Alterations in intracellular signaling pathways are important in treating complex neuropsychiatric disorders 1 . Signaling pathway components, including several protein kinases and phosphatases, are direct targets of some of the most effective medications for treating these disorders. For example, lithium, one of the most established treatments for bipolar disorder (BPD), is believed to exert its therapeutic effects by directly modulating the activity of inositol monophosphatase and of glycogen synthase kinase 3β (GSK3β) 1 . It is still unclear, however, whether abnormalities in signaling pathways are central to the pathophysiology of psychiatric illnesses, including schizophrenia. Nevertheless, genes regulating such signaling cascades, especially those affecting synaptic transmission and plasticity, are good candidate susceptibility genes. RESULTS Low levels of AKT1 in individuals with schizophreniaIt is well established that most of the central nervous system (CNS) protein kinases and phosphatases are involved in a wide variety of cellular functions and are expressed in diverse cell types including peripheral blood lymphocytes. We speculated that alterations in brain levels or activity of protein kinases and phosphatases may contribute to schizophrenia susceptibility in humans and that this might be observed in the peripheral tissues of individuals with schizophrenia. We examined the abundance of several kinases implicated in synaptic plasticity (PRKACA, AKT1, PRKC, MAP3K, GSK3β, PIK3CB and PIK3R2) 2-4 . We assessed changes in protein levels because they are more likely to be cell-autonomous and less likely to be influenced by the cellular environment when compared, for example, with rapid and reversible activity modifications through phosphorylation. Protein extracts from lymphocyte-derived cell lines from individuals with schizophrenia (n = 28) and unaffected controls (n = 28) were subjected to SDS-PAGE and immunoblot analysis. Levels of protein kinase AKT1 were 68% lower in individuals with schizophrenia than in controls (Fig. 1a, P = 0.014, Mann-Whitney test, corrected for eight tests). In contrast, we observed no differences in the levels of the other kinases tested.This initial exploratory analysis may have been confounded by an imperfect match of cases and controls and the influence of Epstein-Barr virus (EBV) transformation on the levels of AKT1. Nonetheless, the specificity of our findings together with the fact that the same cellular pathway has been implicated in mood disorders 1 prompted us to follow up our initial observation using four complementary approaches. First, we attempted to verify a reduction in AKT1 levels in postmortem frontal cortex, one of the primary sites of disease pathology. Second, we examined whether the reduction in AKT1 levels was reflected by a reduction in substrate phosphorylation in both peripheral lymphocytes and postmortem frontal cortex. Third, we tested whether certain variants of the gene encoding AKT1 were preferentially transmitted in individuals with schizophreni...

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We evaluated a possible association between the brain-derived neurotrophic factor (BDNF) gene and susceptibility to obsessive-compulsive disorder (OCD) by genotyping a number of single-nucleotide polymorphisms (SNPs) and one microsatellite marker from the extended BDNF locus in 164 triads with OCD. Extensive background linkage disequilibrium was observed at this locus. Single-locus transmission-distortion tests revealed significant evidence of association with the disease for all the BDNF gene markers tested, including a Val66Met variation affecting the sequence of the proBDNF protein. Analysis of multi-SNP haplotypes provided similar results. Haplotype transmission comparisons in this and previous studies point to a functionally distinct BDNF haplotype uniquely marked by the rare Met66 allele, which is undertransmitted and likely confers a protective effect in OCD and other psychiatric disorders.

Schizophrenia is a severe psychiatric disorder characterized by a complex mode of inheritance. Forebrain-specific CNB knockout mice display a spectrum of behavioral abnormalities related to altered behaviors observed in schizophrenia patients. To examine whether calcineurin dysfunction is involved in schizophrenia etiology, we undertook studies of an initial subset of calcineurinrelated genes, prioritizing ones that map to loci previously implicated in schizophrenia by linkage studies. Transmission disequilibrium studies in a large sample of affected families detected association of the PPP3CC gene, which encodes the calcineurin ␥ catalytic subunit, with disease. Our results identify PPP3CC, located at 8p21.3, as a potential schizophrenia susceptibility gene and support the proposal that alterations in calcineurin signaling contribute to schizophrenia pathogenesis. S chizophrenia is a severe psychiatric condition that affects Ϸ1% of the population worldwide (1). Studies of the inheritance of schizophrenia have revealed that it is a multifactorial disease characterized by multiple genetic susceptibility elements, each contributing a modest increase in risk (2). Family linkage studies and studies of chromosomal abnormalities associated with schizophrenia have identified a number of schizophrenia susceptibility loci (2, 3). Such loci provide a basis for higher resolution genetic studies and a criterion for assessment of potential candidate genes. In addition to direct genetic analysis, a longstanding body of pharmacological studies has led to the prevailing hypotheses that dysfunction of dopaminergic or Nmethyl-D-aspartate (NMDA) receptor-mediated signaling are major contributing factors in schizophrenia pathogenesis (4-6).Calcineurin is a calcium-dependent serine͞threonine protein phosphatase that is highly expressed in the CNS (7,8). Calcineurin consists of a heterodimer composed of a regulatory subunit, CNB, and a catalytic subunit, CNA (7, 8). There are three different CNA isoforms encoded by distinct genes. Calcineurin activity plays a key role in the downstream regulation of dopaminergic signal transduction (9) and in the induction of certain forms of N-methyl-D-aspartate receptor-dependent synaptic plasticity (10, 11). Thus, calcineurin function could comprise a critical link between dopaminergic and glutamatergic signaling.In an accompanying study, we report that forebrain-specific CNB knockout mice display a spectrum of behavioral abnormalities that is strikingly reminiscent of altered behaviors observed in schizophrenia patients (12). Based on these findings, we decided to further investigate the potential involvement of calcineurin dysfunction in schizophrenia etiology by directly testing for genetic association of calcineurin-related candidate genes with schizophrenia. We prioritized examination of genes encoding calcineurin subunits or calcineurin-related molecules that map to schizophrenia susceptibility loci. We present direct genetic evidence for association of the PPP3CC gene with schizophrenia. PP...

We report on our initial genetic linkage studies of schizophrenia in the genetically isolated population of the Afrikaners from South Africa. A 10-cM genomewide scan was performed on 143 small families, 34 of which were informative for linkage. Using both nonparametric and parametric linkage analyses, we obtained evidence for a small number of disease loci on chromosomes 1, 9, and 13. These results suggest that few genes of substantial effect exist for schizophrenia in the Afrikaner population, consistent with our previous genealogical tracing studies. The locus on chromosome 1 reached genomewide significance levels (nonparametric LOD score of 3.30 at marker D1S1612, corresponding to an empirical P value of.012) and represents a novel susceptibility locus for schizophrenia. In addition to providing evidence for linkage for chromosome 1, we also identified a proband with a uniparental disomy (UPD) of the entire chromosome 1. This is the first time a UPD has been described in a patient with schizophrenia, lending further support to involvement of chromosome 1 in schizophrenia susceptibility in the Afrikaners.

Here we characterize and compare the contribution of three recently identified strong candidate schizophrenia susceptibility genes; G72, neuregulin 1 (NRG1) and dystrobrevin-binding protein 1 (DTNBP1) in two independent datasets of patients with distinct genetic backgrounds. On the basis of corrected P-values from single-and multilocus transmission distortion tests our analysis provides no support for a contribution of G72, NRG1 or DTNBP1 in the tested samples. When transmission of individual haplotypes was considered, a picture more consistent with the original studies emerged, where transmission distortions in the same direction as the original samples and involving the same core haplotypes were observed for G72 and NRG1. Interestingly, whereas the NRG1 gene analysis was dominated by the presence of over-transmitted haplotypes, the G72 gene analysis was consistently dominated in both datasets by under-transmissions. Negative transmissions involved a core haplotype complementary to the originally detected over-transmitted haplotype, suggesting the presence of a protective variant within the G72 locus.

We examined neural network ontogeny using microelectrode array (MEA) recordings made in multiwell MEA (mwMEA) plates over the first 12 days in vitro (DIV). In primary cortical cultures, action potential spiking activity developed rapidly between DIV 5 and 12. Spiking was sporadic and unorganized at early DIV, and became progressively more organized with time, with bursting parameters, synchrony, and network bursting increasing between DIV 5 and 12. We selected 12 features to describe network activity; principal components analysis using these features demonstrated segregation of data by age at both the well and plate levels. Using random forest classifiers and support vector machines, we demonstrated that four features (coefficient of variation [CV] of within-burst interspike interval, CV of interburst interval, network spike rate, and burst rate) could predict the age of each well recording with >65% accuracy. When restricting the classification to a binary decision, accuracy improved to as high as 95%. Further, we present a novel resampling approach to determine the number of wells needed for comparing different treatments. Overall, these results demonstrate that network development on mwMEA plates is similar to development in single-well MEAs. The increased throughput of mwMEAs will facilitate screening drugs, chemicals, or disease states for effects on neurodevelopment.

Samples containing highly unbalanced DNA mixtures from two individuals commonly occur both in forensic mixed stains and in peripheral blood DNA microchimerism induced by pregnancy or following organ transplant. Because of PCR amplification bias, the genetic identification of a DNA that contributes trace amounts to a mixed sample represents a tremendous challenge. This means that standard genetic markers, namely microsatellites, also referred as short tandem repeats (STR), and single-nucleotide polymorphism (SNP) have limited power in addressing common questions of forensic and medical genetics. To address this issue, we developed a molecular marker, named DIP–STR that relies on pairing deletion–insertion polymorphisms (DIP) with STR. This novel analytical approach allows for the unambiguous genotyping of a minor component in the presence of a major component, where DIP–STR genotypes of the minor were successfully procured at ratios up to 1:1,000. The compound nature of this marker generates a high level of polymorphism that is suitable for identity testing. Here, we demonstrate the power of the DIP–STR approach on an initial set of nine markers surveyed in a Swiss population. Finally, we discuss the limitations and potential applications of our new system including preliminary tests on clinical samples and estimates of their performance on simulated DNA mixtures.