VPS41, a protein involved in lysosomal trafficking, is protective in Caenorhabditis elegans and mammalian cellular models of Parkinson's disease.

VPS41 is a protein identified as a potential therapeutic target for Parkinson's disease (PD) as a result of a high-throughput RNAi screen in Caenorhabditis elegans. VPS41 has a plausible mechanistic link to the pathogenesis of PD, as in yeast it is known to participate in trafficking of proteins to the lysosomal system and several recent lines of evidence have pointed to the importance of lysosomal system dysfunction in the neurotoxicity of alpha-synuclein (alpha-syn). We found that expression of the human form of VPS41 (hVPS41) prevents dopamine (DA) neuron loss induced by alpha-syn overexpression and 6-hydroxydopamine (6-OHDA) neurotoxicity in C. elegans. In SH-SY5Y neuroblastoma cell lines stably transfected with hVPS41, we determined that presence of this protein conferred protection against the neurotoxins 6-OHDA and rotenone. Overexpression of hVPS41 did not alter the mitochondrial membrane depolarization induced by these neurotoxins. hVPS41 did, however, block downstream events in the apoptotic cascade including activation of caspase-9 and caspase-3, and PARP cleavage. We also observed that hVPS41 reduced the accumulation of insoluble high-molecular weight forms of alpha-syn in SH-SY5Y cells after treatment with rotenone. These data show that hVPS41 is protective against both alpha-syn and neurotoxic-mediated injury in invertebrate and cellular models of PD. These protective functions may be related to enhanced clearance of misfolded or aggregated protein, including alpha-syn. Our studies indicate that hVPS41 may be a useful target for developing therapeutic strategies for human PD.

Investigating bacterial sources of toxicity as an environmental contributor to dopaminergic neurodegeneration.

Parkinson disease (PD) involves progressive neurodegeneration, including loss of dopamine (DA) neurons from the substantia nigra. Select genes associated with rare familial forms of PD function in cellular pathways, such as the ubiquitin-proteasome system (UPS), involved in protein degradation. The misfolding and accumulation of proteins, such as alpha-synuclein, into inclusions termed Lewy Bodies represents a clinical hallmark of PD. Given the predominance of sporadic PD among patient populations, environmental toxins may induce the disease, although their nature is largely unknown. Thus, an unmet challenge surrounds the discovery of causal or contributory neurotoxic factors that could account for the prevalence of sporadic PD. Bacteria within the order Actinomycetales are renowned for their robust production of secondary metabolites and might represent unidentified sources of environmental exposures. Among these, the aerobic genera, Streptomyces, produce natural proteasome inhibitors that block protein degradation and may potentially damage DA neurons. Here we demonstrate that a metabolite produced by a common soil bacterium, S. venezuelae, caused DA neurodegeneration in the nematode, Caenorhabditis elegans, which increased as animals aged. This metabolite, which disrupts UPS function, caused gradual degeneration of all neuronal classes examined, however DA neurons were particularly vulnerable to exposure. The presence of DA exacerbated toxicity because neurodegeneration was attenuated in mutant nematodes depleted for tyrosine hydroxylase (TH), the rate-limiting enzyme in DA production. Strikingly, this factor caused dose-dependent death of human SH-SY5Y neuroblastoma cells, a dopaminergic line. Efforts to purify the toxic activity revealed that it is a highly stable, lipophilic, and chemically unique small molecule. Evidence of a robust neurotoxic factor that selectively impacts neuronal survival in a progressive yet moderate manner is consistent with the etiology of age-associated neurodegenerative diseases. Collectively, these data suggest the potential for exposures to the metabolites of specific common soil bacteria to possibly represent a contributory environmental component to PD.

The Differential Effects of R580A Mutation on Transamidation and GTP Binding Activity of Rat and Human Type 2 Transglutaminase.

Type 2 transglutaminase (TG2) is an acyltransferase, which also undergoes a GTP-binding/GTPase cycle, with guanine nucleotide and calcium binding reciprocally regulating its transamidation (TG) activity. TG2 is expressed ubiquitously throughout the human body and is the predominant neuronal transglutaminase. Given a postulated role for TG2 in a number of physiological and pathological processes including neurodegenerative diseases, it is of critical importance to understand how TG2 and its enzymatic activities are regulated in the cells. The various aspects of TG2 regulation are addressed by using rat and human TG2 proteins, however, despite their homologous structure, regulation of their enzymatic activities may differ, especially in the cellular context. Here, we evaluate the role of Arg580 in human TG2 and Arg579 in rat TG2 in modulating GTP binding and TG activities in vitro and in situ. We confirm the importance of Arg580 and Arg579 in TG2 for GTP binding as their mutation to Ala completely abolished GTP binding activity in both human (R580A) and rat TG2 (R579A). Next, we showed that in transfected human embryonic kidney (HEK) 293 cells, basal in situ TG activity of human R580A TG2 and rat R579A TG2 was significantly greater than their wild-type (WT) counterparts. However, TG activity of the mutants and WT TG2 became equivalent when the intracellular calcium concentration was maximally increased with maitotoxin. Also, in vitro TG activity assay revealed an intriguing difference between rat and human TG2; at a calcium concentration when their activities were maximum, the protein level of human R580A TG2 was lower than its WT counterpart, whereas rat R579A and WT TG2 protein levels were similar. Taken together, our study underscores an essential role of Arg580 in human TG2 and Arg579 in rat TG2 for their GTP binding ability and also describes for the first time that these amino acid residues differentially influence the TG activity of human or rat TG2 by calcium in vitro and in situ.

Type 2 transglutaminase differentially modulates striatal cell death in the presence of wild type or mutant huntingtin.

Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt), is characterized by extensive loss of striatal neurons. The dysregulation of type 2 transglutaminase (TG2) has been proposed to contribute to the pathogenesis in HD as TG2 is up-regulated in HD brain and knocking out TG2 in mouse models of HD ameliorates the disease process. To understand the role of TG2 in the pathogenesis of HD, immortalized striatal cells established from mice in which mutant htt with a polyglutamine stretch of 111 Gln had been knocked-in and wild type (WT) littermates, were stably transfected with human TG2 in a tetracycline inducible vector. Overexpression of TG2 in the WT striatal cells resulted in significantly greater cell death under basal conditions as well as in response to thapsigargin treatment, which causes increased intracellular calcium concentrations. Furthermore, in WT striatal cells TG2 overexpression potentiated mitochondrial membrane depolarization, intracellular reactive oxygen species production, and apoptotic cell death in response to thapsigargin. In contrast, in mutant striatal cells, TG2 overexpression did not increase cell death, nor did it potentiate thapsigargin-induced mitochondrial membrane depolarization or intracellular reactive oxygen species production. Instead, TG2 overexpression in mutant striatal cells attenuated the thapsigargin-activated apoptosis. When in situ transglutaminase activity was quantitatively analyzed in these cell lines, we found that in response to thapsigargin treatment TG2 was activated in WT, but not mutant striatal cells. These data suggest that mutant htt alters the activation of TG2 in response to certain stimuli and therefore differentially modulates how TG2 contributes to cell death processes.

Transglutaminase 2 in neurodegenerative disorders.

Type 2 transglutaminase (TG2) is a calcium-dependent acyltransferase which also undergoes a GTP-binding/GTPase cycle even though it lacks any obvious sequence similarity with canonical GTP-binding (G) proteins. As an enzyme which is responsible for the majority of transglutaminase (TG) activity in the brain, TG2 is likely to play a modulatory role in nervous system development and has regulatory effect on neuronal cell death as well. Most importantly, numerous studies have presented data demonstrating that dysregulation of TG2 may contribute to the pathogenesis of many neurodegenerative disorders, including Huntington's disease, Alzheimer's disease, Parkinson's disease and amyotrophic lateral sclerosis as well as nervous system injuries. Although TG2's involvement in these disease conditions is strongly suggested by various findings, such as the increase of TG2 mRNA expression, protein level and TG activity in the pathological process of these neurodegenerative disorders, as well as the therapeutic effect of TG2 genetic deletion in animal models of Huntington's disease, the precise mechanism underlying TG2's role remain unclear. TG2 was originally proposed to contribute to the pathogenesis of these diseases by facilitating the formation of insoluble protein aggregates, however recent findings clearly indicate that this is likely not the case. Nonetheless, there is data to suggest that TG2 may play a role in neurodegenerative processes by stabilizing toxic oligomers of the disease-relevant proteins, although further studies are needed to validate these initial in vitro findings.

Striatal cells from mutant huntingtin knock-in mice are selectively vulnerable to mitochondrial complex II inhibitor-induced cell death through a non-apoptotic pathway.

Extensive striatal neuronal loss occurs in Huntington's disease (HD), which is caused by an expanded polyglutamine tract in huntingtin (htt). Evidence suggests that mutant htt directly or indirectly compromises mitochondrial function, contributing to the neuronal loss. To determine the role of compromised mitochondrial function in the neuronal cell death in HD, immortalized striatal cells established from Hdh(Q7) (wild-type) and Hdh(Q111) (mutant) mouse knock-in embryos were treated with 3-nitropropionic acid (3-NP), a mitochondrial complex II toxin. 3-NP treatment caused significantly greater cell death in mutant striatal cells compared with wild-type cells. In contrast, the extent of cell death induced by rotenone, a complex I inhibitor, was similar in both cell lines. Although evidence of apoptosis was present in 3-NP-treated wild-type striatal cells, it was absent in 3-NP-treated mutant cells. 3-NP treatment caused a greater loss of mitochondrial membrane potential (deltapsim) in mutant striatal cells compared with wild-type cells. Cyclosporine A, an inhibitor of mitochondrial permeability transition pore (PTP), and ruthenium red, an inhibitor of the mitochondrial calcium uniporter, both rescued mutant striatal cells from 3-NP-induced cell death and prevented the loss of deltapsim. These data show that mutant htt specifically increases cell vulnerability to mitochondrial complex II inhibition and further switched the type of cell death induced by complex II inhibition from apoptosis to a non-apoptotic form, caused by mitochondrial membrane depolarization, probably initiated by mitochondrial calcium overload and subsequent PTP opening. These findings suggest that impaired mitochondrial complex II function in HD may contribute to non-apoptotic neuronal cell death.