Hua She

Regulation of Neuronal Survival Factor MEF2D by Chaperone-Mediated Autophagy

Chaperone-mediated autophagy controls the degradation of selective cytosolic proteins and may protect neurons against degeneration. In a neuronal cell line, we found that chaperone-mediated autophagy regulated the activity of myocyte enhancer factor 2D (MEF2D), a transcription factor required for neuronal survival. MEF2D was observed to continuously shuttle to the cytoplasm, interact with the chaperone Hsc70, and undergo degradation. Inhibition of chaperone-mediated autophagy caused accumulation of inactive MEF2D in the cytoplasm. MEF2D levels were increased in the brains of α-synuclein transgenic mice and patients with Parkinsons disease. Wild-type α-synuclein and a Parkinsons disease–associated mutant disrupted the MEF2D-Hsc70 binding and led to neuronal death. Thus, chaperone-mediated autophagy modulates the neuronal survival machinery, and dysregulation of this pathway is associated with Parkinsons disease.

Alternative Mitochondrial Electron Transfer as a Novel Strategy for Neuroprotection

Neuroprotective strategies, including free radical scavengers, ion channel modulators, and anti-inflammatory agents, have been extensively explored in the last 2 decades for the treatment of neurological diseases. Unfortunately, none of the neuroprotectants has been proved effective in clinical trails. In the current study, we demonstrated that methylene blue (MB) functions as an alternative electron carrier, which accepts electrons from NADH and transfers them to cytochrome c and bypasses complex I/III blockage. A de novo synthesized MB derivative, with the redox center disabled by N-acetylation, had no effect on mitochondrial complex activities. MB increases cellular oxygen consumption rates and reduces anaerobic glycolysis in cultured neuronal cells. MB is protective against various insults in vitro at low nanomolar concentrations. Our data indicate that MB has a unique mechanism and is fundamentally different from traditional antioxidants. We examined the effects of MB in two animal models of neurological diseases. MB dramatically attenuates behavioral, neurochemical, and neuropathological impairment in a Parkinson disease model. Rotenone caused severe dopamine depletion in the striatum, which was almost completely rescued by MB. MB rescued the effects of rotenone on mitochondrial complex I-III inhibition and free radical overproduction. Rotenone induced a severe loss of nigral dopaminergic neurons, which was dramatically attenuated by MB. In addition, MB significantly reduced cerebral ischemia reperfusion damage in a transient focal cerebral ischemia model. The present study indicates that rerouting mitochondrial electron transfer by MB or similar molecules provides a novel strategy for neuroprotection against both chronic and acute neurological diseases involving mitochondrial dysfunction.

Direct regulation of complex I by mitochondrial MEF2D is disrupted in a mouse model of Parkinson disease and in human patients

The transcription factors in the myocyte enhancer factor 2 (MEF2) family play important roles in cell survival by regulating nuclear gene expression. Here, we report that MEF2D is present in rodent neuronal mitochondria, where it can regulate the expression of a gene encoded within mitochondrial DNA (mtDNA). Immunocytochemical, immunoelectron microscopic, and biochemical analyses of rodent neuronal cells showed that a portion of MEF2D was targeted to mitochondria via an N-terminal motif and the chaperone protein mitochondrial heat shock protein 70 (mtHsp70). MEF2D bound to a MEF2 consensus site in the region of the mtDNA that contained the gene NADH dehydrogenase 6 (ND6), which encodes an essential component of the complex I enzyme of the oxidative phosphorylation system; MEF2D binding induced ND6 transcription. Blocking MEF2D function specifically in mitochondria decreased complex I activity, increased cellular H(2)O(2) level, reduced ATP production, and sensitized neurons to stress-induced death. Toxins known to affect complex I preferentially disrupted MEF2D function in a mouse model of Parkinson disease (PD). In addition, mitochondrial MEF2D and ND6 levels were decreased in postmortem brain samples of patients with PD compared with age-matched controls. Thus, direct regulation of complex I by mitochondrial MEF2D underlies its neuroprotective effects, and dysregulation of this pathway may contribute to PD.

Phosphorylation of Neuronal Survival Factor MEF2D by Glycogen Synthase Kinase 3β in Neuronal Apoptosis

Glycogen synthase kinase 3β (GSK3β) has been identified to play important roles in neuronal death. Evidence from both in vitro and in vivo studies indicates that increased GSK3β activity contributes to neurodegeneration and to the pathogenesis of Alzheimer disease. But the molecular mechanisms that underlie GSK3β-mediated neurotoxicity remain poorly understood. We reported here that myocyte enhancer factor 2D (MEF2D), a nuclear transcription factor known to promote neuronal survival, is directly phosphorylated by GSK3β. Our data showed that phosphorylation of MEF2D by GSK3β at three specific residues in its transactivation domain inhibits MEF2D transcriptional activity. Withdrawal of neuronal activity in cerebellar granule neurons activated GSK3β in the nucleus, leading to GSK3β-dependent inhibition of MEF2 function. This inhibition contributed to GSK3β-mediated neuronal toxicity. Overexpression of MEF2D mutant that is resistant to GSK3β inhibition protected cerebellar granule neurons from either GSK3β activation- or neuronal activity deprivation-induced toxicity. These results identify survival factor MEF2D as a novel downstream effector targeted by GSK3β and define a molecular link between activation of GSK3β and neuronal survival machinery which may underlie in part GSK3β-mediated neurotoxicity.

Oxidation of Survival Factor MEF2D in Neuronal Death and Parkinson's Disease

AIMS Dysfunction of myocyte enhancer factor 2D (MEF2D), a key survival protein and transcription factor, underlies the pathogenic loss of dopaminergic (DA) neurons in Parkinsons disease (PD). Both genetic factors and neurotoxins associated with PD impair MEF2D function in vitro and in animal models of PD. We investigated whether distinct stress conditions target MEF2D via converging mechanisms. RESULTS We showed that exposure of a DA neuronal cell line to 6-hyroxydopamine (6-OHDA), which causes PD in animals models, led to direct oxidative modifications of MEF2D. Oxidized MEF2D bound to heat-shock cognate protein 70 kDa, the key regulator for chaperone-mediated autophagy (CMA), at a higher affinity. Oxidative stress also increased the level of lysosomal-associated membrane protein 2A (LAMP2A), the rate-limiting receptor for CMA substrate flux, and stimulated CMA activity. These changes resulted in accelerated degradation of MEF2D. Importantly, 6-OHDA induced MEF2D oxidation and increased LAMP2A in the substantia nigra pars compacta region of the mouse brain. Consistently, the levels of oxidized MEF2D were much higher in postmortem PD brains compared with the controls. Functionally, reducing the levels of either MEF2D or LAMP2A exacerbated 6-OHDA-induced death of the DA neuronal cell line. Expression of an MEF2D mutant that is resistant to oxidative modification protected cells from 6-OHDA-induced death. INNOVATION This study showed that oxidization of survival protein MEF2D is one of the pathogenic mechanisms involved in oxidative stress-induced DA neuronal death. CONCLUSION Oxidation of survival factor MEF2D inhibits its function, underlies oxidative stress-induced neurotoxicity, and may be a part of the PD pathogenic process.

Stress Induces p38 MAPK-Mediated Phosphorylation and Inhibition of Drosha-Dependent Cell Survival

MicroRNAs (miRNAs) regulate the translational potential of their mRNA targets and control many cellular processes. The key step in canonical miRNA biogenesis is the cleavage of the primary transcripts by the nuclear RNase III enzyme Drosha. Emerging evidence suggests that the miRNA biogenic cascade is tightly controlled. However, little is known whether Drosha is regulated. Here, we show that Drosha is targeted by stress. Under stress, p38 MAPK directly phosphorylates Drosha at its N terminus. This reduces its interaction with DiGeorge syndrome critical region gene 8 and promotes its nuclear export and degradation by calpain. This regulatory mechanism mediates stress-induced inhibition of Drosha function. Reduction of Drosha sensitizes cells to stress and increases death. In contrast, increase in Drosha attenuates stress-induced death. These findings reveal a critical regulatory mechanism by which stress engages p38 MAPK pathway to destabilize Drosha and inhibit Drosha-mediated cellular survival.

Activation of Transcription Factor MEF2D by Bis(3)-cognitin Protects Dopaminergic Neurons and Ameliorates Parkinsonian Motor Defects

Background: Dysregulation of myocyte enhancer factor 2D (MEF2D) is implicated in the pathogenic process of Parkinson disease (PD). Results: A small molecule bis(3)-cognitin activates MEF2D and protects Parkinsonian impairments. Conclusion: Bis(3)-cognitin provides protection of dopaminergic neurons in a model of PD by reversing MEF2D dysfunction. Significance: Activation of MEF2D by pharmacological approach has the potential to be a novel therapeutic for PD. Parkinson disease (PD) is characterized by the selective demise of dopaminergic (DA) neurons in the substantial nigra pars compacta. Dysregulation of transcriptional factor myocyte enhancer factor 2D (MEF2D) has been implicated in the pathogenic process in in vivo and in vitro models of PD. Here, we identified a small molecule bis(3)-cognitin (B3C) as a potent activator of MEF2D. We showed that B3C attenuated the toxic effects of neurotoxin 1-methyl-4-phenylpyridinium (MPP+) by activating MEF2D via multiple mechanisms. B3C significantly reduced MPP+-induced oxidative stress and potentiated Akt to down-regulate the activity of MEF2 inhibitor glycogen synthase kinase 3β (GSK3β) in a DA neuronal cell line SN4741. Furthermore, B3C effectively rescued MEF2D from MPP+-induced decline in both nucleic and mitochondrial compartments. B3C offered SN4741 cells potent protection against MPP+-induced apoptosis via MEF2D. Interestingly, B3C also protected SN4741 cells from wild type or mutant A53T α-synuclein-induced cytotoxicity. Using the in vivo PD model of C57BL/6 mice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP), we showed that B3C maintained redox homeostasis, promoted Akt function activity, and restored MEF2D level in midbrain neurons. Moreover, B3C greatly prevented the loss of tyrosine hydroxylase signal in substantial nigra pars compacta DA neurons and ameliorated behavioral impairments in mice treated with MPTP. Collectedly, our studies identified B3C as a potent neuroprotective agent whose effectiveness relies on its ability to effectively up-regulate MEF2D in DA neurons against toxic stress in models of PD in vitro and in vivo.

Hypertonic saline reduces lipopolysaccharide-induced mouse brain edema through inhibiting aquaporin 4 expression.

IntroductionThree percent sodium chloride (NaCl) treatment has been shown to reduce brain edema and inhibited brain aquaporin 4 (AQP4) expression in bacterial meningitis induced by Escherichia coli. Lipopolysaccharide (LPS) is the main pathogenic component of E. coli. We aimed to explore the effect of 3% NaCl in mouse brain edema induced by LPS, as well as to elucidate the potential mechanisms of action.MethodsThree percent NaCl was used to treat cerebral edema induced by LPS in mice in vivo. Brain water content, IL-1β, TNFα, immunoglobulin G (IgG), AQP4 mRNA and protein were measured in brain tissues. IL-1β, 3% NaCl and calphostin C (a specific inhibitor of protein kinase C) were used to treat the primary astrocytes in vitro. AQP4 mRNA and protein were measured in astrocytes. Differences in various groups were determined by one-way analysis of variance.ResultsThree percent NaCl attenuated the increase of brain water content, IL-1β, TNFα, IgG, AQP4 mRNA and protein in brain tissues induced by LPS. Three percent NaCl inhibited the increase of AQP4 mRNA and protein in astrocytes induced by IL-1β in vitro. Calphostin C blocked the decrease of AQP4 mRNA and protein in astrocytes induced by 3% NaCl in vitro.ConclusionsOsmotherapy with 3% NaCl ameliorated LPS-induced cerebral edema in vivo. In addition to its osmotic force, 3% NaCl exerted anti-edema effects possibly through down-regulating the expression of proinflammatory cytokines (IL-1β and TNFα) and inhibiting the expression of AQP4 induced by proinflammatory cytokines. Three percent NaCl attenuated the expression of AQP4 through activation of protein kinase C in astrocytes.INTRODUCTION Three percent sodium chloride (NaCl) treatment has been shown to reduce brain edema and inhibited brain aquaporin 4 (AQP4) expression in bacterial meningitis induced by Escherichia coli. Lipopolysaccharide (LPS) is the main pathogenic component of E. coli. We aimed to explore the effect of 3% NaCl in mouse brain edema induced by LPS, as well as to elucidate the potential mechanisms of action. METHODS Three percent NaCl was used to treat cerebral edema induced by LPS in mice in vivo. Brain water content, IL-1β, TNFα, immunoglobulin G (IgG), AQP4 mRNA and protein were measured in brain tissues. IL-1β, 3% NaCl and calphostin C (a specific inhibitor of protein kinase C) were used to treat the primary astrocytes in vitro. AQP4 mRNA and protein were measured in astrocytes. Differences in various groups were determined by one-way analysis of variance. RESULTS Three percent NaCl attenuated the increase of brain water content, IL-1β, TNFα, IgG, AQP4 mRNA and protein in brain tissues induced by LPS. Three percent NaCl inhibited the increase of AQP4 mRNA and protein in astrocytes induced by IL-1β in vitro. Calphostin C blocked the decrease of AQP4 mRNA and protein in astrocytes induced by 3% NaCl in vitro. CONCLUSIONS Osmotherapy with 3% NaCl ameliorated LPS-induced cerebral edema in vivo. In addition to its osmotic force, 3% NaCl exerted anti-edema effects possibly through down-regulating the expression of proinflammatory cytokines (IL-1β and TNFα) and inhibiting the expression of AQP4 induced by proinflammatory cytokines. Three percent NaCl attenuated the expression of AQP4 through activation of protein kinase C in astrocytes.

Neurotoxin‐induced selective ubiquitination and regulation of MEF2A isoform in neuronal stress response

J. Neurochem. (2012) 122, 1203–1210.

Modulation of Neuronal Survival Factor MEF2 by Kinases in Parkinson's Disease.

Parkinson’s disease (PD) is the second most common neurodegenerative disorder due to selective death of neurons in the substantia nigra pars compacta. The cause of cell death remains largely unknown. Myocyte enhancer factor 2 (MEF2) is a group of transcriptional factors required to regulate neuronal development, synaptic plasticity, as well as survival. Recent studies show that MEF2 functions are regulated in multiple subcellular organelles and suggest that dysregulation of MEF2 plays essential roles in the pathogenesis of PD. Many kinases associated with transcription, translation, protein misfolding, autophagy, and cellular energy homeostasis are involved in the neurodegenerative process. Following the first demonstration that mitogen-activated protein kinase p38 (p38 MAPK) directly phosphorylates and activates MEF2 to promote neuronal survival, several other kinase regulators of MEF2s have been identified. These include protein kinase A and extracellular signal regulated kinase 5 as positive MEF2 regulators, and cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase 3β as negative regulators in response to diverse toxic signals relevant to PD. It is clear that MEF2 has emerged as a key point where survival and death signals converge to exert their regulatory effects, and dysregulation of MEF2 function in multiple subcellular organelles may underlie PD pathogenesis. Moreover, several other kinases such as leucine-rich repeat kinase 2 and PTEN-induced putative kinase 1 (PINK1) are of particular interest due to their potential interaction with MEF2.