Dorfin localizes to Lewy bodies and ubiquitylates synphilin-1

Parkinson's disease (PD) is a neurodegenerative disease characterized by loss of nigra dopaminergic neurons. Lewy bodies (LBs) are a characteristic neuronal inclusion in PD brains. In this study, we report that Dorfin, a RING finger-type ubiquityl ligase for mutant superoxide dismutase-1, was localized with ubiquitin in LBs. Recently, synphilin-1 was identified to associate with alpha-synuclein and to be a major component of LBs. We found that overexpression of synphilin-1 in cultured cells led to the formation of large juxtanuclear inclusions, but showed no cytotoxicity. Dorfin colocalized in these large inclusions with ubiquitin and proteasomal components. In contrast to full-length synphilin-1, overexpression of the central portion of synphilin-1, including ankyrin-like repeats, a coiled-coil domain, and an ATP/GTP-binding domain, predominantly led to the formation of small punctate aggregates scattered throughout the cytoplasm and showed cytotoxic effects. Dorfin and ubiquitin did not localize in these small aggregates. Overexpression of the N or C terminus of synphilin-1 did not lead to the formation of any aggregates. Dorfin physically bound and ubiquitylated synphilin-1 through its central portion, but did not ubiquitylate wild-type or mutant alpha-synuclein. These results suggest that the central domain of synphilin-1 has an important role in the formation of aggregates and cytotoxicity and that Dorfin may be involved in the pathogenic process of PD and LB formation by ubiquitylation of synphilin-1.     

X-Linked inhibitor of apoptosis protein is involved in mutant SOD1-mediated neuronal degeneration

Mutations in the superoxide dismutase 1 (SOD1) gene cause the degeneration of motor neurons in familial amyotrophic lateral sclerosis (FALS). An apoptotic process including caspase-1 and -3 has been shown to participate in the pathogenesis of FALS transgenic (Tg) mouse model. Here we report that IAP proteins, potent inhibitors of apoptosis, are involved in the FALS Tg mouse pathologic process. The levels of X-linked inhibitor of apoptosis protein (XIAP) mRNA and protein were significantly decreased in the spinal cord of symptomatic G93A-SOD1 Tg mice compared with littermates. In contrast, the levels of cIAP-1 mRNA and protein were increased in symptomatic G93A-SOD1 Tg mice, whereas the levels of cIAP-2 mRNA and protein were unchanged. In situ hybridization showed that the expression of XIAP was remarkably reduced in the motor neurons of Tg mice, and the expression of cIAP-1 was strongly increased in the reactive astrocytes of Tg mice. Overexpression of XIAP markedly inhibited the cell death and caspase-3 activity in the neuro2a cells expressing mutant SOD1. Deletional mutant analysis revealed that the N-terminal domain of XIAP, the BIR1-2 domains, was essential for this inhibitory activity. These results suggest that XIAP plays a role in the apoptotic mechanism in the progression of disease in mutant SOD1 Tg mice and holds therapeutic possibilities for FALS.     

Microglial overexpression of fALS-linked mutant SOD1 induces SOD1 processing impairment,activation and neurotoxicity and is counteracted by the autophagy inducer trehalose

Amyotrophic lateral sclerosis (ALS) is a fatal motor neuron disease. Mutations in the gene encoding copper/zinc superoxide dismutase-1 (SOD1) are responsible for most familiar cases, but the role of mutant SOD1 protein dysfunction in non-cell autonomous neurodegeneration, especially in relation to microglial activation, is still unclear. Here, we focused our study on microglial cells, which release SOD1 also through exosomes. We observed that in rat primary microglia the overexpression of the most-common SOD1 mutations linked to fALS (G93A and A4V) leads to SOD1 intracellular accumulation, which correlates to autophagy dysfunction and microglial activation. In primary contact co-cultures, fALS mutant SOD1 overexpression by microglial cells appears to be neurotoxic by itself. Treatment with the autophagy-inducer trehalose reduced mutant SOD1 accumulation in microglial cells, decreased microglial activation and abrogated neurotoxicity in the co-culture model. These data suggest that i) the alteration of the autophagic pathway due to mutant SOD1 overexpression is involved in microglial activation and neurotoxicity; ii) the induction of autophagy with trehalose reduces microglial SOD1 accumulation through proteasome degradation and activation, leading to neuroprotection. Our results provide a novel contribution towards better understanding key cellular mechanisms in non-cell autonomous ALS neurodegeneration.     

Dorfin prevents cell death by reducing mitochondrial localizing mutant superoxide dismutase 1 in a neuronal cell model of familial amyotrophic lateral sclerosis

Abstract Dorfin is a RING-finger type ubiquitin ligase for mutant superoxide dismutase 1 (SOD1) that enhances its degradation. Mutant SOD1s cause familial amyotrophic lateral sclerosis (FALS) through the gain of unelucidated toxic properties. We previously showed that the accumulation of mutant SOD1 in the mitochondria triggered the release of cytochrome c, followed by the activation of the caspase cascade and induction of neuronal cell death. In the present study, therefore, we investigated whether Dorfin can modulate the level of mutant SOD1 in the mitochondria and subsequent caspase activation. We showed that Dorfin significantly reduced the amount of mutant SOD1 in the mitochondria, the release of cytochrome c and the activation of the following caspase cascade, thereby preventing eventual neuronal cell death in a neuronal cell model of FALS. These results suggest that reducing the accumulation of mutant SOD1 in the mitochondria may be a new therapeutic strategy for mutant SOD1-associated FALS, and that Dorfin may play a significant role in this.     

Alsin, the product of ALS2 gene, suppresses SOD1 mutant neurotoxicity through RhoGEF domain by interacting with SOD1 mutants

Mutation of the ALS2 gene encoding alsin is linked to the onset of autosomal recessive motor neuron diseases, including juvenile-onset amyotrophic lateral sclerosis (ALS). Alsin long form (LF) belongs to the family of the guanine nucleotide exchanging factor (GEF) for small GTPases. Expression of alsin LF, but not alsin short form, protected motor neuronal cells from toxicity induced by mutants of the Cu/Zn-superoxide dismutase (SOD1) gene, which cause autosomal dominant ALS. In contrast, expression of alsin did not suppress neurotoxicity by other neurodegenerative insults such as Alzheimer's disease-related genes. Deletion analysis of alsin LF demonstrated that the RhoGEF domain is essential for alsin-mediated neuroprotection. Furthermore, we found that alsin LF bound to SOD1 mutants, but not to wtSOD1, via the RhoGEF domain. Such functional and physical interaction between two ALS-related genes will become a promising clue to clarify the pathogenesis of ALS and other motor neuron diseases.     

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.     

SCF TrCP1 activates and ubiquitylates TAp63gamma

p63 is a member of the p53 tumor suppressor family that is critical for epithelial differentiation and also has an important role in cancer progression. Currently, the molecular mechanisms governing regulation of p63 function remain largely unclear. This study identifies a unique E3 ubiquitin ligase for p63, SCF(betaTrCP1). SCF(betaTrCP1) is able to bind p63gamma isoforms, with a higher affinity for the TAp63gamma isoform. Strikingly, co-expression of TAp63gamma and betaTrCP1 leads to the stabilization of TAp63gamma. This stabilization of TAp63gamma leads to up-regulation of p21 at the mRNA and protein level by increased binding of TAp63gamma at the p21 promoter. The up-regulation of p21 causes a subsequent increase in G(1) phase cell cycle arrest. Last, SCF(betaTrCP1) is able to ubiquitylate TAp63gamma, and this ubiquitylation, as well as the increased activity of TAp63gamma, is ablated with the expression of a ubiquitin-deficient mutant of betaTrCP1 (DeltaFbetaTrCP1). Therefore, our study reveals that SCF(betaTrCP1) is an E3 ligase that activates p63 through ubiquitylation.     

Altered macroautophagy in the spinal cord of SOD1 mutant mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by selective loss of motor neurons (MNs). About 20% familial cases of ALS (fALS) carried the Cu, Zn-superoxide dismutase (SOD1) gene mutation, which plays a crucial role in the pathogenesis of fALS. There is evidence suggesting that macroautophagy can degrade mutated SOD1 in vitro. To investigate whether the mutant SOD1 can induce macroautophagy in vivo, we examined the LC3 processing in spinal cord and the activation status of macroautophagy in MNs of SOD1(G93A) transgenic mice at different stages. Our data demonstrated that autophagy was activated in spinal cord of SOD1(G93A) mice indicating a possible role of macroautophagy in the pathogenesis of ALS.     

Familial amyotrophic lateral sclerosis-linked mutant SOD1 aberrantly interacts with tubulin

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause 20-25% of familial amyotrophic lateral sclerosis (ALS). Mutant SOD1 causes motor neuron degeneration through toxic gain-of-function(s). However, the direct molecular targets of mutant SOD1, underlying its toxicity, are not fully understood. In this study, we found that α/β-tubulin is one of the major mutant SOD1-interacting proteins, but that wild-type SOD1 does not interact with it. The interaction between tubulin and mutant SOD1 was detected in the spinal cords of mutant G93A SOD1 transgenic mice before the onset of symptoms. Tubulin interacted with amino acid residues 1-23 and 116-153 of SOD1. Overexpression of mutant SOD1 resulted in the accumulation of tubulin in detergent-insoluble fractions. In a cell-free system, mutant SOD1 modulated tubulin polymerization, while wild-type SOD1 did not. Since tightly regulated microtubule dynamics is essential for neurons to remain viable, α/β-tubulin could be an important direct target of mutant SOD1.     

Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.     

Role of mitochondria in mutant SOD1 linked amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with an adult onset characterized by loss of both upper and lower motor neurons. In ~ 10% of cases, patients developed ALS with an apparent genetic linkage (familial ALS or fALS). Approximately 20% of fALS displays mutations in the SOD1 gene encoding superoxide dismutase 1. There are many proposed cellular and molecular mechanisms among which, mitochondrial dysfunctions occur early, prior to symptoms occurrence. In this review, we modeled the effect of mutant SOD1 protein via the formation of a toxic complex with Bcl2 on mitochondrial bioenergetics. Furthermore, we discuss that the shutdown of ATP permeation through mitochondrial outer membrane could lead to both respiration inhibition and temporary mitochondrial hyperpolarization. Moreover, we reviewed mitochondrial calcium signaling, oxidative stress, fission and fusion, autophagy and apoptosis in mutant SOD1-linked ALS. Functional defects in mitochondria appear early before symptoms are manifested in ALS. Therefore, mitochondrial dysfunction is a promising therapeutic target in ALS. This article is part of a Special Issue entitled: Misfolded Proteins, Mitochondrial Dysfunction, and Neurodegenerative Diseases.     

Cholera toxin prevents Th1-mediated autoimmune disease by inducing immune deviation

Cholera toxin (CT), a major enterotoxin produced by Vibrio cholerae, is known for its properties as a mucosal adjuvant that promotes Th2 or mixed Th1 + Th2 responses. In this study, we explore the ability of CT to act as a systemic adjuvant to counteract the Th1 response leading to experimental autoimmune uveitis. We report that susceptible B10.RIII mice immunized with a uveitogenic regimen of the retinal Ag interphotoreceptor retinoid-binding protein could be protected from disease by a single systemic injection of as little as 2 micro g of CT at the time of immunization. The protected mice were not immunosuppressed, but rather displayed evidence of immune deviation. Subsequent adaptive responses to interphotoreceptor retinoid-binding protein showed evidence of Th2 enhancement, as indicated by reduced delayed-type hypersensitivity in the context of enhanced Ag-specific lymphocyte proliferation and IL-4 production. Ag-specific production of several other cytokines, including IFN-gamma, was not appreciably altered. The inhibitory effect of CT was dependent on the enzymatic A subunit of CT, because the cell-binding B subunit alone could not block disease development. Mice given CT displayed detectable IL-4 levels in their serum within hours of CT administration. This innate IL-4 production was critical for protection, as infusion of neutralizing Ab against IL-4 to mice, given a uveitogenic immunization and treated with CT, counteracted immune deviation and abrogated protection. Our data indicate that systemic administration of CT inhibits experimental autoimmune uveitis by skewing the response to the uveitogenic autoantigen to a nonpathogenic phenotype.     

Prevention of mutant SOD1 motoneuron degeneration by copper chelators in vitro

An animal model of familial amyotrophic lateral sclerosis (FALS) has been generated by overexpression of human CuZn superoxide dismutase (SOD1) containing a substitution of glycine to alanine at position 93 in transgenic G93A mice. The loss of motoneurons shown in this model has been attributed to a dominant gain of function of this mutated enzyme, which might be due to copper toxicity. This hypothesis was tested in purified spinal motoneurons cultures originating from G93A transgenic embryos. Spinal motoneurons were isolated from E13 embryos by several steps including density gradient centrifugation. The effect of copper chelators on survival and neurite growth of motoneurons was investigated. Survival of G93A motoneurons was decreased by 46% as compared to wild-type motoneurons. Moreover, G93A motoneurons showed reduced neurite outgrowth. Copper chelators strikingly increased viability of G93A motoneurons (by over 200%) but had no effect on wild-type cells. Presence of DDC in the medium increases the length of neurites from G93A motoneurons. The present results suggest the capacity of copper chelators to reduce the effect of reverse function of mutated SOD1 on motoneurons.     

Daf-2 signaling modifies mutant SOD1 toxicity in C. elegans

The DAF-2 Insulin/IGF-1 signaling (IIS) pathway is a strong modifier of Caenorhabditis elegans longevity and healthspan. As aging is the greatest risk factor for developing neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS), we were interested in determining if DAF-2 signaling modifies disease pathology in mutant superoxide dismutase 1 (SOD1) expressing C. elegans. Worms with pan-neuronal G85R SOD1 expression demonstrate significantly impaired locomotion as compared to WT SOD1 expressing controls and they develop insoluble SOD1 aggregates. Reductions in DAF-2 signaling, either through a hypomorphic allele or neuronally targeted RNAi, decreases the abundance of aggregated SOD1 and results in improved locomotion in a DAF-16 dependant manner. These results suggest that manipulation of the DAF-2 Insulin/IGF-1 signaling pathway may have therapeutic potential for the treatment of ALS.     

Lysyl-tRNA synthetase is a target for mutant SOD1 toxicity in mitochondria

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting the motor neurons. The majority of familial forms of ALS are caused by mutations in the Cu,Zn-superoxide dismutase (SOD1). In mutant SOD1 spinal cord motor neurons, mitochondria develop abnormal morphology, bioenergetic defects, and degeneration. However, the mechanisms of mitochondrial toxicity are still unclear. One possibility is that mutant SOD1 establishes aberrant interactions with nuclear-encoded mitochondrial proteins, which can interfere with their normal trafficking from the cytosol to mitochondria. Lysyl-tRNA synthetase (KARS), an enzyme required for protein translation that was shown to interact with mutant SOD1 in yeast, is a good candidate as a target for interaction with mutant SOD1 at the mitochondrion in mammals because of its dual cytosolic and mitochondrial localization. Here, we show that in mammalian cells mutant SOD1 interacts preferentially with the mitochondrial form of KARS (mitoKARS). KARS-SOD1 interactions occur also in the mitochondria of the nervous system in transgenic mice. In the presence of mutant SOD1, mitoKARS displays a high propensity to misfold and aggregate prior to its import into mitochondria, becoming a target for proteasome degradation. Impaired mitoKARS import correlates with decreased mitochondrial protein synthesis. Ultimately, the abnormal interactions between mutant SOD1 and mitoKARS result in mitochondrial morphological abnormalities and cell toxicity. mitoKARS is the first described member of a group of mitochondrial proteins whose interaction with mutant SOD1 contributes to mitochondrial dysfunction in ALS.     

Insoluble mutant SOD1 is partly oligoubiquitinated in amyotrophic lateral sclerosis mice

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS) through an unknown gain-of-function mechanism. Mutant SOD1 aggregation may be the toxic property. In fact, proteinaceous inclusions rich in mutant SOD1 have been found in tissues from the familial form of ALS patients and in mutant SOD1 animals, before disease onset. However, very little is known of the constituents and mechanism of formation of aggregates in ALS. We and others have shown that there is a progressive accumulation of detergent-insoluble mutant SOD1 in the spinal cord of G93A SOD1 mice. To investigate the mechanism of SOD1 aggregation, we characterized by proteome technologies SOD1 isoforms in a Triton X-100-insoluble fraction of spinal cord from G93A SOD1 mice at different stages of the disease. This showed that at symptomatic stages of the disease, part of the insoluble SOD1 is unambiguously mono- and oligoubiquitinated, in spinal cord and not in hippocampus, and that ubiquitin branches at Lys(48), the major signal for proteasome degradation. At presymptomatic stages of the disease, only insoluble unmodified SOD1 is recovered. Partial ubiquitination of SOD1-rich inclusions was also confirmed by immunohistochemical and electron microscopy analysis of lumbar spinal cord sections from symptomatic G93A SOD1 mice. On the basis of these results, we propose that ubiquitination occurs only after SOD1 aggregation and that oligoubiquitination may underline alternative mechanisms in disease pathogenesis.