Faculty Bibliography

The brain-derived neurotrophic factor (BDNF) Val66Met polymorphism is a common human single nucleotide polymorphism (SNP) that affects the regulated release of BDNF, and has been implicated in affective disorders and cognitive dysfunction. A decreased activation of the infralimbic medial prefrontal cortex (IL-mPFC), a brain region critical for the regulation of affective behaviors, has been described in BDNF(Met) carriers. However, it is unclear whether and how the Val66Met polymorphism affects the IL-mPFC synapses. Here, we report that spike timing-dependent plasticity (STDP) was absent in the IL-mPFC pyramidal neurons from BDNF(Met/Met) mice, a mouse that recapitulates the specific phenotypic properties of the human BDNF Val66Met polymorphism. Also, we observed a decrease in NMDA and GABA receptor-mediated synaptic transmission in the pyramidal neurons of BDNF(Met/Met) mice. While BDNF enhanced non-NMDA receptor transmission and depressed GABA receptor transmission in the wild-type mice, both effects were absent in BDNF(Met/Met) mice after BDNF treatment. Indeed, exogenous BDNF reversed the deficits in STDP and NMDA receptor transmission in BDNF(Met/Met) neurons. BDNF-mediated selective reversal of the deficit in plasticity and NMDA receptor transmission, but its lack of effect on GABA and non-NMDA receptor transmission in BDNF(Met/Met) mice, suggests separate mechanisms of Val66Met polymorphism upon synaptic transmission. The effect of the Val66Met polymorphism on synaptic transmission and plasticity in the IL-mPFC represents a mechanism to account for this impact of SNP on affective disorders and cognitive dysfunction.

Herpes simplex virus type-1 (HSV-1) establishes latency in peripheral neurons, creating a permanent source of recurrent infections. The latent genome is assembled into chromatin and lytic cycle genes are silenced. Processes that orchestrate reentry into productive replication (reactivation) remain poorly understood. We have used latently infected cultures of primary superior cervical ganglion (SCG) sympathetic neurons to profile viral gene expression following a defined reactivation stimulus. Lytic genes are transcribed in two distinct phases, differing in their reliance on protein synthesis, viral DNA replication and the essential initiator protein VP16. The first phase does not require viral proteins and has the appearance of a transient, widespread de-repression of the previously silent lytic genes. This allows synthesis of viral regulatory proteins including VP16, which accumulate in the cytoplasm of the host neuron. During the second phase, VP16 and its cellular cofactor HCF-1, which is also predominantly cytoplasmic, concentrate in the nucleus where they assemble an activator complex on viral promoters. The transactivation function supplied by VP16 promotes increased viral lytic gene transcription leading to the onset of genome amplification and the production of infectious viral particles. Thus regulated localization of de novo synthesized VP16 is likely to be a critical determinant of HSV-1 reactivation in sympathetic neurons.

Regulation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for adaptation to environmental changes. The principle regulator of the HPA axis is corticotrophin-releasing hormone (CRH), which is made in the parventricular nucleus and is an important target of negative feedback by glucocorticoids. However, the molecular mechanisms that regulate CRH are not fully understood. Disruption of normal HPA axis activity is a major risk factor of neuropsychiatric disorders in which decreased expression of the glucocorticoid receptor (GR) has been documented. To investigate the role of the GR in CRH neurons, we have targeted the deletion of the GR, specifically in the parventricular nucleus. Impairment of GR function in the parventricular nucleus resulted in an enhancement of CRH expression and an up-regulation of hypothalamic levels of BDNF and disinhibition of the HPA axis. BDNF is a stress and activity-dependent factor involved in many activities modulated by the HPA axis. Significantly, ectopic expression of BDNF in vivo increased CRH, whereas reduced expression of BDNF, or its receptor TrkB, decreased CRH expression and normal HPA functions. We find the differential regulation of CRH relies upon the cAMP response-element binding protein coactivator CRTC2, which serves as a switch for BDNF and glucocorticoids to direct the expression of CRH.

HYPOTHESIS: Reactivation of herpes simplex virus type 1 (HSV-1) in geniculate ganglion neurons (GGNs) is an etiologic mechanism of Bell's palsy (BP) and delayed facial palsy (DFP) after otologic surgery. BACKGROUND: Several clinical studies, including temporal bone studies, antibody, titers, and intraoperative studies, suggest that reactivation of HSV-1 from latently infected GGNs may lead to both BP and DFP. However, it is difficult to study these processes in humans or live animals. METHODS: Primary cultures of GGNs were latently infected with Patton strain HSV-1 expressing a green fluorescent protein-late lytic gene chimera. Four days later, these cultures were treated with trichostatin A (TSA), a known chemical reactivator of HSV-1 in other neurons. Cultures were monitored daily by fluorescent microscopy. Titers of media from lytic, latent, and latent/TSA treated GGN cultures were obtained using plaque assays on Vero cells. RNA was harvested from latently infected GGN cultures and examined for the presence of viral transcripts using reverse transcription-polymerase chain reaction. RESULTS: Latently infected GGN cultures displayed latency-associated transcripts only, whereas lytically infected and reactivated latent cultures produced other viral transcripts, as well. The GGN cultures displayed a reactivation rate of 65% after treatment with TSA. Media from latently infected cultures contained no detectable infectious HSV-1, whereas infectious virus was observed in both lytically and latently infected/TSA-treated culture media. CONCLUSION: We have shown that cultured GGNs can be latently infected with HSV-1, and HSV-1 in these latently infected neurons can be reactivated using TSA, yielding infectious virus. These results have implications for the cause of both BP and DFP

Growth of axons and dendrites is a dynamic process that involves guidance molecules, adhesion proteins, and neurotrophic factors. Although neurite extension is stimulated by the neurotrophin nerve growth factor (NGF), we found that the precursor of NGF, proNGF, induced acute collapse of growth cones of cultured hippocampal neurons. This retraction was initiated by an interaction between the p75 neurotrophin receptor (p75(NTR)) and the sortilin family member SorCS2 (sortilin-related VPS10 domain-containing receptor 2). Binding of proNGF to the p75(NTR)-SorCS2 complex induced growth cone retraction by initiating the dissociation of the guanine nucleotide exchange factor Trio from the p75(NTR)-SorCS2 complex, resulting in decreased Rac activity and, consequently, growth cone collapse. The actin-bundling protein fascin was also inactivated, contributing to the destabilization and collapse of actin filaments. These results identify a bifunctional signaling mechanism by which proNGF regulates actin dynamics to acutely modulate neuronal morphology

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling

Acetylcholinesterase inhibitors are first-line therapies for Alzheimer's disease. These drugs increase cholinergic tone in the target areas of the cholinergic neurons of the basal forebrain. Basal forebrain cholinergic neurons are dependent upon trophic support by nerve growth factor (NGF) through its neurotrophin receptor, TrkA. In the present study, we investigated whether the acetylcholinesterase inhibitors donepezil and galantamine could influence neurotrophin receptor signaling in the brain. Acute administration of donepezil (3 mg/kg, i.p.) led to the rapid autophosphorylation of TrkA and TrkB neurotrophin receptors in the adult mouse hippocampus. Similarly, galantamine dose-dependently (3, 9 mg/kg, i.p.) increased TrkA and TrkB phosphorylation in the mouse hippocampus. Both treatments also increased the phosphorylation of transcription factor CREB and tended to increase the phosphorylation of AKT kinase but did not alter the activity of MAPK42/44. Chronic treatment with galantamine (3 mg/kg, i.p., 14 days), did not induce changes in hippocampal NGF and BDNF synthesis or protein levels. Our findings show that acetylcholinesterase inhibitors are capable of rapidly activating hippocampal neurotrophin signaling and thus suggest that therapies targeting Trk signaling may already be in clinical use in the treatment of AD

BDNF is produced from many transcripts that display distinct subcellular localization, suggesting that spatially restricted effects occur as a function of genetic and physiological regulation. Different BDNF 5' splice variants give a restricted localization in the cell body or the proximal and distal compartments of dendrites; however, the functional consequences are not known. Silencing individual endogenous transcripts or overexpressing BDNF-GFP transcripts in cultured neurons demonstrated that whereas some transcripts (1 and 4) selectively affected proximal dendrites, others (2C and 6) affected distal dendrites. Moreover, segregation of BDNF transcripts resulted in a highly selective activation of the BDNF TrkB receptor. These studies indicate that spatial segregation of BDNF transcripts enables BDNF to differentially shape distinct dendritic compartments