Faculty Bibliography

Activation of nuclear factor-kappaB (NF-kappaB), a key feature of the neurotrophin signaling, has been shown to be critical for neuronal survival under pathologic settings. However, the precise mechanism by which neurotrophins activate NF-kappaB is not well understood. Here we report that the Ankyrin-rich Membrane Spanning (ARMS/Kidins220) protein, a novel transmembrane substrate of tropomyosin receptor kinase B (TrkB), plays an important role in NF-kappaB signaling elicited by brain-derived neurotrophic factor (BDNF). Accordingly, depletion of ARMS by specific RNA interference, or disruption of ARMS-TrkB interaction with expression of dominant-negative ARMS mutant, abolished BDNF-induced signaling to NF-kappaB. Our data further suggests that ARMS may promote NF-kappaB signaling via activation of mitogen-activated kinase (MAPK) and IkappaB kinase (IKK), thereby facilitating phosphorylation of RelA (major NF-kappaB subunit) at an IKK-sensitive site. The results shown here identify ARMS as a major factor that links neurotrophin signaling to NF-kappaB

In addition to its role as a neurotransmitter, dopamine can stimulate neurite outgrowth and morphological effects upon primary neurons. To investigate the signal transduction mechanisms used by dopamine in developing striatal neurons, we focused upon the effects of activating the dopamine D1 receptor. Using the D1 receptor agonist, SKF38393, we found that Trk neurotrophin receptors were activated in embryonic (E) day 18 striatal neurons. K-252a, a Trk tyrosine kinase inhibitor, and a dopamine D1 receptor antagonist could block the effects of SKF38393. The increase in TrkB phosphorylation was not the result of increased neurotrophin production. Induction of TrkB activity by D1 agonist was accompanied by the phosphorylation of several Trk signaling proteins, including PLCgamma, Akt and MAP kinase. Biotinylation experiments followed by immunostaining by phospho-TrkB specific antibodies indicated that the mechanism involved increased TrkB surface expression by dopamine D1 receptor activation. This increase in cell surface TrkB expression was dependent upon an increase in intracellular Ca2+. These results indicate that stimulation of dopamine D1 receptors can be coupled to the neurotrophin receptor signaling to mediate dopamine's effects upon striatal neurons

Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we have demonstrated that Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we have applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by BDNF (brain derived neurotrophic factor), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitates (pY IPs) comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase (RTK) for BDNF, and others such as Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are proteins known to regulate intracellular trafficking of RTKs. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics

Glucocorticoids (GCs) display both protective and destructive effects in the nervous system. In excess, GCs produce neuronal damage after stress or brain injury; however, the neuroprotective effects of adrenal steroids also have been reported. The mechanisms that account for the positive actions are not well understood. Here we report that GCs can selectively activate Trk receptor tyrosine kinases after in vivo administration in the brain and in cultures of hippocampal and cortical neurons. Trk receptors are normally activated by neurotrophins, such as NGF and brain-derived neurotrophic factor, but the activation of Trk receptors by GCs does not depend on increased production of neurotrophins. Other tyrosine kinase receptors, such as EGF and FGF receptors, were not activated by GCs. The ability of GCs to increase Trk receptor activity resulted in the neuroprotection of neurons deprived of trophic support and could be modulated by steroid-converting enzymes. Pharmacological and shRNA experiments indicate that Trk receptor activation by GCs depends on a genomic action of the GC receptor. The ability of GCs to promote Trk receptor activity represents a molecular mechanism that integrates the actions of GCs and neurotrophins

Neurogenesis, the division, migration, and differentiation of new neurons, occurs throughout life. Brain derived neurotrophic factor (BDNF) has been identified as a potential signaling molecule regulating neurogenesis in the subventricular zone (SVZ), but its functional consequences in vivo have not been well defined. We report marked and unexpected deficits in survival but not proliferation of newly born cells of adult knock-in mice containing a variant form of BDNF [a valine (Val) to methionine (Met) substitution at position 66 in the prodomain of BDNF (Val66Met)], a genetic mutation shown to lead to a selective impairment in activity-dependent BDNF secretion. Utilizing knock-out mouse lines, we identified BDNF and tyrosine receptor kinase B (TrkB) as the critical molecules for the observed impairments in neurogenesis, with p75 knock-out mice showing no effect on cell proliferation or survival. We then localized the activated form of TrkB to a discrete population of cells, type A migrating neuroblasts, and demonstrate a decrease in TrkB phosphorylation in the SVZ of Val66Met mutant mice. With these findings, we identify TrkB signaling, potentially through activity dependent release of BDNF, as a critical step in the survival of migrating neuroblasts. Utilizing a behavioral task shown to be sensitive to disruptions in olfactory bulb neurogenesis, we identified specific impairments in spontaneous olfactory discrimination, but not general olfactory sensitivity or habituation to olfactory stimuli in BDNF mutant mice. Through these observations, we have identified novel links between genetic variant BDNF and adult neurogenesis in vivo, which may contribute to significant impairments in olfactory function