Deletion of PLC??1 in GABAergic neurons increases seizure susceptibility in aged mice

Synaptic inhibition plays a fundamental role in the information processing of neural circuits. It sculpts excitatory signals and prevents hyperexcitability of neurons. Owing to these essential functions, dysregulated synaptic inhibition causes a plethora of neurological disorders, including epilepsy, autism, and schizophrenia. Among these disorders, epilepsy is associated with abnormal hyperexcitability of neurons caused by the deficits of GABAergic neuron or decreased GABAergic inhibition at synapses. Although many antiepileptic drugs are intended to improve GABA-mediated inhibition, the molecular mechanisms of synaptic inhibition regulated by GABAergic neurons are not fully understood. Increasing evidence indicates that phospholipase C??1 (PLC??1) is involved in the generation of seizure, while the causal relationship between PLC??1 and seizure has not been firmly established yet. Here, we show that genetic deletion of PLC??1 in GABAergic neurons leads to handling-induced seizure in aged mice. In addition, aged Plcg1F/F; Dlx5/6-Cre mice exhibit other behavioral alterations, including hypoactivity, reduced anxiety, and fear memory deficit. Notably, inhibitory synaptic transmission as well as the number of inhibitory synapses are decreased in the subregions of hippocampus. These findings suggest that PLC??1 may be a key determinant of maintaining both inhibitory synapses and synaptic transmission, potentially contributing to the regulation of E/I balance in the hippocampus

Rapid, experience-dependent translation of neurogranin enables memory encoding

Experience induces de novo protein synthesis in the brain and protein synthesis is required for long-term memory. It is important to define the critical temporal window of protein synthesis and identify newly synthesized proteins required for memory formation. Using a behavioral paradigm that temporally separates the contextual exposure from the association with fear, we found that protein synthesis during the transient window of context exposure is required for contextual memory formation. Among an array of putative activity-dependent translational neuronal targets tested, we identified one candidate, a schizophrenia-associated candidate mRNA, neurogranin (Ng, encoded by the Nrgn gene) responding to novel-context exposure. The Ng mRNA was recruited to the actively translating mRNA pool upon novel-context exposure, and its protein levels were rapidly increased in the hippocampus. By specifically blocking activity-dependent translation of Ng using virus-mediated molecular perturbation, we show that experience-dependent translation of Ng in the hippocampus is required for contextual memory formation. We further interrogated the molecular mechanism underlying the experience-dependent translation of Ng, and found that fragile-X mental retardation protein (FMRP) interacts with the 3′UTR of the Nrgn mRNA and is required for activity-dependent translation of Ng in the synaptic compartment and contextual memory formation. Our results reveal that FMRP-mediated, experience-dependent, rapid enhancement of Ng translation in the hippocampus during the memory acquisition enables durable context memory encoding. Keywords: hippocampus; contextual memory; dentate gyrus; ASD; schizophreni

Molecular mechanisms of synaptic plasticity

Thesis: Ph. D. in Biological Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2016.Cataloged from PDF version of thesis. Vita.Includes bibliographical references.Synaptic plasticity serves as a central molecular mechanism underlying learning and memory formation in the brain. An increase in intracellular calcium concentrations triggered by neuronal activity induces synaptic plasticity, and calmodulin is a key protein that detects the elevated calcium levels and propagates downstream signaling. Neurogranin is a neuron-specific protein that binds to calmodulin and regulates the availability of calmodulin in the postsynaptic compartments of excitatory neurons. Dysregulation of neurogranin has been reported to cause altered synaptic plasticity as well as impairment in hippocampus-dependent learning, and is also associated with the higher risk of developing neurodegenerative and psychiatric diseases. Therefore, it is critical to understand how neurogranin regulates the induction of synaptic plasticity in the brain at the molecular level. The focus of this thesis is to examine how the changes in neurogranin expression levels contribute to the induction of synaptic plasticity in the hippocampus with a spike-timing-dependent plasticity paradigm and to understand the underlying molecular mechanisms. Using lentivirus-mediated manipulations of neurogranin levels in hippocampal CAl neurons, we found that increasing neurogranin levels in CAI neurons prolongs the timing window for spike-timing-dependent long-term potentiation (LTP), whereas acute knockdown of neurogranin inhibits the expression of LTP via regulating PP2B activity. We have also found that neurogranin interferes with calcium-dependent inactivation of neuronal L-type calcium channels and allows a sustained influx of calcium during the membrane depolarization in hippocampal neurons. Our results indicate that dynamic changes in neurogranin levels play a crucial role in setting the threshold for inducing LTP in spike-timing-dependent plasticity in the hippocampus.by Hongik Hwang.Ph. D. in Biological Chemistr

Physical and Functional Interaction between 5-HT6 Receptor and Nova-1

5-HT6 receptor (5-HT6R) is implicated in cognitive dysfunction, mood disorder, psychosis, and eating disorders. However, despite its significant role in regulating the brain functions, regulation of 5-HT6R at the molecular level is poorly understood. Here, using yeast two-hybrid assay, we found that human 5-HT6R directly binds to neuro-oncological ventral antigen 1 (Nova-1), a brain-enriched splicing regulator. The interaction between 5-HT6R and Nova-1 was confirmed using GST pull-down and co-immunoprecipitation assays in cell lines and rat brain. The splicing activity of Nova-1 was decreased upon overexpression of 5-HT6R, which was examined by detecting the spliced intermediates of gonadotropin-releasing hormone (GnRH), a known pre-mRNA target of Nova-1, using RT-PCR. In addition, overexpression of 5-HT6R induced the translocation of Nova-1 from the nucleus to cytoplasm, resulting in the reduced splicing activity of Nova-1. In contrast, overexpression of Nova-1 reduced the activity and the total protein levels of 5-HT6R. Taken together, these results indicate that when the expression levels of 5-HT6R or Nova-1 protein are not properly regulated, it may also deteriorate the function of the other. Copyright © Experimental Neurobiology 2019.1

Effect of the Gintonin-Enriched Fraction on Glucagon-Like-Protein-1 Release

Ginseng-derived gintonin reportedly contains functional lysophosphatidic acids (LPAs) as LPA receptor ligands. The effect of the gintonin-enriched fraction (GEF) on in vitro and in vivo glucagon-like protein-1 (GLP-1) secretion, which is known to stimulate insulin secretion, via LPA receptor(s) remains unclear. Accordingly, we examined the effects of GEF on GLP-1 secretion using human enteroendocrine NCI-H716 cells. The expression of several of LPA receptor subtypes in NCI-H716 cells using qPCR and Western blotting was examined. LPA receptor subtype expression was in the following order: LPA6 > LPA2 > LPA4 > LPA5 > LPA1 (qPCR), and LPA6 > LPA4 > LPA2 > LPA1 > LPA3 > LPA5 (Western blotting). GEF-stimulated GLP-1 secretion occurred in a dose- and time-dependent manner, which was suppressed by cAMP-Rp, a cAMP antagonist, but not by U73122, a phospholipase C inhibitor. Furthermore, silencing the human LPA6 receptor attenuated GEF-mediated GLP-1 secretion. In mice, low-dose GEF (50 mg/kg, peroral) increased serum GLP-1 levels; this effect was not blocked by Ki16425 co-treatment. Our findings indicate that GEF-induced GLP-1 secretion could be achieved via LPA6 receptor activation through the cAMP pathway. Hence, GEF-induced GLP secretion via LPA6 receptor regulation might be responsible for its beneficial effects on human endocrine physiology

Autophagy Modulation in Aggresome Formation: Emerging Implications and Treatments of Alzheimer’s Disease

Alzheimer’s disease (AD) is one of the most prevailing neurodegenerative diseases in the world, which is characterized by memory dysfunction and the formation of tau and amyloid β (Aβ) aggregates in multiple brain regions, including the hippocampus and cortex. The formation of senile plaques involving tau hyperphosphorylation, fibrillar Aβ, and neurofibrillary tangles (NFTs) is used as a pathological marker of AD and eventually produces aggregation or misfolded protein. Importantly, it has been found that the failure to degrade these aggregate-prone proteins leads to pathological consequences, such as synaptic impairment, cytotoxicity, neuronal atrophy, and memory deficits associated with AD. Recently, increasing evidence has suggested that the autophagy pathway plays a role as a central cellular protection system to prevent the toxicity induced by aggregation or misfolded proteins. Moreover, it has also been revealed that AD-related protein aggresomes could be selectively degraded by autophagosome and lysosomal fusion through the autophagy pathway, which is known as aggrephagy. Therefore, the regulation of autophagy serve as a useful approach to modulate the formation of aggresomes associated with AD. This review focuses on the recent improvements in the application of natural compounds and small molecules as a potential therapeutic approach for AD prevention and treatment via aggrephagy

Shank Proteins Differentially Regulate Synaptic Transmission

Shank proteins, one of the principal scaffolds in the postsynaptic density (PSD) of the glutamatergic synapses, have been associated with autism spectrum disorders and neuropsychiatric diseases. However, it is not known whether different Shank family proteins have distinct functions in regulating synaptic transmission, and how they differ from other scaffold proteins in this aspect. Here, we investigate the role of Shanks in regulating glutamatergic synaptic transmission at rat hippocampal SC-CA1 synapses, using lentivirus-mediated knockdown and molecular replacement combined with dual whole-cell patch clamp in hippocampal slice culture. In line with previous findings regarding PSD-MAGUK scaffold manipulation, we found that loss of scaffold proteins via knockdown of Shank1 or Shank2, but not Shank3, led to a reduction of the number but not the unitary response of AMPAR containing synapses. Only when both Shank1 and Shank2 were knocked down, were both the number and the unitary response of active synapses reduced. This reduction was accompanied by a decrease in NMDAR-mediated synaptic response, indicating more profound deficits in synaptic transmission. Molecular replacement with Shank2 and Shank3c rescued the synaptic transmission to the basal level, and the intact sterile _-motif (SAM) of Shank proteins is required for maintaining glutamatergic synaptic transmission. We also found that altered neural activity did not influence the effect of Shank1 or Shank2 knockdown on AMPAR synaptic transmission, in direct contrast to the activity dependence of the effect of PSD-95 knockdown, revealing differential interaction between activity-dependent signaling and scaffold protein families in regulating synaptic AMPAR function. Keywords: ASD; bicuculline; excitatory; hippocampus; mini; Prosap1National Institutes of Health (U.S.) (Grant MH080310