergic and glutamatergic gene promoters. Here, we show that the behavioral deficits and the increased 5MC and 5HMC at glutamic acid decarboxylase 67 (Gad1), reelin (Reln) and brain-derived neurotrophic factor (Bdnf) promoters and the reduced expression of the messenger RNAs (mRNAs) and proteins corresponding to these genes in FC of adult PRS mice is reversed by treatment with clozapine (5 mg kg(-1) twice a day for 5 days) but not by haloperidol (1 mg kg(-1) twice a day for 5 days). Interestingly, clozapine had no effect on either the behavior, promoter methylation or the expression of these mRNAs and proteins when administered to offspring of nonstressed pregnant mice. Clozapine, but not haloperidol, reduced the elevated levels of DNMT1 and TET1, as well as the elevated levels of DNMT1 binding to Gad1, Reln and Bdnf promoters in PRS mice suggesting that clozapine, unlike haloperidol, may limit DNA methylation by interfering with DNA methylation dynamics. We conclude that the PRS mouse model may be useful preclinically in screening for the potential efficacy of antipsychotic drugs acting on altered epigenetic mechanisms. Furthermore, PRS mice may be invaluable for understanding the etiopathogenesis of SZ and BP disorder and for predicting treatment responses at early stages of the illness allowing for early detection and remedial intervention.

RELN encodes a large, secreted glycoprotein integral to proper neuronal positioning during development and regulation of synaptic function postnatally. Rare, homozygous, null mutations lead to lissencephaly with cerebellar hypoplasia (LCH), accompanied by developmental delay and epilepsy. Until recently, little was known about the frequency or consequences of heterozygous mutations. Several lines of evidence from multiple studies now implicate heterozygous mutations in RELN in autism spectrum disorders (ASD). RELN maps to the AUTS1 locus on 7q22, and at this time over 40 distinct mutations have been identified that would alter the protein sequence, four of which are de novo. The RELN mutations that are most clearly consequential are those that are predicted to inactivate the signaling function of the encoded protein and those that fall in a highly conserved RXR motif found at the core of the 16 Reelin subrepeats. Despite the growing evidence of RELN dysfunction in ASD, it appears that these mutations in isolation are insufficient and that secondary genetic or environmental factors are likely required for a diagnosis.

Over the last decade, finding a reliable biomarker for the early detection of schizophrenia (Scz) has been a topic of interest. The main goal of the current review is to provide a comprehensive view of the brain, blood, cerebrospinal fluid (CSF), and serum biomarkers of Scz disease. Imaging studies have demonstrated that the volumes of the corpus callosum, thalamus, hippocampal formation, subiculum, parahippocampal gyrus, superior temporal gyrus, prefrontal and orbitofrontal cortices, and amygdala-hippocampal complex were reduced in patients diagnosed with Scz. It has been revealed that the levels of interleukin 1β (IL-1β), IL-6, IL-8, and TNF-α were increased in patients with Scz. Decreased mRNA levels of brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), neurotrophin-3 (NT-3), nerve growth factor (NGF), and vascular endothelial growth factor (VEGF) genes have also been reported in Scz patients. Genes with known strong relationships with this disease include BDNF, catechol-O-methyltransferase (COMT), regulator of G-protein signaling 4 (RGS4), dystrobrevin-binding protein 1 (DTNBP1), neuregulin 1 (NRG1), Reelin (RELN), Selenium-binding protein 1 (SELENBP1), glutamic acid decarboxylase 67 (GAD 67), and disrupted in schizophrenia 1 (DISC1). The levels of dopamine, tyrosine hydroxylase (TH), serotonin or 5-hydroxytryptamine (5-HT) receptor 1A and B (5-HTR1A and 5-HTR1B), and 5-HT1B were significantly increased in Scz patients, while the levels of gamma-aminobutyric acid (GABA), 5-HT transporter (5-HTT), and 5-HT receptor 2A (5-HTR2A) were decreased. The increased levels of SELENBP1 and Glycogen synthase kinase 3 subunit α (GSK3α) genes in contrast with reduced levels of B-cell translocation gene 1 (BTG1), human leukocyte antigen DRB1 (HLA-DRB1), heterogeneous nuclear ribonucleoprotein A3 (HNRPA3), and serine/arginine-rich splicing factor 1 (SFRS1) genes have also been reported. This review covers various dysregulation of neurotransmitters and also highlights the strengths and weaknesses of studies attempting to identify candidate biomarkers.

Reelin (Reln) is expressed in specific GABAergic neurons in layer I and II of neocortex, and is secreted into the extracellular matrix where it surrounds dendrites, spines and neurite arborizations, and binds to integrin receptors located on post-synaptic densities of apical dendritic spines. Experiments in rodents (including wild type or reeler heterozygous mice) and non-human primates suggest the Reln secreted in the extracellular matrix of neocortex, via integrin receptors, modulates the function of the adaptor protein DAB1(drosophila disable-gene) homologous product) thereby participating in dynamic processes associated with plasticity changes in dendrites, dendritic spines and their synapses. A local protein synthesis at dendritic spines (ie the activity regulated cytoskeleton associated protein, Arc) probably acts as a signal for plastic modulatory activities in synapses operative in neural group interactions. A research strategy directed toward identifying specific neurochemical markers operative in the etiopathology of psychotic disorders lead to the identification of a downregulation (30-50%) of Reln and glutamic acid decarboxylase 67(GAD67) expression in prefrontal cortex and other brain areas of schizoprenia and bipolar disorder patients with psychosis. These downregulations were not due to neuronal damage, postmortem interval, or antipsychotic medication. The dysfunction of GABAergic interneurons observed in psychotic brains in combination with reduced Reln expression and downregulation of Reln-integrin receptor interaction, may provide an explanation for the reported decrease in neuropile expression including dendritic spine density reduction, in neocortex of schizophrenia patients. This downregulation of neuropile plasticity may be a factor to be considered in the etiology of the disintegration of consciousness, which is one of the primary signs of psychosis.

Autism is one of the most genetically influenced neuropsychiatric disorders. However, its detailed genetic basis is far from being clear. Genome-wide association studies have revealed a number of candidate genes, mostly related to synaptogenesis and various neuroendocrine pathways. In our study we have focused on oxytocin (OT), oxytocin receptor (OXTR), GABA receptor gamma 3 (GABRG3), neuroligin (NLGN4X), and reelin (RELN). After signed consent, 90 autistic boys and 85 healthy controls were enrolled in the study. Polymorphisms of OT (rs2740204), OXTR (rs2228485), GABRG3 (rs28431127), and NLGN4X (rs5916338) were analyzed using restriction fragment length polymorphism. (GGC)n STR polymorphism in the 5' UTR of the RELN gene was genotyped using fragment analysis. The only significant association in autistic boys in Slovakia was found with higher number of GGC repeats in the RELN gene (P=0.001) potentially explaining lower RELN levels in blood and brain of autistic patients.

A novel secretory pathway has been identified in the study of mice homozygous for the Reln(Orl) mutation, a line characterised by the defective secretion of the large extracellular matrix glycoprotein Reelin. By using both light and electron microscopy, immunohistochemical studies for Reelin in these mutants identified morphological changes in their Cajal-Retzius cells (CR cells). The CR cells of the mutant displayed the characteristic features of bipolar, tangentially elongated neurons with a dendritic proximal pole and an axonal cone at the opposite end of the soma. At either pole, cisterns of prominent rough endoplasmic reticulum (RER) were found to be rich in Reelin. However, the Reelin-positive RER cisterns of the axonal cones were hugely dilated in homozygous Reln(Orl) mice as compared with their wild type counterparts. CR cell axons displayed beads throughout their length, each contained a smooth spheroidal cistern filled with Reelin-immunoreactive fibrillar material, and were increased in number and size in Reln(Orl) mice. RER phenotype was rescued in the Reln(Alb2) mice, a mutation in which no Reelin protein is produced. We propose that the RER dilations viewed in the Reln(Orl) mutation are due to the accumulation of the defective Reelin protein, and the large axonal beads in Reln(Orl) mice reflect the accumulation of truncated Reelin as the result of defects in its secretion. These observations point to an original, hitherto unrecognised, mechanism of secretion by bulk transport in smooth cisterns from the axonal cone into the axon, followed by secretion in the cortical marginal zone from the axonal cisterns that we have named axonal reelin reservoirs.