Wenming Li

Chaperone-mediated autophagy: machinery, regulation and biological consequences

Degradation of dysfunctional intracellular components in the lysosome system can occur through three different pathways, i.e., macroautophagy, microautophagy and chaperone-mediated autophagy (CMA). In this review, we focus on CMA, a type of autophagy distinct from the other two autophagic pathways owing to its selectivity, saturability and competitivity by which a subset of long-lived cytosolic soluble proteins are directly delivered into the lysosomal lumen via specific receptors. CMA participates in quality control to maintain normal cell functions by clearing “old” proteins and provides energy to cells under nutritional stress. Deregulation of CMA has recently been shown to underlie some diseases, especially neurodegenerative disorders for which the decline with age in the activity of CMA may become a major aggravating factor. Therefore, targeting aberrant alteration in CMA under pathological conditions could serve as a potential therapeutic strategy for treating related diseases.

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.

Cdk5: Mediator of neuronal development, death and the response to DNA damage

In the central nervous system, cyclin-dependent kinase 5 (Cdk5), an unusual member of the Cdk family, is implicated in the regulation of various physiological processes ranging from neuronal survival, migration and differentiation, to synaptogenesis, synaptic plasticity and neurotransmission. Dysregulation of this kinase has been demonstrated to play a critical role in the pathogenic process of neurodegenerative disorders. DNA damage is emerging as an important pathological component in various neurodegenerative conditions. In this review, we discuss the recent progress regarding the regulation and roles of Cdk5 under physiological conditions, and its dysregulation under pathological conditions, especially in neuronal death mediated by DNA damage.

Chaperone-mediated autophagy and neurodegeneration: connections, mechanisms, and therapeutic implications

Lysosomes degrade dysfunctional intracellular components via three pathways: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA). Unlike the other two, CMA degrades cytosolic proteins with a recognized KFERQ-like motif in lysosomes and is important for cellular homeostasis. CMA activity declines with age and is altered in neurodegenerative diseases. Its impairment leads to the accumulation of aggregated proteins, some of which may be directly tied to the pathogenic processes of neurodegenerative diseases. Its induction may accelerate the clearance of pathogenic proteins and promote cell survival, representing a potential therapeutic approach for the treatment of neurodegenerative diseases. In this review, we summarize the current findings on how CMA is involved in neurodegenerative diseases, especially in Parkinson’s disease.

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.

Phosphorylation of LAMP2A by p38 MAPK couples ER stress to chaperone-mediated autophagy

Endoplasmic reticulum (ER) and lysosomes coordinate a network of key cellular processes including unfolded protein response (UPR) and autophagy in response to stress. How ER stress is signaled to lysosomes remains elusive. Here we find that ER disturbance activates chaperone-mediated autophagy (CMA). ER stressors lead to a PERK-dependent activation and recruitment of MKK4 to lysosomes, activating p38 MAPK at lysosomes. Lysosomal p38 MAPK directly phosphorylates the CMA receptor LAMP2A at T211 and T213, which causes its membrane accumulation and active conformational change, activating CMA. Loss of ER stress-induced CMA activation sensitizes cells to ER stress-induced death. Neurotoxins associated with Parkinson’s disease fully engages ER-p38 MAPK–CMA pathway in the mouse brain and uncoupling it results in a greater loss of SNc dopaminergic neurons. This work identifies the coupling of ER and CMA as a critical regulatory axis fundamental for physiological and pathological stress response.The endoplasmic reticulum (ER) and lysosome are central to cellular stress responses, but it is unclear how ER stress is signaled to lysosomes. Here the authors show that ER stress activates chaperone-mediated autophagy (CMA) via direct phosphorylation of the CMA receptor LAMP2A by the lysosomal p38 MAPK.

Calpain-mediated Degradation of Myocyte Enhancer Factor 2D Contributes to Excitotoxicity by Activation of Extrasynaptic N-Methyl-d-aspartate Receptors

Background: Myocyte enhancer factor 2D (MEF2D) plays important roles in neuronal survival. Results: Activation of extrasynaptic NMDAR causes calpain-mediated cleavage of MEF2D. Conclusion: Extrasynaptic NMDA receptors-induced excitotoxicity is in part mediated by degradation of MEF2D. Significance: Learning how MEF2D is dysregulated by excessive NMDA-activated calpain may provide a therapeutic strategy by inhibiting MEF2D degradation for excitotoxicity-associated diseases. Synaptic and extrasynaptic NMDA receptors (NMDARs) appear to play opposite roles in neuronal survival and death. Here we report the new findings on the dysregulation of survival factor, myocyte enhancer factor 2D (MEF2D), by extrasynaptic NMDARs. Excitotoxicity led to the NMDAR-dependent degradation of MEF2D protein and inhibition of its transactivation activity in mature cortical neurons. The activation of extrasynaptic NMDARs alone was sufficient for degradation of MEF2D. Calpain directly cleaved MEF2D in vitro and blocking this protease activity greatly attenuated NMDAR signaled degradation of MEF2D in neurons. Consistently, inhibition of calpain protected cortical neurons from NMDA-induced excitotoxicity. Furthermore, knockdown of MEF2D sensitized neurons to NMDA-induced excitotoxicity, which was not protected by calpain inhibition. Collectively, these findings suggest that dysregulation of MEF2D by calpain may mediate excitotoxicity via an extrasynaptic NMDAR-dependent manner.

Chaperone-mediated autophagy: Advances from bench to bedside

Protein homeostasis or proteostasis is critical for proper cellular function and survival. It relies on the balance between protein synthesis and degradation. Lysosomes play an important role in degrading and recycling intracellular components via autophagy. Among the three types of lysosome-based autophagy pathways, chaperone-mediated autophagy (CMA) selectively degrades cellular proteins with KFERQ-like motif by unique machinery. During the past several years, significant advances have been made in our understanding of how CMA itself is modulated and what physiological and pathological processes it may be involved in. One particularly exciting discovery is how other cellular stress organelles such as ER signal to CMA. As more proteins are identified as CMA substrates, CMA function has been associated with an increasing number of important cellular processes, organelles, and diseases, including neurodegenerative diseases. Here we will summarize the recent advances in CMA biology, highlight ER stress-induced CMA, and discuss the role of CMA in diseases.