Inhibition of retrograde transport modulates misfolded protein accumulation and clearance in motoneuron diseases

Motoneuron diseases, like spinal bulbar muscular atrophy (SBMA) and amyotrophic lateral sclerosis (ALS), are associated with proteins that because of gene mutation or peculiar structures, acquire aberrant (misfolded) conformations toxic to cells. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Here, using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrate that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome, rather than by autophagy and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner.     

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.     

p62 accumulates and enhances aggregate formation in model systems of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive neurode-generative disease characterized by motor neuron death. A hallmark of the disease is the appearance of protein aggregates in the affected motor neurons. We have found that p62, a protein implicated in protein aggregate formation, accumulated progressively in the G93A mouse spinal cord. The accumulation of p62 was in parallel to the increase of polyubiquitinated proteins and mutant SOD1 aggregates. Immunostaining studies showed that p62, ubiquitin, and mutant SOD1 co-localized in the protein aggregates in affected cells in G93A mouse spinal cord. The p62 protein selectively interacted with familial ALS mutants, but not WT SOD1. When p62 was co-expressed with SOD1 in NSC34 cells, it greatly enhanced the formation of aggregates of the ALS-linked SOD1 mutants, but not wild-type SOD1. Cell viability was measured in the presence and absence of overexpressed p62, and the results suggest that the large aggregates facilitated by p62 were not directly toxic to cells under the conditions in this study. Deletion of the ubiquitin-association (UBA) domain of p62 significantly decreased the p62-facilitated aggregate formation, but did not completely inhibit it. Further protein interaction experiments also showed that the truncated p62 with the UBA domain deletion remained capable of interacting with mutant SOD1. The findings of this study show that p62 plays a critical role in forming protein aggregates in familial ALS, likely by linking misfolded mutant SOD1 molecules and other cellular proteins together.     

S-nitrosylated GAPDH mediates neuronal apoptosis induced by amyotrophic lateral sclerosis-associated mutant SOD1G93A

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with the selective loss of motor neurons in the brain, brain stem, and spinal cord. A number of the mutants of the human gene for superoxide dismutase 1 (SOD1) have been shown to cause familial ALS as a result of gain-of-function toxicity by an unknown mechanism. In this study, we show that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a critical mediator of the apoptotic cell death signaling cascade induced by the ALS-associated G93A mutant of human SOD1 [SOD1(G93A)]. We observed that SOD1(G93A) induces S-nitrosylation of GAPDH and the subsequent binding of GAPDH and Siah1 in NSC34 motor neuron-like cells. Furthermore, SOD1(G93A) promoted nuclear translocation of S-nitrosylated GAPDH in the cells. In addition, SOD1(G93A)-induced apoptotic cell death was inhibited by deprenyl, a chemical inhibitor of GAPDH S-nitrosylation, in NSC34 cells. Taken together, our findings suggest that S-nitrosylation of GAPDH plays a critical role in SOD1(G93A)-induced neuronal apoptosis.     

IFNγ triggers a LIGHT-dependent selective death of motoneurons contributing to the non-cell-autonomous effects of mutant SOD1

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that primarily affects motoneurons in the brain and spinal cord. Dominant mutations in superoxide dismutase-1 (SOD1) cause a familial form of ALS. Mutant SOD1-damaged glial cells contribute to ALS pathogenesis by releasing neurotoxic factors, but the mechanistic basis of the motoneuron-specific elimination is poorly understood. Here, we describe a motoneuron-selective death pathway triggered by activation of lymphotoxin-β receptor (LT-βR) by LIGHT, and operating by a novel signaling scheme. We show that astrocytes expressing mutant SOD1 mediate the selective death of motoneurons through the proinflammatory cytokine interferon-γ (IFNγ), which activates the LIGHT-LT-βR death pathway. The expression of LIGHT and LT-βR by motoneurons in vivo correlates with the preferential expression of IFNγ by motoneurons and astrocytes at disease onset and symptomatic stage in ALS mice. Importantly, the genetic ablation of Light in an ALS mouse model retards progression, but not onset, of the disease and increases lifespan. We propose that IFNγ contributes to a cross-talk between motoneurons and astrocytes causing the selective loss of some motoneurons following activation of the LIGHT-induced death pathway.     

Transgenic mouse models of amyotrophic lateral sclerosis

The discovery of missense mutations in the gene coding for the Cu/Zn superoxide dismutase 1 (SOD1) in subsets of familial cases was rapidly followed by the generation of transgenic mice expressing various forms of SOD1 mutants. The mice overexpressing high levels of mutant SOD1 mRNAs do develop motor neuron disease but unraveling the mechanisms of pathogenesis has been very challenging. Studies with mouse lines suggest that the toxicity of mutant SOD1 is unrelated to copper-mediated catalysis but rather to propensity of a subfraction of mutant SOD1 proteins to form misfolded protein species and aggregates. However, the mechanism of toxicity of SOD1 mutants remains to be elucidated. Involvement of cytoskeletal components in ALS pathogenesis is supported by several mouse models of motor neuron disease with neurofilament abnormalities and with genetic defects in microtubule-based transport. Here, we describe how transgenic mouse models have been used for understanding pathogenic pathways of motor neuron disease and for pre-clinical drug testing.     

Degradation of amyotrophic lateral sclerosis-linked mutant Cu,Zn-superoxide dismutase proteins by macroautophagy and the proteasome

Mutations in the Cu,Zn-superoxide dismutase (SOD1) gene cause approximately 20% of familial cases of amyotrophic lateral sclerosis (fALS). Accumulating evidence indicates that a gain of toxic function of mutant SOD1 proteins is the cause of the disease. It has also been shown that the ubiquitin-proteasome pathway plays a role in the clearance and toxicity of mutant SOD1. In this study, we investigated the degradation pathways of wild-type and mutant SOD1 in neuronal and nonneuronal cells. We provide here the first evidence that wild-type and mutant SOD1 are degraded by macroautophagy as well as by the proteasome. Based on experiments with inhibitors of these degradation pathways, the contribution of macroautophagy to mutant SOD1 clearance is comparable with that of the proteasome pathway. Using assays that measure cell viability and cell death, we observed that under conditions where expression of mutant SOD1 alone does not induce toxicity, macroautophagy inhibition induced mutant SOD1-mediated cell death, indicating that macroautophagy reduces the toxicity of mutant SOD1 proteins. We therefore propose that both macroautophagy and the proteasome are important for the reduction of mutant SOD1-mediated neurotoxicity in fALS. Inhibition of macroautophagy also increased SOD1 levels in detergent-soluble and -insoluble fractions, suggesting that both detergent-soluble and -insoluble SOD1 are degraded by macroautophagy. These findings may provide further insights into the mechanisms of pathogenesis of fALS.     

Rilmenidine promotes MTOR-independent autophagy in the mutant SOD1 mouse model of amyotrophic lateral sclerosis without slowing disease progression

Macroautophagy/autophagy is the main intracellular catabolic pathway in neurons that eliminates misfolded proteins, aggregates and damaged organelles associated with ageing and neurodegeneration. Autophagy is regulated by both MTOR-dependent and -independent pathways. There is increasing evidence that autophagy is compromised in neurodegenerative disorders, which may contribute to cytoplasmic sequestration of aggregation-prone and toxic proteins in neurons. Genetic or pharmacological modulation of autophagy to promote clearance of misfolded proteins may be a promising therapeutic avenue for these disorders. Here, we demonstrate robust autophagy induction in motor neuronal cells expressing SOD1 or TARDBP/TDP-43 mutants linked to amyotrophic lateral sclerosis (ALS). Treatment of these cells with rilmenidine, an anti-hypertensive agent and imidazoline-1 receptor agonist that induces autophagy, promoted autophagic clearance of mutant SOD1 and efficient mitophagy. Rilmenidine administration to mutant SOD1^G93A mice upregulated autophagy and mitophagy in spinal cord, leading to reduced soluble mutant SOD1 levels. Importantly, rilmenidine increased autophagosome abundance in motor neurons of SOD1^G93A mice, suggesting a direct action on target cells. Despite robust induction of autophagy in vivo, rilmenidine worsened motor neuron degeneration and symptom progression in SOD1^G93A mice. These effects were associated with increased accumulation and aggregation of insoluble and misfolded SOD1 species outside the autophagy pathway, and severe mitochondrial depletion in motor neurons of rilmenidine-treated mice. These findings suggest that rilmenidine treatment may drive disease progression and neurodegeneration in this mouse model due to excessive mitophagy, implying that alternative strategies to beneficially stimulate autophagy are warranted in ALS.     

State of the art and the dark side of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a disorder that involves the degeneration of motor neurons, muscle atrophy, and paralysis. In a few familiar forms of ALS, mutations in the superoxide dismutase-1 (SOD1) gene have been held responsible for the degeneration of motor neurons. Nevertheless, after the discovery of the SOD1 mutations no consensus has emerged as to which cells, tissues and pathways are primarily implicated in the pathogenic events that lead to ALS. Ubiquitous overexpression of mutant SOD1 in transgenic animals recapitulates the pathological features of ALS. However, the toxicity of mutant SOD1 is not necessarily limited to the central nervous system. Views about ALS pathogenesis are now enriched by the recent discovery of mutations in a pair of DNA/RNA-binding proteins called TDP-43 and FUS/TLS as causes of familial and sporadic forms of ALS. Although the steps that lead to the pathological state are well defined, several fundamental issues are still controversial: are the motor neurons the first direct targets of ALS; and what is the contribution of non-neuronal cells, if any, to the pathogenesis of ALS? The state of the art of ALS pathogenesis and the open questions are discussed in this review.     

Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome

Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.     

Effects of Cellular Pathway Disturbances on Misfolded Superoxide Dismutase-1 in Fibroblasts Derived from ALS Patients

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.     

Expression of the protein chaperone, clusterin, in spinal cord cells constitutively and following cellular stress, and upregulation by treatment with Hsp90 inhibitor

Clusterin, a protein chaperone found at high levels in physiological fluids, is expressed in nervous tissue and upregulated in several neurological diseases. To assess relevance to amyotrophic lateral sclerosis (ALS) and other motor neuron disorders, clusterin expression was evaluated using long-term dissociated cultures of murine spinal cord and SOD1^G93A transgenic mice, a model of familial ALS. Motor neurons and astrocytes constitutively expressed nuclear and cytoplasmic forms of clusterin, and secreted clusterin accumulated in culture media. Although clusterin can be stress inducible, heat shock failed to increase levels in these neural cell compartments despite robust upregulation of stress-inducible Hsp70 (HspA1) in non-neuronal cells. In common with HSPs, clusterin was upregulated by treatment with the Hsp90 inhibitor, geldanamycin, and thus could contribute to the neuroprotection previously identified for such compounds in disease models. Clusterin expression was not altered in cultured motor neurons expressing SOD1^G93A by gene transfer or in presymptomatic SOD1^G93A transgenic mice; however, clusterin immunolabeling was weakly increased in lumbar spinal cord of overtly symptomatic mice. More striking, mutant SOD1 inclusions, a pathological hallmark, were strongly labeled by anti-clusterin. Since secreted, as well as intracellular, mutant SOD1 contributes to toxicity, the extracellular chaperoning property of clusterin could be important for folding and clearance of SOD1 and other misfolded proteins in the extracellular space. Evaluation of chaperone-based therapies should include evaluation of clusterin as well as HSPs, using experimental models that replicate the control mechanisms operant in the cells and tissue of interest.     

A role of small heat shock protein B8 (HspB8) in the autophagic removal of misfolded proteins responsible for neurodegenerative diseases

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motorneurons. As with other age-dependent neurodegenerative disorders, ALS is linked to the presence of misfolded proteins that may perturb several intracellular mechanisms and trigger neurotoxicity. Misfolded proteins aggregate intracellularly generating insoluble inclusions that are a key neuropathological hallmark of ALS. Proteins involved in the intracellular degradative systems, signalling pathways and the human TAR DNA-binding protein TDP-43 are major components of these inclusions. While their role and cytotoxicity are still largely debated, aggregates represent a powerful marker to follow protein misfolding in the neurodegenerative processes. Using in vitro and in vivo models of mutant SOD1 associated familial ALS (fALS), we and other groups demonstrated that protein misfolding perturbs one of the major intracellular degradative pathways, the ubiquitin proteasome system, giving rise to a vicious cycle that leads to the further deposit of insoluble proteins and finally to the formation of inclusions. The aberrant response to mutated SOD1 thus leads to the activation of the cascade of events ultimately responsible for cell death. Hence, our idea is that, by assisting protein folding, we might reduce protein aggregation, restore a fully functional proteasome activity and/or other cascades of events triggered by the mutant proteins responsible for motorneuron death in ALS. This could be obtained by stimulating mutant protein turnover, using an alternative degradative pathway that could clear mutant SOD1, namely autophagy.     

D-serine is a key determinant of glutamate toxicity in amyotrophic lateral sclerosis

Excitotoxicity has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS). More recently, glial involvement has been shown to be essential for ALS-related motoneuronal death. Here, we identified an N-methyl-D-aspartate (NMDA) receptor co-agonist, D-serine (D-Ser), as a glia-derived enhancer of glutamate (Glu) toxicity to ALS motoneurons. Cell death assay indicated that primary spinal cord neurons from ALS mice were more vulnerable to NMDA toxicity than those from control mice, in a D-Ser-dependent manner. Levels of D-Ser and its producing enzyme, serine racemase, in spinal cords of ALS mice were progressively elevated, dominantly in glia, with disease progression. In vitro, expression of serine racemase was induced not only by an extracellular pro-inflammatory factor, but also by transiently expressed G93A-superoxide dismutase1 in microglial cells. Furthermore, increases of D-Ser levels were also observed in spinal cords of both familial and sporadic ALS patients. Collectively, Glu toxicity enhanced by D-Ser overproduced in glia is proposed as a novel mechanism underlying ALS motoneuronal death, and this mechanism may be regarded as a potential therapeutic target for ALS.     

Links between electrophysiological and molecular pathology of amyotrophic lateral sclerosis

Multiple deficits have been described in amyotrophic lateral sclerosis (ALS), from the first changes in normal functioning of the motoneurons and glia to the eventual loss of spinal and cortical motoneurons. In this review, current results, including changes in size, and electrical properties of motoneurons, glutamate excitotoxicity, calcium buffering, deficits in mitochondrial and cellular transport, impediments to proteostasis which lead to stress of the endoplasmic reticulum (ER), and glial contributions to motoneuronal vulnerability are recapitulated. Results are mainly drawn from the mutant SOD1 mouse model of ALS, and emphasis is placed on early changes that precede the onset of symptoms and the interplay between molecular and electrical processes.     

Amyotrophic lateral sclerosis mutations have the greatest destabilizing effect on the apo- and reduced form of SOD1, leading to unfolding and oxidative aggregation

Mutant forms of Cu,Zn-superoxide dismutase (SOD1) that cause familial amyotrophic lateral sclerosis (ALS) exhibit toxicity that promotes the death of motor neurons. Proposals for the toxic properties typically involve aberrant catalytic activities or protein aggregation. The striking thermodynamic stability of mature forms of the ALS mutant SOD1 (Tm>70 degrees C) is not typical of protein aggregation models that involve unfolding. Over 44 states of the polypeptide are possible, depending upon metal occupancy, disulfide status, and oligomeric state; however, it is not clear which forms might be responsible for toxicity. Recently the intramolecular disulfide has been shown to be required for SOD1 activity, leading us to examine these states of several disease-causing SOD1 mutants. We find that ALS mutations have the greatest effect on the most immature form of SOD1, destabilizing the metal-free and disulfide-reduced polypeptide to the point that it is unfolded at physiological temperatures (Tm<37 degrees C). We also find that immature states of ALS mutant (but not wild type) proteins readily form oligomers at physiological concentrations. Furthermore, these oligomers are more susceptible to mild oxidative stress, which promotes incorrect disulfide cross-links between conserved cysteines and drives aggregation. Thus it is the earliest disulfide-reduced polypeptides in the SOD1 assembly pathway that are most destabilized with respect to unfolding and oxidative aggregation by ALS-causing mutations.     

A mutation in dynein rescues axonal transport defects and extends the life span of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition characterized by motoneuron degeneration and muscle paralysis. Although the precise pathogenesis of ALS remains unclear, mutations in Cu/Zn superoxide dismutase (SOD1) account for approximately 20-25% of familial ALS cases, and transgenic mice overexpressing human mutant SOD1 develop an ALS-like phenotype. Evidence suggests that defects in axonal transport play an important role in neurodegeneration. In Legs at odd angles (Loa) mice, mutations in the motor protein dynein are associated with axonal transport defects and motoneuron degeneration. Here, we show that retrograde axonal transport defects are already present in motoneurons of SOD1(G93A) mice during embryonic development. Surprisingly, crossing SOD1(G93A) mice with Loa/+ mice delays disease progression and significantly increases life span in Loa/SOD1(G93A) mice. Moreover, there is a complete recovery in axonal transport deficits in motoneurons of these mice, which may be responsible for the amelioration of disease. We propose that impaired axonal transport is a prime cause of neuronal death in neurodegenerative disorders such as ALS.