Mitochondrial dysfunction induced by knockdown of mortalin is rescued by Parkin

Mutations in the parkin gene are the most common cause of autosomal recessive Parkinson’s disease (PD). As an E3-ubiquitin ligase, Parkin is associated with mitochondrial dynamics and mitophagy. Mortalin, a molecular chaperone, is located primarily in mitochondria, where it functions to maintain mitochondrial homeostasis and antagonize oxidative stress injury. A reduced expression level of mortalin has been observed in the affected brain regions of PD patients. Mortalin also interacts with a variety of PD-related proteins and plays an indispensible role in helping native protein refolding and importing proteins into the mitochondrial matrix. Thus, the main aims of the present study were to investigate mitochondrial dysfunction induced by knockdown of mortalin and to test whether Parkin overexpression could rescue this effect. We found that lentivirus-mediated knockdown of mortalin in HeLa cells resulted in a collapse of mitochondrial membrane potential, an abnormal accumulation of reactive oxygen species and apparent alterations in mitochondrial morphology under H₂O₂-induced stress conditions. Remarkably, Parkin overexpression rescued these mitochondrial abnormalities. In HeLa cells expressing Parkin, co-immunoprecipitation of endogenous mortalin and wild-type Parkin was detected when they were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP). In conclusion, we indicate that the relatively decreased mortalin expression level and its impaired interaction with Parkin could affect its roles in mitochondrial function.     

Mitochondrial proteolytic stress induced by loss of mortalin function is rescued by Parkin and PINK1

The mitochondrial chaperone mortalin was implicated in Parkinson''s disease (PD) because of its reduced levels in the brains of PD patients and disease-associated rare genetic variants that failed to rescue impaired mitochondrial integrity in cellular knockdown models. To uncover the molecular mechanisms underlying mortalin-related neurodegeneration, we dissected the cellular surveillance mechanisms related to mitochondrial quality control, defined the effects of reduced mortalin function at the molecular and cellular levels and investigated the functional interaction of mortalin with Parkin and PINK1, two PD-related proteins involved in mitochondrial homeostasis. We found that reduced mortalin function leads to: (1) activation of the mitochondrial unfolded protein response (UPR(mt)), (2) increased susceptibility towards intramitochondrial proteolytic stress, (3) increased autophagic degradation of fragmented mitochondria and (4) reduced mitochondrial mass in human cells in vitro and ex vivo. These alterations caused increased vulnerability toward apoptotic cell death. Proteotoxic perturbations induced by either partial loss of mortalin or chemical induction were rescued by complementation with native mortalin, but not disease-associated mortalin variants, and were independent of the integrity of autophagic pathways. However, Parkin and PINK1 rescued loss of mortalin phenotypes via increased lysosomal-mediated mitochondrial clearance and required intact autophagic machinery. Our results on loss of mortalin function reveal a direct link between impaired mitochondrial proteostasis, UPR(mt) and PD and show that effective removal of dysfunctional mitochondria via either genetic (PINK1 and Parkin overexpression) or pharmacological intervention (rapamycin) may compensate mitochondrial phenotypes.     

Proteomic analysis of gamma-butyrolactone-treated mouse thalamus reveals dysregulated proteins upon absence seizure

Absence seizure has been of interest because the symptom is related to sensory processing. However, the mechanism that causes the disease is not understood yet. To better understand the molecular mechanism related to the disease progress at protein level, we performed proteomic studies using the thalamus of mice for which absence seizure was induced by gamma-butyrolactone (GBL). Differential proteome expression between GBL-treated mice and control mice was examined by fluorescence 2D difference gel electrophoresis (DIGE) at three different time points (5, 10, and 30 min) after GBL-administration. We identified 16 proteins differentially expressed by >1.4-fold at any of the three time points. All proteins besides the serine protease inhibitor EIA were down-regulated in absence seizure-induced mice. The down-regulated proteins can be classified into five groups by their biological functions: cytoskeleton rearrangement, neuroprotection, neurotransmitter secretion, calcium binding, and metabolism. The maximum level of change was reached by 10 min after GBL-treatment, with the expression level returning back to the original at 30 min when mice were awakened from absence seizure thereby demonstrating the proteomic response is reversible. Our results suggest that absence seizures are associated with restricted functional sets of proteins, whose down-regulation may interfere with general function of neuronal cells.     

Down-regulation of Mortalin Exacerbates Aβ-mediated Mitochondrial Fragmentation and Dysfunction

Mitochondrial dynamics greatly influence the biogenesis and morphology of mitochondria. Mitochondria are particularly important in neurons, which have a high demand for energy. Therefore, mitochondrial dysfunction is strongly associated with neurodegenerative diseases. Until now various post-translational modifications for mitochondrial dynamic proteins and several regulatory proteins have explained complex mitochondrial dynamics. However, the precise mechanism that coordinates these complex processes remains unclear. To further understand the regulatory machinery of mitochondrial dynamics, we screened a mitochondrial siRNA library and identified mortalin as a potential regulatory protein. Both genetic and chemical inhibition of mortalin strongly induced mitochondrial fragmentation and synergistically increased Aβ-mediated cytotoxicity as well as mitochondrial dysfunction. Importantly we determined that the expression of mortalin in Alzheimer disease (AD) patients and in the triple transgenic-AD mouse model was considerably decreased. In contrast, overexpression of mortalin significantly suppressed Aβ-mediated mitochondrial fragmentation and cell death. Taken together, our results suggest that down-regulation of mortalin may potentiate Aβ-mediated mitochondrial fragmentation and dysfunction in AD.     

Enhanced phosphorylation of a 65 kDa protein is associated with rapid induction of stress proteins in 9L rat brain tumor cells

Induction of heat-shock proteins and glucose-regulated proteins in 9L rat brain tumor cells can be differentially elicited by sodium arsenite, cadmium chloride, zinc chloride, copper sulfate, sodium fluoride, and L-azetidine-2-carboxylic acid. The kinds of stress protein induced by the above chemicals varied considerably, mainly determined by the nature and the concentration of the chemicals, as well as the treatment protocols. In addition, at the concentrations where stress proteins can be induced, the above chemicals were able to suppress general protein synthesis and were cytotoxic. Enhanced phosphorylation of a protein with an apparent molecular weight of 65 kDa was detected during the induction of stress proteins except in azetidine treatments during which uptake of phosphate by the cells was impaired after prolonged incubation. The phosphate moiety on the 65 kDa phosphoprotein appeared to be alkaline-stable and two-dimensional gel electrophoresis revealed that the phosphoprotein resolved into four isoforms with isoelectric points ranging from 5.1 to 5.6. Enhanced phosphorylation of the same protein was also detected in heat-shocked and withangulatin A-treated 9L cells in which stress proteins were induced. It is suggested that this phosphoprotein may be a common target for heat stress response-stimulated phosphorylation and important in the further metabolic responses of the cell to stress. © 1993 Wiley-Liss, Inc.     

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.     

The alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptor trafficking regulator "stargazin" is related to the claudin family of proteins by Its ability to mediate cell-cell adhesion

Mutations in the Cacng2 gene encoding the neuronal transmembrane protein stargazin result in recessively inherited epilepsy and ataxia in "stargazer" mice. Functional studies suggest a dual role for stargazin, both as a modulatory gamma subunit for voltage-dependent calcium channels and as a regulator of post-synaptic membrane targeting for alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors. Co-immunoprecipitation experiments demonstrate that stargazin can bind proteins of either complex in vivo, but it remains unclear whether it can associate with both complexes simultaneously. Cacng2 is one of eight closely related genes (Cacng1-8) encoding proteins with four transmembrane segments, cytoplasmic termini, and molecular masses between 25 and 44 kDa. This group of Cacng genes constitutes only one branch of a larger monophyletic assembly dominated by over 20 genes encoding proteins known as claudins. Claudins regulate cell adhesion and paracellular permeability as fundamental components of non-neuronal tight junctions. Because stargazin is structurally similar to claudins, we hypothesized that it might also have retained claudin-like functions inherited from a common ancestor. Here, we report that expression of stargazin in mouse L-fibroblasts results in cell aggregation comparable with that produced by claudins, and present evidence that the interaction is heterotypic and calcium dependent. The data suggest that the cell adhesion function of stargazin preceded its current role in neurons as a regulator of either voltage-dependent calcium channels or AMPA receptors. We speculate these complexes may have co-opted the established presence of stargazin at sites of close cell-cell contact to facilitate their own evolving intercellular signaling functions.     

Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo

The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.     

Reactivation of protein aggregates by mortalin and Tid1—the human mitochondrial Hsp70 chaperone system

The mitochondrial 70-kDa heat shock protein (mtHsp70), also known in humans as mortalin, is a central component of the mitochondrial protein import motor and plays a key role in the folding of matrix-localized mitochondrial proteins. MtHsp70 is assisted by a member of the 40-kDa heat shock protein co-chaperone family named Tid1 and a nucleotide exchange factor. Whereas, yeast mtHsp70 has been extensively studied in the context of protein import in the mitochondria, and the bacterial 70-kDa heat shock protein was recently shown to act as an ATP-fuelled unfolding enzyme capable of detoxifying stably misfolded polypeptides into harmless natively refolded proteins, little is known about the molecular functions of the human mortalin in protein homeostasis. Here, we developed novel and efficient purification protocols for mortalin and the two spliced versions of Tid1, Tid1-S, and Tid1-L and showed that mortalin can mediate the in vitro ATP-dependent reactivation of stable-preformed heat-denatured model aggregates, with the assistance of Mge1 and either Tid1-L or Tid1-S co-chaperones or yeast Mdj1. Thus, in addition of being a central component of the protein import machinery, human mortalin together with Tid1, may serve as a protein disaggregating machine which, for lack of Hsp100/ClpB disaggregating co-chaperones, may carry alone the scavenging of toxic protein aggregates in stressed, diseased, or aging human mitochondria.     

Identification and Functional Characterization of Nuclear Mortalin in Human Carcinogenesis

The Hsp70 family protein mortalin is an essential chaperone that is frequently enriched in cancer cells and exists in various subcellular sites, including the mitochondrion, plasma membrane, endoplasmic reticulum, and cytosol. Although the molecular mechanisms underlying its multiple subcellular localizations are not yet clear, their functional significance has been revealed by several studies. In this study, we examined the nuclear fractions of human cells and found that the malignantly transformed cells have more mortalin than the normal cells. We then generated a mortalin mutant that lacked a mitochondrial targeting signal peptide. It was largely localized in the nucleus, and, hence, is called nuclear mortalin (mot-N). Functional characterization of mot-N revealed that it efficiently protects cancer cells against endogenous and exogenous oxidative stress. Furthermore, compared with the full-length mortalin overexpressing cancer cells, mot-N derivatives showed increased malignant properties, including higher proliferation rate, colony forming efficacy, motility, and tumor forming capacity both in in vitro and in vivo assays. We demonstrate that mot-N promotes carcinogenesis and cancer cell metastasis by inactivation of tumor suppressor protein p53 functions and by interaction and functional activation of telomerase and heterogeneous ribonucleoprotein K (hnRNP-K) proteins.     

Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteomics

Apoptosis contributes to cell death after cerebral ischaemia. A quantitative proteomics approach has been employed to define alterations in protein levels in apoptosis induced with staurosporine (STS). Human neuroblastoma derived SH-SY5Y cells were treated with STS (500 nM for 6 h) to induce apoptosis. Quantitative 2-DE was used to determine the changing protein levels with MALDI-TOF MS identification of proteins. Of the 154 proteins analysed, 13 proteins were significantly altered as a result of the apoptotic stimulus; ten of the proteins showed an increase in level with STS and were identified as heat shock cognate 71 (Hsc71), two isoforms of heat shock protein 70 (Hsp70), glucose regulated protein 78 (GRP78), F-actin capping protein, stress-induced phosphoprotein 1, chromatin assembly factor 1 (CAF-1), protein disulphide isomerase A3 (PDI A3) precursor, transitional ER ATPase and actin interacting protein 1 (AIP 1). Three proteins which displayed significant decrease in levels with STS were identified as tubulin, vimentin and glucose regulated protein 94 (GRP94). The functional roles and subcellular locations of these proteins collectively indicate that STS-induced apoptosis provokes induces an unfolded protein response involving molecular chaperones, cochaperones and structural proteins indicative of ER stress.     

Proteomic analysis of hearts from frataxin knockout mice: marked rearrangement of energy metabolism, a response to cellular stress and altered expression of proteins involved in cell structure, motility and metabolism

A frequent cause of death in Friedreich's ataxia patients is cardiomyopathy, but the molecular alterations underlying this condition are unknown. We performed 2-DE to characterize the changes in protein expression of hearts using the muscle creatine kinase frataxin conditional knockout (KO) mouse. Pronounced changes in protein expression profile were observed in 9 week-old KO mice with severe cardiomyopathy. In contrast, only several proteins showed altered expression in asymptomatic 4 week-old KO mice. In hearts from frataxin KO mice, components of the iron-dependent complex-I and -II of the mitochondrial electron transport chain and enzymes involved in ATP homeostasis (creatine kinase, adenylate kinase) displayed decreased expression. Interestingly, the KO hearts exhibited increased expression of enzymes involved in the citric acid cycle, catabolism of branched-chain amino acids, ketone body utilization and pyruvate decarboxylation. This constitutes evidence of metabolic compensation due to decreased expression of electron transport proteins. There was also pronounced up-regulation of proteins involved in stress protection, such as a variety of chaperones, as well as altered expression of proteins involved in cellular structure, motility and general metabolism. This is the first report of the molecular changes at the protein level which could be involved in the cardiomyopathy of the frataxin KO mouse.     

Arsenite‐induced changes in hepatic protein abundance in cynomolgus monkeys (Macaca fascicularis)

Arsenic is an environmental pollutant, and its liver toxicity has long been recognized. The effect of arsenic on liver protein expression was analyzed using a proteomic approach in monkeys. Monkeys were orally administered sodium arsenite (SA) for 28 days. As shown by 2D‐PAGE in combination with MS, the expression levels of 16 proteins were quantitatively changed in SA‐treated monkey livers compared to control‐treated monkey livers. Specifically, the levels of two proteins, mortalin and tubulin beta chain, were increased, and 14 were decreased, including plastin‐3, cystathionine‐beta‐synthase, selenium‐binding protein 1, annexin A6, alpha‐enolase, phosphoenolpyruvate carboxykinase‐M, erlin‐2, and arginase‐1. In view of their functional roles, differential expression of these proteins may contribute to arsenic‐induced liver toxicity, including cell death and carcinogenesis. Among the 16 identified proteins, four were selected for validation by Western blot and immunohistochemistry. Additional Western blot analyses indicated arsenic‐induced dysregulation of oxidative stress related, genotoxicity‐related, and glucose metabolism related proteins in livers from SA‐treated animals. Many changes in the abundance of toxicity‐related proteins were also demonstrated in SA‐treated human hepatoma cells. These data on the arsenic‐induced regulation of proteins with critical roles may help elucidate the specific mechanisms underlying arsenic‐induced liver toxicity.     

Circulating mortalin autoantibody—a new serological marker of liver cirrhosis

Mortalin is a stress chaperone belonging to the Hsp70 family of proteins. Frequently enriched in cancers, it is a multifunctional protein and regulates cell proliferation, apoptosis, mitochondrial functions, and the activity of tumor suppressor protein p53. In the present study, we investigated circulating mortalin and its autoantibody in normal, cirrhosis, and cancerous liver. We found that although mortalin is enriched in liver cancer cells and tumors, it is not detected in the serum of either the liver cirrhosis or cancer patients. In contrast, mortalin autoantibody was detected in patients’ sera and showed significant correlation with the occurrence of cirrhosis. It is suggested as a potential noninvasive marker for liver cirrhosis.     

Three-dimensional structure of an AMPA receptor without associated stargazin/TARP proteins

Most excitatory synaptic transmissions in the central nervous system are mediated by the neurotransmitter glutamate. Binding of glutamate released from the presynaptic membrane causes glutamate receptors in the postsynaptic membrane to open, which results in a transient depolarization of the postsynaptic membrane. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) subtype of glutamate receptors is responsible for the majority of excitatory postsynaptic currents and is thought to play a central role in synaptic plasticity. Because modulation of glutamate receptors is believed to be involved in the basic mechanism underlying information storage in the brain, the molecular architecture of native AMPA receptors (AMPA-Rs) is of great interest. Previously, we have shown that AMPA-Rs purified from the brain are tightly associated with members of the stargazin/TARP (transmembrane AMPA receptor regulatory protein) family of membrane proteins [Nakagawa et al., Nature 433 (2005), pp. 545-549]. Here, we present a three-dimensional (3D) density map of the hetero-tetrameric AMPA-R without associated stargazin/TARP proteins as determined by cryo-negative stain single-particle electron microscopy. In the absence of stargazin/TARP proteins, the density representing the transmembrane region of the AMPA-R particles is substantially smaller, corroborating our previous analysis that was based solely on projection images.     

Stress chaperone mortalin regulates human melanogenesis

In order to identify the cellular factors involved in human melanogenesis, we carried out shRNA-mediated loss-of-function screening in conjunction with induction of melanogenesis by 1-oleoyl-2-acetyl-glycerol (OAG) in human melanoma cells using biochemical and visual assays. Gene targets of the shRNAs (that caused loss of OAG-induced melanogenesis) and their pathways, as determined by bioinformatics, revealed involvement of proteins that regulate cell stress response, mitochondrial functions, proliferation, and apoptosis. We demonstrate, for the first time, that the mitochondrial stress chaperone mortalin is crucial for melanogenesis. Upregulation of mortalin was closely associated with melanogenesis in in vitro cell-based assays and clinical samples of keloids with hyperpigmentation. Furthermore, its knockdown resulted in compromised melanogenesis. The data proposed mortalin as an important protein that may be targeted to manipulate pigmentation for cosmetic and related disease therapeutics.     

Proteomic identification of a stress protein, mortalin/mthsp70/GRP75: relevance to Parkinson disease

Functional impairment of mitochondria and proteasomes and increased oxidative damage comprise the main pathological phenotypes of Parkinson disease (PD). Using an unbiased quantitative proteomic approach, we compared nigral mitochondrial proteins of PD patients with those from age-matched controls. 119 of 842 identified proteins displayed significant differences in their relative abundance (increase/decrease) between the two groups. We confirmed that one of these, mortalin (mthsp70/GRP75, a mitochondrial stress protein), is substantially decreased in PD brains as well as in a cellular model of PD. In addition, nine candidate mortalin-binding partners were identified as potential mediators of PD pathology. Manipulations of mortalin level in dopaminergic neurons resulted in significant changes in sensitivity to PD phenotypes via pathways involving mitochondrial and proteasomal function as well as oxidative stress.     

Hippocampal electrical activity and gamma-aminobutyrate metabolism in brain tissue following administration of homocysteine

Abstract: The concentration of γ-aminobutyrate (GABA) and the activity of glutamate decarboxylase and GABA-transaminase were measured in extracts of mouse brain before the onset and during the course of generalized seizures induced by systemic administration of homocysteine thiolactone. The results indicate that whole brain GABA metabolism is unaffected by subconvulsive and convulsive doses of homocysteine at all stages of the generalized seizure. Electroencephalographic monitoring of rat brain electrical activity via hippocampal electrode implantation allowed the course of homocysteine-induced seizures to be followed and afforded a means of quantifying such seizures.