Anti-PSD95 monoclonal antibody, clone 7E3-1B8

Opioid drugs are highly addictive and their abuse has a strong genetic load. Dopamine-glutamate interactions are hypothesized to be important for regulating neural systems central for addiction vulnerability. Balanced dopamine-glutamate interaction is mediated through several functional associations, including a physical link between discs, large homolog 4 (Drosophila) (DLG4, PSD-95) and dopamine receptor 1 (DRD1) within the postsynaptic density to regulate DRD1 trafficking. To address whether genetic associations with heroin abuse exist in relation to dopamine and glutamate and their potential interactions, we evaluated single-nucleotide polymorphisms of key genes within these systems in three populations of opiate abusers and controls, totaling 489 individuals from Europe and the United States. Despite significant differences in racial makeup of the separate samples, polymorphisms of DRD1 and DLG4 were found to be associated with opiate abuse. In addition, a strong gene-gene interaction between homer 1 homolog (Drosophila) (HOMER1) and DRD1 was predicted to occur in Caucasian subjects. This interaction was further analyzed by evaluating DRD1 genotype in relation to HOMER1b/c protein expression in postmortem tissue from a subset of Caucasian subjects. DRD1 rs265973 genotype correlated with HOMER1b/c levels in the striatum, but not cortex or amygdala; the correlation was inversed in opiate abusers as compared with controls. Cumulatively, these results support the hypothesis that there may be significant, genetically influenced interactions between glutamatergic and dopaminergic pathways in opiate abusers.

Most mammalian genes produce multiple mRNA isoforms derived from alternative pre-mRNA splicing, with each alternative exon controlled by a complex network of regulatory factors. The identification of these regulators can be laborious and is usually carried out one factor at a time. We have developed a broadly applicable high-throughput screening method that simultaneously identifies multiple positive and negative regulators of a particular exon. Two minigene reporters were constructed: One produces green fluorescent protein (GFP) from the mRNA including an exon, and red fluorescent protein (RFP) from the mRNA lacking the exon; the other switches these fluorescent products of exon inclusion and exclusion. Combining results from these two reporters eliminates many false positives and greatly enriches for true splicing regulators. After extensive optimization of this method, we performed a gain-of-function screen of 15,779 cDNA clones and identified 40 genes affecting exon 18 of Discs large homolog 4 (Dlg4; also known as post-synaptic density protein 95 [Psd-95]). We confirmed that 28 of the 34 recoverable clones alter reporter splicing in RT-PCR assays. Remarkably, 18 of the identified genes encode splicing factors or RNA binding proteins, including PTBP1, a previously identified regulator of this exon. Loss-of-function experiments examining endogenous Dlg4 transcripts validated the effects of five of eight genes tested in independent cell lines, and two genes were further confirmed to regulate Dlg4 splicing in primary neurons. These results identify multiple new regulators of Dlg4 splicing, and validate an approach to isolating splicing regulators for almost any cassette exon from libraries of cDNAs, shRNAs, or small molecules.