ABSTRACT
The three neurexins genes (NRXN1/2/3) encode polymorphic synaptic membrane proteins that are involved in cognitive functioning. Neurexins' selectivity of function is presumably conferred through differential use of 2 promoters and 5 alternative splicing sites (SS#1/2/3/4/5). In day-old rat brain neurons grown in culture, activation (depolarization) induces reversible, calcium dependent, repression of NRXN2α SS#3 insert. The effects of depolarization on NRXN1/2/3α splicing and biochemical pathways mediating them were further studied in these neurons. NRXN1/2/3α splicing in the course of memory formation in vivo was also explored, using fear conditioning paradigm in rats in which the animals were trained to associate an aversive stimulus (electrical shock) with a neutral context (a tone), resulting in the expression of fear responses to the neutral context.In the cultured neurons depolarization induced, beside NRXN2α SS#3, repression of SS#3 and SS#4 exons in NRXN3α but not NRXN1α. The repressions were mediated by the calcium/protein kinase C/Rho-associated protein kinase (ROCK) pathway. Fear conditioning induced significant and transient repressions of the NRXN1/2/3α SS#4 exons in the rat hippocampus. ROCK inhibition prior to training attenuated the behavioral fear response, the NRXN1/2/3α splicing repressions and subsequent recovery and the levels of excitatory (PSD95) and inhibitory (gephyrin) synaptic proteins in the hippocampus. No such effects were observed in the prefrontal cortex. Significant correlations existed between the fear response and hippocampal NRXN3α and NRXN2α SS#4 inserts as well as PSD95 protein levels. Hippocampal NRXN1α SS#4 insert and gephyrin levels did not correlate with the behavioral response but were negatively correlated with each other.These results show for the first time dynamic, experience related changes in NRXN1/2/3α alternative splicing in the rat brain and a role for ROCK in them. Specific neurexins' transcripts may be involved in synaptic remodeling occurring at an intermediate (hours) time scale in the course of memory formation.
Subject(s)
Alternative Splicing , Membrane Proteins/genetics , Memory , Protein Kinases/metabolism , Animals , Hippocampus/physiopathology , Neurons/physiology , RatsABSTRACT
Neurexins are polymorphic synaptic membrane proteins generated by alternative splicing of transcripts from six promoters in three genes (two promoters in each gene) at five canonical sites. Neurexins and factors regulating their alternative splicing may orchestrate coordinated presynaptic and postsynaptic development. Bioinformatic analysis revealed several sequence elements that could be involved in the regulation of alternative splicing at splice site 4 (SS#4); among them elements suspected as intronic binding sites of polypyrimidine tract binding protein (PTB), and its neuron specific analog nPTB. The role of nPTB in neurexin-2alpha and beta SS#4 splicing were studied using small interfering RNA (siRNA) to silence nPTB expression. Transformed rat retinal ganglion (RGC-5) cells expressed nPTB, PTB and neurexin-2alpha (mostly exon 20 excluded form) but not neurexin-2beta. Mouse spinal-cord glioma hybrid (NSC-34) cells expressed nPTB, PTB and both neurexin-2alpha and beta (mostly exon 20 included form). nPTB-siRNA inhibited nPTB but not PTB expression and significantly enhanced neurexin-2alpha SS# 4 exon-excluded transcript in both cell types. Neurexin-2beta SS#4 splicing was not affected by nPTB-siRNA. These results show a role for nPTB in neurexin-2alpha alternative splicing. The differential enhancement of SS# 4 exon exclusion in neurexin-2alpha suggests that the effects of nPTB are mediated via additional site(s) present in neurexin-2alpha but not in the shorter, beta form.
Subject(s)
Alternative Splicing/genetics , Gene Expression Regulation/physiology , Nerve Tissue Proteins/genetics , Polypyrimidine Tract-Binding Protein/physiology , Alternative Splicing/drug effects , Animals , Cell Line, Transformed , Computational Biology/methods , Gene Expression Regulation/drug effects , Humans , Mice , Nerve Tissue Proteins/physiology , RNA, Small Interfering/pharmacology , RatsABSTRACT
Nuclear localization of androgen receptors (ARs) is essential for their activity. Melatonin induces AR nuclear exclusion via increase in cGMP, calcium, and protein kinase C (PKC) activation, presumably through G-protein(s). The effects of regulators of G-protein signaling (RGS) on AR localization were studied in AR-expressing PC3 cells. Gi-specific RGS10 inhibited melatonin but not cGMP-induced AR nuclear exclusion, independent of androgen. No evidence for Gq activation by melatonin was found. However, Gi/Gq-selective RGS4 inhibited AR nuclear exclusion downstream of PKC activation--an effect that was abrogated by constitutively active Gq. RGS10 and RGS4, but not RGS2, ablated the inhibitory effects of melatonin on AR reporter gene activity. For the first time, these data show regulation by Gi and Gi-specific RGS protein-mediated AR nuclear exclusion, which is potentially important in the treatment of AR-dependent cancers and neurodegenerative disorders. They also reveal a role for a Gq protein downstream of PKC activation in AR nuclear localization.
Subject(s)
Cell Nucleus/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , RGS Proteins/metabolism , Receptors, Androgen/metabolism , Animals , Cell Line , Cyclic GMP/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/genetics , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Genes, Reporter , Humans , Ionomycin/metabolism , Ionophores/metabolism , Melatonin/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RGS Proteins/genetics , Receptors, Androgen/genetics , Tetradecanoylphorbol Acetate/metabolismABSTRACT
BACKGROUND: Prostate cancer is one of the most frequently diagnosed cancers in males. Autocrine/paracrine growth factors for the epidermal growth factor receptor (EGFR) have been identified in prostate tumors suggesting a role for EGFR in the progression of prostate cancer. The androgen-dependent prostate cancer cell line, LNCaP, expresses the EGFR as well as two additional members of the family; ErbB-2 and ErbB-3, which can be activated by neuregulin (NRG) isoforms. The effect of ErbB ligands on the viability of LNCaP cells was studied. METHODS: In the present study, we examined the effect of NRG on LNCaP cell growth and survival in the absence of androgen mimetic by the MTT assay, FACS analysis, nuclei staining, and Western blotting. RESULTS: Our results demonstrate that NRG activates ErbB-2/ErbB-3 heterodimers and induces cell death of LNCaP cells. By contrast, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers and induces cell growth and survival. Interestingly, LNCaP cells treated with PI3K inhibitor underwent cell death but cells treated with both NRG and PI3K inhibitor survived as the control cells, indicating that the PI3K pathway may mediate NRG-induced cell death. NRG-induced cell death was not inhibited by the broad-spectrum caspases inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD-FMK). However, NRG-induced cell death was inhibited by type II cell death inhibitor, 3-methyladenine. CONCLUSIONS: These results suggest that NRG induces type II cell death of LNCaP cells through PI3K-dependent pathway.
