The legs at odd angles (Loa) mutation in cytoplasmic dynein ameliorates mitochondrial function in SOD1G93A mouse model for motor neuron disease.
ABSTRACT: Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute approximately 10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15-20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1(G93A). In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1(G93A) motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1(G93A) motor neurons.
Project description:Retrograde axonal transport of cellular signals driven by dynein is vital for neuronal survival. Mouse models with defects in the retrograde transport machinery, including the Loa mouse (point mutation in dynein) and the Tg(dynamitin) mouse (overexpression of dynamitin), exhibit mild neurodegenerative disease. Transport defects have also been observed in more rapidly progressive neurodegeneration, such as that observed in the SOD1(G93A) transgenic mouse model for familial amyotrophic lateral sclerosis (ALS). Here, we test the hypothesis that alterations in retrograde signaling lead to neurodegeneration. In vivo, in vitro, and live-cell imaging motility assays show misregulation of transport and inhibition of retrograde signaling in the SOD1(G93A) model. However, similar inhibition is also seen in the Loa and Tg(dynamitin) mouse models. Thus, slowing of retrograde signaling leads only to mild degeneration and cannot explain ALS etiology. To further pursue this question, we used a proteomics approach to investigate dynein-associated retrograde signaling. These data indicate a significant decrease in retrograde survival factors, including P-Trk (phospho-Trk) and P-Erk1/2, and an increase in retrograde stress factor signaling, including P-JNK (phosphorylated c-Jun N-terminal kinase), caspase-8, and p75(NTR) cleavage fragment in the SOD1(G93A) model; similar changes are not seen in the Loa mouse. Cocultures of motor neurons and glia expressing mutant SOD1 (mSOD1) in compartmentalized chambers indicate that inhibition of retrograde stress signaling is sufficient to block activation of cellular stress pathways and to rescue motor neurons from mSOD1-induced toxicity. Hence, a shift from survival-promoting to death-promoting retrograde signaling may be key to the rapid onset of neurodegeneration seen in ALS.
Project description:Dominant mutations in cytoplasmic dynein (Loa or Cra) have been reported to provoke selective, age-dependent killing of motor neurons, while paradoxically slowing degeneration and death of motor neurons in one mouse model of an inherited form of ALS. Examination of Loa animals reveals no degeneration of large caliber alpha-motor neurons beyond an age-dependent loss (initiating only after 18 months) that was comparable in Loa and wild-type littermates. Absence of Loa-mediated alpha-motor neuron loss contrasted with dramatic, sustained, mutant dynein-mediated postnatal loss of lumbar proprioceptive sensory axons, accompanied by decreased excitatory glutamatergic inputs to motor neurons. In mouse models of inherited ALS caused by mutations in superoxide dismutase (SOD1), mutant dynein modestly prolonged survival in the one mouse model with the most extensive motor neuron loss (SOD(G93A)) while showing marginal (SOD(G85R)) or no (SOD(G37R)) benefit in models with higher numbers of surviving motor neurons at end stage. These findings support a noncell autonomous, excitotoxic contribution from proprioceptive sensory neurons that modestly accelerates disease onset in inherited ALS.
Project description:Mutations in copper-zinc superoxide dismutase (SOD1) have been linked to a subset of familial amytrophic lateral sclerosis (fALS), a fatal neurodegenerative disease characterized by progressive motor neuron death. An increasing amount of evidence supports that mitochondrial dysfunction and apoptosis activation play a critical role in the fALS etiology, but little is known about the mechanisms by which SOD1 mutants cause the mitochondrial dysfunction and apoptosis. In this study, we use proteomic approaches to identify the mitochondrial proteins that are altered in the presence of a fALS-causing mutant G93A-SOD1. A comprehensive characterization of mitochondrial proteins from NSC34 cells, a motor neuron-like cell line, was achieved by two independent proteomic approaches. Four hundred seventy unique proteins were identified in the mitochondrial fraction collectively, 75 of which are newly discovered proteins that previously had only been reported at the cDNA level. Two-dimensional gel electrophoresis was subsequently used to analyze the differences between the mitochondrial proteomes of NSC34 cells expressing wild-type and G93A-SOD1. Nine and 36 protein spots displayed elevated and suppressed abundance respectively in G93A-SOD1-expressing cells. The 45 spots were identified by MS, and they include proteins involved in mitochondrial membrane transport, apoptosis, the respiratory chain, and molecular chaperones. In particular, alterations in the post-translational modifications of voltage-dependent anion channel 2 (VDAC2) were found, and its relevance to regulating mitochondrial membrane permeability and activation of apoptotic pathways is discussed. The potential role of other proteins in the mutant SOD1-mediated fALS is also discussed. This study has produced a short list of mitochondrial proteins that may hold the key to the mechanisms by which SOD1 mutants cause mitochondrial dysfunction and neuronal death. It has laid the foundation for further detailed functional studies to elucidate the role of particular mitochondrial proteins, such as VDAC2, in the pathogenesis of familial ALS.
Project description:Reduced axonal mitochondrial transport has been observed in major neurodegenerative diseases, including fALS patients and SOD1(G93A) mice. However, it is unclear whether this defect plays a critical role in axonal degeneration or simply reflects sequelae of general transport alteration. Using genetic mouse models combined with time-lapse imaging of live neurons, we previously discovered that axon-targeted syntaphilin (SNPH) acts as a docking receptor specific for axonal mitochondria. Deletion of the snph gene in mice results in a substantially higher proportion of axonal mitochondria in the mobile state without any effect on the transport of other axonal organelles. Here we address whether increased (rescued) axonal mitochondrial mobility changes the disease course by crossing fALS-linked transgenic SOD1(G93A) and snph(-/-) knock-out mice. We found that a 2-fold increase in axonal mitochondrial mobility in SOD1(G93A)/snph(-/-) mice did not affect the onset of ALS-like symptoms. Both SOD1(G93A) and SOD1(G93A)/snph(-/-) mice exhibit similar weight loss, deterioration in motor function and motor neuron loss, significant gliosis, and a lifespan of 152-154 days. Thus, for the first time, our study provides genetic and pathological evidence that the impairment of mitochondrial transport seen in SOD1(G93A) mice plays a minimal role in the rapid-onset of fALS-linked pathology.
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, progressive neurodegenerative disorder with no known cure. Cu/Zn-superoxide dismutase (SOD1) was the first identified protein associated with familial ALS (fALS). Recently, TAR DNA-binding protein 43 (TDP-43) has been found to be a principal component of ubiquitinated cytoplasmic inclusions in neurons and glia in ALS. However, it remains unclear whether these ALS-linked proteins partly have a shared pathogenesis. Here, we determine the association between mutant SOD1 and the modification of TDP-43 and the relationship of pathologic TDP-43 to neuronal cytotoxicity in SOD1 ALS. In this work, using animal model, human tissue, and cell models, we provide the evidence that the association between the TDP-43 modification and the pathogenesis of SOD1 fALS. We demonstrated an age-dependent increase in TDP-43 C-terminal fragments and phosphorylation in motor neurons and glia of SOD1 mice and SOD1G85S ALS patient. Cytoplasmic TDP-43 was also observed in iPSC-derived motor neurons from SOD1G17S ALS patient. Moreover, we observed that mutant SOD1 interacts with TDP-43 in co-immunoprecipitation assays with G93A hSOD1-transfected cell lines. Mutant SOD1 overexpression led to an increase in TDP-43 modification in the detergent-insoluble fraction in the spinal cord of SOD1 mice and fALS patient. Additionally, we showed cellular apoptosis in response to the interaction of mutant SOD1 and fragment forms of TDP-43. These findings suggest that mutant SOD1 could affect the solubility/insolubility of TDP-43 through physical interactions and the resulting pathological modifications of TDP-43 may be involved in motor neuron death in SOD1 fALS.
Project description:One pathological hallmark in ALS motor neurons (MNs) is axonal accumulation of damaged mitochondria. A fundamental question remains: does reduced degradation of those mitochondria by an impaired autophagy-lysosomal system contribute to mitochondrial pathology? We reveal MN-targeted progressive lysosomal deficits accompanied by impaired autophagic degradation beginning at asymptomatic stages in fALS-linked hSOD1(G93A) mice. Lysosomal deficits result in accumulation of autophagic vacuoles engulfing damaged mitochondria along MN axons. Live imaging of spinal MNs from the adult disease mice demonstrates impaired dynein-driven retrograde transport of late endosomes (LEs). Expressing dynein-adaptor snapin reverses transport defects by competing with hSOD1(G93A) for binding dynein, thus rescuing autophagy-lysosomal deficits, enhancing mitochondrial turnover, improving MN survival, and ameliorating the disease phenotype in hSOD1(G93A) mice. Our study provides a new mechanistic link for hSOD1(G93A)-mediated impairment of LE transport to autophagy-lysosomal deficits and mitochondrial pathology. Understanding these early pathological events benefits development of new therapeutic interventions for fALS-linked MN degeneration.
Project description:An important consequence of protein misfolding related to neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS), is the formation of proteinaceous inclusions or aggregates within the central nervous system. We have previously shown that several familial ALS-linked copper-zinc superoxide dismutase (SOD1) mutants (A4V, G85R, and G93A) interact and co-localize with the dynein-dynactin complex in cultured cells and affected tissues of ALS mice. In this study, we report that the interaction between mutant SOD1 and the dynein motor plays a critical role in the formation of large inclusions containing mutant SOD1. Disruption of the motor by overexpression of the p50 subunit of dynactin in neuronal and non-neuronal cell cultures abolished the association between aggregation-prone SOD1 mutants and the dynein-dynactin complex. The p50 overexpression also prevented mutant SOD1 inclusion formation and improved the survival of cells expressing A4V SOD1. Furthermore, we observed that two ALS-linked SOD1 mutants, H46R and H48Q, which showed a lower propensity to interact with the dynein motor, also produced less aggregation and fewer large inclusions. Overall, these data suggest that formation of large inclusions depends upon association of the abnormal SOD1s with the dynein motor. Whether the misfolded SOD1s directly perturb axonal transport or impair other functional properties of the dynein motor, this interaction could propagate a toxic effect that ultimately causes motor neuron death in ALS.
Project description:Mutations in the Cu/Zn Superoxide Dismutase (SOD1) gene cause an inherited form of ALS with upper and lower motor neuron loss. The mechanism underlying mutant SOD1-mediated motor neuron degeneration remains unclear. While defects in mitochondrial dynamics contribute to neurodegeneration, including ALS, previous reports remain conflicted. Here, we report an improved technique to isolate, transfect, and culture rat spinal cord motor neurons. Using this improved system, we demonstrate that mutant SOD1(G93A) triggers a significant decrease in mitochondrial length and an accumulation of round fragmented mitochondria. The increase of fragmented mitochondria coincides with an arrest in both anterograde and retrograde axonal transport and increased cell death. In addition, mutant SOD1(G93A) induces a reduction in neurite length and branching that is accompanied with an abnormal accumulation of round mitochondria in growth cones. Furthermore, restoration of the mitochondrial fission and fusion balance by dominant-negative dynamin-related protein 1 (DRP1) expression rescues the mutant SOD1(G93A)-induced defects in mitochondrial morphology, dynamics, and cell viability. Interestingly, both SIRT3 and PGC-1? protect against mitochondrial fragmentation and neuronal cell death by mutant SOD1(G93A). This data suggests that impairment in mitochondrial dynamics participates in ALS and restoring this defect might provide protection against mutant SOD1(G93A)-induced neuronal injury.
Project description:Mutations in the superoxide dismutase 1 (sod1) gene cause familial amyotrophic lateral sclerosis (FALS), likely due to the toxic properties of misfolded mutant SOD1 protein. Here we demonstrated that, starting from the pre-onset stage of FALS, misfolded SOD1 species associates specifically with kinesin-associated protein 3 (KAP3) in the ventral white matter of SOD1(G93A)-transgenic mouse spinal cord. KAP3 is a kinesin-2 subunit responsible for binding to cargos including choline acetyltransferase (ChAT). Motor axons in SOD1(G93A)-Tg mice also showed a reduction in ChAT transport from the pre-onset stage. By employing a novel FALS modeling system using NG108-15 cells, we showed that microtubule-dependent release of acetylcholine was significantly impaired by misfolded SOD1 species. Furthermore, such impairment was able to be normalized by KAP3 overexpression. KAP3 was incorporated into SOD1 aggregates in human FALS cases as well. These results suggest that KAP3 sequestration by misfolded SOD1 species and the resultant inhibition of ChAT transport play a role in the dysfunction of ALS.
Project description:BACKGROUND:MicroRNAs (miRNAs) are endogenous non-coding RNAs that regulate gene expression at the post-transcriptional level and are key modulators in neurodegenerative diseases. Overexpressed miRNAs play an important role in ALS; however, the pathogenic mechanisms of deregulated miRNAs are still unclear. METHODS:We aimed to assess the dysfunction of RNAs or miRNAs in fALS (SOD1 mutations). We compared the RNA-seq of subcellular fractions in NSC-34 WT (hSOD1) and MT (hSOD1 (G93A)) cells to find altered RNAs or miRNAs. We identified that Hif1? and Mef2c were upregulated, and Mctp1 and Rarb were downregulated in the cytoplasm of NSC-34 MT cells. RESULTS:SOD1 mutations decreased the level of miR-18b-5p. Induced Hif1? which is the target for miR-18b increased Mef2c expression as a transcription factor. Mef2c upregulated miR-206 as a transcription factor. Inhibition of Mctp1 and Rarb which are targets of miR-206 induces intracellular Ca2+ levels and reduces cell differentiation, respectively. We confirmed that miR-18b-5p pathway was also observed in G93A Tg, fALS (G86S) patient, and iPSC-derived motor neurons from fALS (G17S) patient. CONCLUSIONS:Our data indicate that SOD1 mutation decreases miR-18b-5p, which sequentially regulates Hif1?, Mef2c, miR-206, Mctp1 and Rarb in fALS-linked SOD1 mutation. These results provide new insights into the downregulation of miR-18b-5p dependent pathogenic mechanisms of ALS.