Postsynaptic Density-95 Isoform Abnormalities in Schizophrenia.

Background: Postsynaptic density-95 (PSD-95) protein expression is dysregulated in schizophrenia in a variety of brain regions. We have designed experiments to examine PSD-95 mRNA splice variant expression in the dorsolateral prefrontal cortex from subjects with schizophrenia. Methods: We performed quantitative PCR and western blot analysis to measure PSD-95 expression in schizophrenia vs control subjects, rodent haloperidol treatment studies, rodent postmortem interval studies, and GluN1 knockdown (KD) mice vs controls. Results: We found decreased mRNA expression of beta (t = 4.506, df = 383, P < .0001) and truncated (t = 3.378, df = 383, P = .0008) isoforms of PSD-95, whereas alpha was unchanged. Additionally, we found decreased PSD-95 protein expression in schizophrenia (t = 2.746, df = 71, P = .0076). We found no correlation between PSD-95 protein and alpha, beta, or truncated mRNA isoforms in schizophrenia. PSD-95 beta transcript was increased (t = 3.346, df = 14, P < .05) in the GluN1 KD mouse model of schizophrenia. There was an increase in PSD-95 alpha mRNA expression (t = 2.905, df = 16, P < .05) in rats following long-term haloperidol administration. Conclusions: Our findings describe a unique pathophysiology of specific PSD-95 isoform dysregulation in schizophrenia, chronic neuroleptic treatment, and a genetic lesion mouse model of drastically reduced N-methyl-d-aspartate receptor (NMDAR) complex expression. These data indicate that regulation of PSD-95 is multifaceted, may be isoform specific, and biologically relevant for synaptic signaling function. Specifically, NMDAR-mediated synaptic remodeling, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor trafficking and interaction may be impaired in schizophrenia by decreased PSD-95 beta and truncated expression (respectively). Further, increased PSD-95 beta transcript in the GluN1 KD mouse model poses a potential compensatory rescue of NMDAR-mediated function via increased postsynaptic throughput of the severely reduced GluN1 signal. Together, these data propose that disruption of excitatory signaling complexes through genetic (GluN1 KD), pharmacologic (antipsychotics), or disease (schizophrenia) mechanisms specifically dysregulates PSD-95 expression.

GM1 ganglioside enhances Ret signaling in striatum.

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell-derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co-receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration- and time-dependent manner. GM1-induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration-dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP-mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits. It has been proposed that the ganglioside GM1 promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. We provide evidence that the GM1 enhances the activity of Ret tyrosine kinase receptor for glia cell-derived neurotrophic factor (GDNF) in the striatum in situ and in vivo, and propose that this might be a mechanism for GM1's neurotrophic actions on dopaminergic neurons. Ret activation is followed by Tyr1062 and Tyr981 phosphorylation and recruitment of PI3-K/Akt, Erk, and Src signaling. GM1 apparently acts by increasing the binding of endogenous GDNF to GFRα1 co-receptor, which is required for the GM1 effect on Ret.

Transcript-specific associations of SLC12A5 (KCC2) in human prefrontal cortex with development, schizophrenia, and affective disorders.

The neuron-specific K(+)-Cl(-) cotransporter SLC12A5, also known as KCC2, helps mediate the electrophysiological effects of GABA. The pattern of KCC2 expression during early brain development suggests that its upregulation drives the postsynaptic switch of GABA from excitation to inhibition. We previously found decreased expression of full-length KCC2 in the postmortem hippocampus of patients with schizophrenia, but not in the dorsolateral prefrontal cortex (DLPFC). Using PCR and rapid amplification of cDNA ends, we discovered several previously unrecognized alternative KCC2 transcripts in both human adult and fetal brain in addition to the previously identified full-length (NM_020708.3) and truncated (AK098371) transcripts. We measured the expression levels of four relatively abundant truncated splice variants, including three novel transcripts (ΔEXON6, EXON2B, and EXON6B) and one previously described transcript (AK098371), in a large human cohort of nonpsychiatric controls across the lifespan, and in patients with schizophrenia and affective disorders. In SH-SY5Y cell lines, these transcripts were translated into proteins and expressed at their predicted sizes. Expression of the EXON6B transcript is increased in the DLPFC of patients with schizophrenia (p = 0.03) but decreased in patients with major depression (p = 0.04). The expression of AK098371 is associated with a GAD1 single nucleotide polymorphism (rs3749034) that previously has been associated with GAD67 expression and risk for schizophrenia. Our data confirm the developmental regulation of KCC2 expression, and provide evidence that KCC2 transcripts are differentially expressed in schizophrenia and affective disorders. Alternate transcripts from KCC2 may participate in the abnormal GABA signaling in the DLPFC associated with schizophrenia.

DISC1 splice variants are upregulated in schizophrenia and associated with risk polymorphisms.

Disrupted-In-Schizophrenia-1 (DISC1) is a promising susceptibility gene for major mental illness, but the mechanism of the clinical association is unknown. We searched for DISC1 transcripts in adult and fetal human brain and tested whether their expression is altered in patients with schizophrenia and is associated with genetic variation in DISC1. Many alternatively spliced transcripts were identified, including groups lacking exon 3 (Delta3), exons 7 and 8 (Delta7Delta8), an exon 3 insertion variant (extra short variant-1, Esv1), and intergenic splicing between TSNAX and DISC1. Isoforms Delta7Delta8, Esv1, and Delta3, which encode truncated DISC1 proteins, were expressed more abundantly during fetal development than during postnatal ages, and their expression was higher in the hippocampus of patients with schizophrenia. Schizophrenia risk-associated polymorphisms [non-synonymous SNPs rs821616 (Cys704Ser) and rs6675281 (Leu607Phe), and rs821597] were associated with the expression of Delta3 and Delta7Delta8. Moreover, the same allele at rs6675281, which predicted higher expression of these transcripts in the hippocampus, was associated with higher expression of DISC1Delta7Delta8 in lymphoblasts in an independent sample. Our results implicate a molecular mechanism of genetic risk associated with DISC1 involving specific alterations in gene processing.