Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

alpha-Synuclein affects the MAPK pathway and accelerates cell death

Insoluble alpha-synuclein accumulates in Parkinson's disease, diffuse Lewy body disease, and multiple system atrophy. However, the relationship between its accumulation and pathogenesis is still unclear. Recently, we reported that overexpression of alpha-synuclein affects Elk-1 phosphorylation in cultured cells, which is mainly performed by mitogen-activated protein kinases (MAPKs). We further examined the relationship between MAPK signaling and the effects of alpha-synuclein expression on ecdysone-inducible neuro2a cell lines and found that cells expressing alpha-synuclein had less phosphorylated MAPKs. Moreover, they showed significant cell death when the concentration of serum in the culture medium was reduced. Under normal serum conditions, the addition of the MAPK inhibitor U0126 also caused cell death in alpha-synuclein-expressing cells. Transfection of constitutively active MEK-1 resulted in MAPK phosphorylation in alpha-synuclein-expressing cells and improved cell viability even under reduced serum conditions. Thus, we conclude that alpha-synuclein regulates the MAPK pathway by reducing the amount of available active MAPK. Our findings suggest a mechanism for pathogenesis and thus offer therapeutic insight into synucleinopathies.     

SUMO-1 interacts with mutant ataxin-1 and colocalizes to its aggregates in Purkinje cells of SCA1 transgenic mice

Spinocerebellar ataxia type 1 (SCA1) is one of several progressive neurodegenerative diseases caused by the expanded polyglutamine tract in ataxin-1, the SCA1 gene product. In SCA1 patients and transgenic mice, the affected neuronal cells contain a large ubiquitin-positive aggregate which is derived from the mutant ataxin-1. Small ubiquitin-like modifier-1 (SUMO-1) is one of the most intriguing ubiquitin-like modifiers being conjugated to target proteins and modulating a number of cellular pathways. Recent findings that the aggregates from several neurodegenerative diseases are SUMO-1-positive prompted us to examine the implication of SUMO-1 in SCA1 pathogenesis. In our yeast two-hybrid experiments using mutant ataxin-1 as bait, we identified a SUMO-1 protein that directly binds to ataxin-1 protein. Interestingly, we found that most of the mutant ataxin-1-derived aggregates were SUMO-1-positive both in Purkinje cells of SCA1 transgenic mice and in HeLa cells, but not wild-type ataxin-1 in HeLa cells. In addition, the aggregates in Purkinje cells of SCA1 transgenic mice were positive against both anti-SUMO-1 and anti-ubiquitin antibodies. These results show that the SUMO-1 protein interacts with mutant ataxin-1 and colocalizes with its aggregates which suggests the involvement of the SUMO-1 system in the pathogenesis of SCA1 disease.     

Interaction between domains of a plant NBS-LRR protein in disease resistance-related cell death

Many plant disease resistance (R) genes encode proteins predicted to have an N-terminal coiled-coil (CC) domain, a central nucleotide-binding site (NBS) domain and a C-terminal leucine-rich repeat (LRR) domain. These CC-NBS-LRR proteins recognize specific pathogen-derived products and initiate a resistance response that often includes a type of cell death known as the hypersensitive response (HR). Co-expression of the potato CC-NBS-LRR protein Rx and its elicitor, the PVX coat protein (CP), results in a rapid HR. Surprisingly, co-expression of the LRR and CC-NBS as separate domains also resulted in a CP-dependent HR. Likewise, the CC domain complemented a version of Rx lacking this domain (NBS- LRR). Correspondingly, the LRR domain interacted physically in planta with the CC-NBS, as did CC with NBS-LRR. Both interactions were disrupted in the presence of CP. However, the interaction between CC and NBS-LRR was dependent on a wild-type P-loop motif, whereas the interaction between CC-NBS and LRR was not. We propose that activation of Rx entails sequential disruption of at least two intramolecular interactions.     

Full-length expanded ataxin-3 enhances mitochondrial-mediated cell death and decreases Bcl-2 expression in human neuroblastoma cells

Machado-Joseph disease (MJD) is an autosomal dominant spinocerebellar degeneration characterized by a wide range of clinical manifestations. An unstable CAG trinucleotide repeat expansion in MJD gene on long arm of chromosome 14 has been identified as the pathologic mutation of MJD and apoptosis was previously shown to be responsible for the neuronal cell death of the disease. In this study, we utilized human neuronal SK-N-SH cells stably transfected with HA-tagged full-length MJD with 78 polyglutamine repeats to examine the effects of polyglutamine expansion on neuronal cell survival in the early stage of disease. Various pro-apoptotic agents were used to assess the tolerance of the mutant cells and to compare the differences between cells with and without mutant ataxin-3. Concentration- and time-dependent experiments showed that the increase in staurosporine-induced cell death was more pronounced and accelerated in cells containing expanded ataxin-3 via MTS assays. Interestingly, under basal conditions, Western blot and immunocytochemical analyses showed a significant decrease of Bcl-2 protein expression and an increase of cytochrome c in cells containing expanded ataxin-3 when compared with those of the parental cells. The same reduction of Bcl-2 was further confirmed in fibroblast cells with mutant ataxin-3. In addition, exogenous expression of Bcl-2 desensitized SK-N-SH-MJD78 cells to poly-Q toxicity. These results indicated that mitochondrial-mediated cell death plays a role in the pathogenesis of MJD. In our cellular model, full-length expanded ataxin-3 that leads to neurodegenerative disorders significantly impaired the expression of Bcl-2 protein, which may be, at least in part, responsible for the weak tolerance to polyglutamine toxicity at the early stage of disease and ultimately resulted in an increase of stress-induced cell death upon apoptotic stress.     

Interaction between the keratin cytoskeleton and eEF1Bgamma affects protein synthesis in epithelial cells

Eukaryotic elongation factor-1 (eEF1) is essential for peptide-chain elongation during translation. We report that its gamma subunit (eEF1Bgamma) specifically binds, and bundles, keratin intermediate filaments. Disrupting this interaction depresses translation by approximately 20% and selectively increases 80S ribosomes in epithelial cells, an outcome recapitulated by RNA interference-mediated silencing of eEF1Bgamma. These findings extend the emerging relationship between keratin proteins and the translational machinery.