Nataliya Rybalchenko

Progesterone Increases the Release of Brain-Derived Neurotrophic Factor from Glia via Progesterone Receptor Membrane Component 1 (Pgrmc1)-Dependent ERK5 Signaling

Progesterone (P4) is cytoprotective in various experimental models, but our understanding of the mechanisms involved is still incomplete. Our laboratory has implicated brain-derived neurotrophic factor (BDNF) signaling as an important mediator of P4s protective actions. We have shown that P4 increases the expression of BDNF, an effect mediated by the classical P4 receptor (PR), and that the protective effects of P4 were abolished using inhibitors of Trk receptor signaling. In an effort to extend our understanding of the interrelationship between P4 and BDNF signaling, we determined whether P4 influenced BDNF release and examined the role of the classical PR and a putative membrane PR, progesterone receptor membrane component-1 (Pgrmc1), as mediators of this response. Given recent data from our laboratory that supported the role of ERK5 in BDNF release, we also tested whether P4-induced BDNF release was mediated by ERK5. In this study, we found that P4 and the membrane-impermeable P4 (P4-BSA) both induced BDNF release from cultured C6 glial cells and primary astrocytes. Both these cells lack the classical nuclear/intracellular PR but express high levels of membrane-associated PR, including Pgrmc1. Using RNA interference-mediated knockdown of Pgrmc1 expression, we determined that P4-induced BDNF release was dependent on the expression of Pgrmc1, although pharmacological inhibition of the PR failed to alter the effects of P4. Furthermore, the BDNF release elicited by P4 was mediated by ERK5, and not ERK1/2. Collectively, our data describe that P4 elicits an increase in BDNF release from glia via a Pgrmc1-induced ERK5 signaling mechanism and identify Pgrmc1 as a potential therapeutic target for future hormone-based drug development for the treatment of such degenerative diseases as Alzheimers disease as well as other diseases wherein neurotrophin dysregulation is noted.

The N-terminus of presenilin-2 increases single channel activity of brain ryanodine receptors through direct protein-protein interaction.

Presenilin-1 (PS1) and presenilin-2 (PS2) form the catalytic core in gamma-secretase complexes and mutations in these proteins result in aberrant cleavage of amyloid precursor protein leading to accumulation of the beta-amyloid in the brain of familial Alzheimer Disease patients. PS2 possesses a hydrophilic cytoplasmic N-terminal domain (PS2 NTF1-87) dispensable for gamma-secretase activity with physiological functions yet to be determined. The effects of this soluble 87 amino acid fragment of mouse PS2 on single channel activity of mouse brain ryanodine receptors (RyR) were determined. PS2 NTF1-87 application to the cytoplasmic side of the RyR significantly increased single channel activity by favoring higher sublevel openings. The Ca(2+) activation and desensitization ranges for RyRs were unchanged. We demonstrate facilitation of RyR gating by PS2 NTF1-87, which might represent a general mechanism of RyR regulation by presenilins potentially prone to be affected by mutations or external stimuli contributing to the development of neurodegenerative diseases.

ERK1/2 and ERK5 have distinct roles in the regulation of brain-derived neurotrophic factor expression.

Neurotrophins play essential roles in the development, differentiation, and survival of neuronal and nonneuronal cells. Alterations in neurotrophin expression have been implicated in a variety of neurodegenerative disorders. Dysregulation of brain‐derived neurotrophic factor (BDNF) has been implicated in deficits of long‐term potentiation and cognition and may contribute to the development of Alzheimers disease (AD). In this study, we used complementary pharmacological and molecular approaches to evaluate the role of ERK1/2 and ERK5, two members of the MAPK pathway associated with neuroprotection, in regulating BDNF expression in C6 glial cells and primary astrocytes. Our data revealed that U0126, an inhibitor of both ERK5 and ERK1/2, increased the levels of BDNF mRNA, whereas the MEK1/2‐specific inhibitor PD184352 did not, suggesting that ERK5 exerts negative control over BDNF expression. This was supported by experiments in which RNAi‐mediated depletion of ERK5 led to an increase in BDNF. In contrast, transfection with constitutively active MEK5 resulted in an inhibition of BDNF expression, confirming the inhibitory role of ERK5 in the regulation of BDNF. Interestingly, transfection with the dominant active mutant of MEK1 (MEKR4F), the upstream activator of ERK1/2, resulted in a modest increase in BDNF levels. Collectively, our data suggest that ERK5 and ERK1/2 exert opposite effects on BDNF expression and support the hypothesis that an imbalance of these two signaling pathways may contribute to the pathology of diseases in which neurotrophin dysregulation is noted.

ERK5/KLF4 signaling as a common mediator of the neuroprotective effects of both nerve growth factor and hydrogen peroxide preconditioning

Oxidative stress has long been implicated in the pathogenesis of various neurodegenerative disorders such as Alzheimer’s disease and stroke. While high levels of oxidative stress are generally associated with cell death, a slight rise of reactive oxygen species (ROS) levels can be protective by “preconditioning” cells to develop a resistance against subsequent challenges. However, the mechanisms underlying such preconditioning (PC)-induced protection are still poorly understood. Previous studies have supported a role of ERK5 (mitogen-activated protein [MAP] kinase 5) in neuroprotection and ischemic tolerance in the hippocampus. In agreement with these findings, our data suggest that ERK5 mediates both hydrogen peroxide (H2O2)-induced PC as well as nerve growth factor (NGF)-induced neuroprotection. Activation of ERK5 partially rescued pheochromocytoma PC12 cells as well as primary hippocampal neurons from H2O2-caused death, while inhibition of ERK5 abolished NGF or PC-induced protection. These results implicate ERK5 signaling as a common downstream pathway for NGF and PC. Furthermore, both NGF and PC increased the expression of the transcription factor, KLF4, which can initiate an anti-apoptotic response in various cell types. Induction of KLF4 by NGF or PC was blocked by siERK5, suggesting that ERK5 is required in this process. siKLF4 can also attenuate NGF- or PC-induced neuroprotection. Overexpression of active MEK5 or KLF4 in H2O2-stressed cells increased Bcl-2/Bax ratio and the expression of NAIP (neuronal apoptosis inhibitory protein). Taken together, our data suggest that ERK5/KLF4 cascade is a common signaling pathway shared by at least two important mechanisms by which neurons can be protected from cell death.

Cell Models for the Study of Sex Steroid Hormone Neurobiology.

To date many aspects of neurons and glia biology remain elusive, due in part to the cellular and molecular complexity of the brain. In recent decades, cell models from different brain areas have been established and proven invaluable toward understanding this complexity. In the field of steroid hormone neurobiology, an important question is: what is the profile of steroid hormone receptor expression in these specific cell lines? Currently, a clear summary of such receptor profiling is lacking. For this reason, we summarized in this review the expression of estrogen, progesterone, and androgen receptors in several widely used cell lines (glial and neuronal) derived from the forebrain and midbrain, based on our own data and that from the literature. Such information will aid in the selection of specific cell lines used to test hypotheses related to the biology of estrogens, progestins, and/or androgens.

The unliganded long isoform of estrogen receptor beta stimulates brain ryanodine receptor single channel activity alongside with cytosolic Ca2

Ca2+ release from intracellular stores mediated by endoplasmic reticulum membrane ryanodine receptors (RyR) plays a key role in activating and synchronizing downstream Ca2+-dependent mechanisms, in different cells varying from apoptosis to nuclear transcription and development of defensive responses. Recently discovered, atypical “nongenomic” effects mediated by estrogen receptors (ER) include rapid Ca2+ release upon estrogen exposure in conditions implicitly suggesting involvement of RyRs. In the present study, we report various levels of colocalization between RyR type 2 (RyR2) and ER type β (ERβ) in the neuronal cell line HT-22, indicating a possible functional interaction. Electrophysiological analyses revealed a significant increase in single-channel ionic currents generated by mouse brain RyRs after application of the soluble monomer of the long form ERβ (ERβ 1). The effect was due to a strong increase in open probability of RyR higher open channel sublevels at cytosolic [Ca2+] concentrations of 100 nM, suggesting a synergistic action of ERβ1 and Ca2+ in RyR activation, and a potential contribution to Ca2+-induced Ca2+ release rather than to basal intracellular Ca2+ concentration level at rest. This RyR/ERβ interaction has potential effects on cellular physiology, including roles of shorter ERβ isoforms and modulation of the RyR/ERβ complexes by exogenous estrogens.