Increased axonal mitochondrial mobility does not slow amyotrophic lateral sclerosis (ALS)-like disease in mutant SOD1 mice

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.     

Disulfide Scrambling Describes the Oligomer Formation of Superoxide Dismutase (SOD1) Proteins in the Familial Form of Amyotrophic Lateral Sclerosis

Dominant mutations in Cu,Zn-superoxide dismutase (SOD1) are a cause of a familial form of amyotrophic lateral sclerosis. Wild-type SOD1 forms a highly conserved intra-molecular disulfide bond, whereas pathological SOD1 proteins are cross-linked via intermolecular disulfide bonds and form insoluble oligomers. A thiol-disulfide status in SOD1 will thus play a regulatory role in determining its folding/misfolding pathways; however, it remains unknown how pathogenic mutations in SOD1 affect the thiol-disulfide status to facilitate the protein misfolding. Here, we show that the structural destabilization of SOD1 scrambles a disulfide bond among four Cys residues in an SOD1 molecule. The disulfide scrambling produces SOD1 monomers with distinct electrophoretic mobility and also reproduces the formation of disulfide-linked oligomers. We have also found that the familial form of amyotrophic lateral sclerosis-causing mutations facilitate the disulfide scrambling in SOD1. Based upon our results, therefore, scrambling of the conserved disulfide bond will be a key event to cause the pathological changes in disease-associated mutant SOD1 proteins.     

Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic amyotrophic lateral sclerosis (ALS) model mice

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.     

DJ-1 Is a Copper Chaperone Acting on SOD1 Activation

Lack of oxidative stress control is a common and often prime feature observed in many neurodegenerative diseases. Both DJ-1 and SOD1, proteins involved in familial Parkinson disease and amyotrophic lateral sclerosis, respectively, play a protective role against oxidative stress. Impaired activity and modified expression of both proteins have been observed in different neurodegenerative diseases. A potential cooperative action of DJ-1 and SOD1 in the same oxidative stress response pathway may be suggested based on a copper-mediated interaction between the two proteins reported here. To investigate the mechanisms underlying the antioxidative function of DJ-1 in relation to SOD1 activity, we investigated the ability of DJ-1 to bind copper ions. We structurally characterized a novel copper binding site involving Cys-106, and we investigated, using different techniques, the kinetics of DJ-1 binding to copper ions. The copper transfer between the two proteins was also examined using both fluorescence spectroscopy and specific biochemical assays for SOD1 activity. The structural and functional analysis of the novel DJ-1 copper binding site led us to identify a putative role for DJ-1 as a copper chaperone. Alteration of the coordination geometry of the copper ion in DJ-1 may be correlated to the physiological role of the protein, to a potential failure in metal transfer to SOD1, and to successive implications in neurodegenerative etiopathogenesis.     

Mutant Copper-Zinc Superoxide Dismutase (SOD1) Induces Protein Secretion Pathway Alterations and Exosome Release in Astrocytes: IMPLICATIONS FOR DISEASE SPREADING AND MOTOR NEURON PATHOLOGY IN AMYOTROPHIC LATERAL SCLEROSIS*

Amyotrophic lateral sclerosis is the most common motor neuron disease and is still incurable. The mechanisms leading to the selective motor neuron vulnerability are still not known. The interplay between motor neurons and astrocytes is crucial in the outcome of the disease. We show that mutant copper-zinc superoxide dismutase (SOD1) overexpression in primary astrocyte cultures is associated with decreased levels of proteins involved in secretory pathways. This is linked to a general reduction of total secreted proteins, except for specific enrichment in a number of proteins in the media, such as mutant SOD1 and valosin-containing protein (VCP)/p97. Because there was also an increase in exosome release, we can deduce that astrocytes expressing mutant SOD1 activate unconventional secretory pathways, possibly as a protective mechanism. This may help limit the formation of intracellular aggregates and overcome mutant SOD1 toxicity. We also found that astrocyte-derived exosomes efficiently transfer mutant SOD1 to spinal neurons and induce selective motor neuron death. We conclude that the expression of mutant SOD1 has a substantial impact on astrocyte protein secretion pathways, contributing to motor neuron pathology and disease spread.     

Mitochondrial Dysfunction and Decrease in Body Weight of a Transgenic Knock-in Mouse Model for TDP-43

The majority of amyotrophic lateral sclerosis (ALS) cases as well as many patients suffering from frontotemporal lobar dementia (FTLD) with ubiquitinated inclusion bodies show TDP-43 pathology, the protein encoded by the TAR DNA-binding protein (Tardbp) gene. We used recombinase-mediated cassette exchange to introduce an ALS patient cDNA into the mouse Tdp-43 locus. Expression levels of human A315T TDP-43 protein were 300% elevated in heterozygotes, whereas the endogenous mouse Tdp-43 was decreased to 20% of wild type levels as a result of disturbed feedback regulation. Heterozygous TDP-43^A315TKi mutants lost 10% of their body weight and developed insoluble TDP-43 protein starting as early as 3 months after birth, a pathology that was exacerbated with age. We analyzed the splicing patterns of known Tdp-43 target genes as well as genome-wide gene expression levels in different tissues that indicated mitochondrial dysfunction. In heterozygous mutant animals, we observed a relative decrease in expression of Parkin (Park2) and the fatty acid transporter CD36 along with an increase in fatty acids, HDL cholesterol, and glucose in the blood. As seen in transmission electron microscopy, neuronal cells in motor cortices of TDP-43^A315TKi animals had abnormal neuronal mitochondrial cristae formation. Motor neurons were reduced to 90%, but only slight motoric impairment was detected. The observed phenotype was interpreted as a predisease model, which might be valuable for the identification of further environmental or genetic triggers of neurodegeneration.     

HDAC6 Regulates Mutant SOD1 Aggregation through Two SMIR Motifs and Tubulin Acetylation

Histone deacetylase 6 (HDAC6) is a tubulin deacetylase that regulates protein aggregation and turnover. Mutations in Cu/Zn superoxide dismutase (SOD1) linked to familial amyotrophic lateral sclerosis (ALS) make the mutant protein prone to aggregation. However, the role of HDAC6 in mutant SOD1 aggregation and the ALS etiology is unclear. Here we report that HDAC6 knockdown increased mutant SOD1 aggregation in cultured cells. Different from its known role in mediating the degradation of poly-ubiquitinated proteins, HDAC6 selectively interacted with mutant SOD1 via two motifs similar to the SOD1 mutant interaction region (SMIR) that we identified previously in p62/sequestosome 1. Expression of the aggregation-prone mutant SOD1 increased α-tubulin acetylation, and the acetylation-mimicking K40Q α-tubulin mutant promoted mutant SOD1 aggregation. Our results suggest that ALS-linked mutant SOD1 can modulate HDAC6 activity and increase tubulin acetylation, which, in turn, facilitates the microtubule- and retrograde transport-dependent mutant SOD1 aggregation. HDAC6 impairment might be a common feature in various subtypes of ALS.     

Copper and zinc metallation status of copper-zinc superoxide dismutase from amyotrophic lateral sclerosis transgenic mice

Mutations in the metalloenzyme copper-zinc superoxide dismutase (SOD1) cause one form of familial amyotrophic lateral sclerosis (ALS), and metals are suspected to play a pivotal role in ALS pathology. To learn more about metals in ALS, we determined the metallation states of human wild-type or mutant (G37R, G93A, and H46R/H48Q) SOD1 proteins from SOD1-ALS transgenic mice spinal cords. SOD1 was gently extracted from spinal cord and separated into insoluble (aggregated) and soluble (supernatant) fractions, and then metallation states were determined by HPLC inductively coupled plasma MS. Insoluble SOD1-rich fractions were not enriched in copper and zinc. However, the soluble mutant and WT SOD1s were highly metallated except for the metal-binding-region mutant H46R/H48Q, which did not bind any copper. Due to the stability conferred by high metallation of G37R and G93A, it is unlikely that these soluble SOD1s are prone to aggregation in vivo, supporting the hypothesis that immature nascent SOD1 is the substrate for aggregation. We also investigated the effect of SOD1 overexpression and disease on metal homeostasis in spinal cord cross-sections of SOD1-ALS mice using synchrotron-based x-ray fluorescence microscopy. In each mouse genotype, except for the H46R/H48Q mouse, we found a redistribution of copper between gray and white matters correlated to areas of high SOD1. Interestingly, a disease-specific increase of zinc was observed in the white matter for all mutant SOD1 mice. Together these data provide a picture of copper and zinc in the cell as well as highlight the importance of these metals in understanding SOD1-ALS pathology.     

Mitochondrial calcium uptake regulates rapid calcium transients in skeletal muscle during excitation-contraction (E-C) coupling

Defective coupling between sarcoplasmic reticulum and mitochondria during control of intracellular Ca(2+) signaling has been implicated in the progression of neuromuscular diseases. Our previous study showed that skeletal muscles derived from an amyotrophic lateral sclerosis (ALS) mouse model displayed segmental loss of mitochondrial function that was coupled with elevated and uncontrolled sarcoplasmic reticulum Ca(2+) release activity. The localized mitochondrial defect in the ALS muscle allows for examination of the mitochondrial contribution to Ca(2+) removal during excitation-contraction coupling by comparing Ca(2+) transients in regions with normal and defective mitochondria in the same muscle fiber. Here we show that Ca(2+) transients elicited by membrane depolarization in fiber segments with defective mitochondria display an ~10% increased amplitude. These regional differences in Ca(2+) transients were abolished by the application of 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, a fast Ca(2+) chelator that reduces mitochondrial Ca(2+) uptake. Using a mitochondria-targeted Ca(2+) biosensor (mt11-YC3.6) expressed in ALS muscle fibers, we monitored the dynamic change of mitochondrial Ca(2+) levels during voltage-induced Ca(2+) release and detected a reduced Ca(2+) uptake by mitochondria in the fiber segment with defective mitochondria, which mirrored the elevated Ca(2+) transients in the cytosol. Our study constitutes a direct demonstration of the importance of mitochondria in shaping the cytosolic Ca(2+) signaling in skeletal muscle during excitation-contraction coupling and establishes that malfunction of this mechanism may contribute to neuromuscular degeneration in ALS.     

Calcium Ions Promote Superoxide Dismutase 1 (SOD1) Aggregation into Non-fibrillar Amyloid: A LINK TO TOXIC EFFECTS OF CALCIUM OVERLOAD IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)?*

Imbalance in metal ion homeostasis is a hallmark in neurodegenerative conditions involving protein deposition, and amyotrophic lateral sclerosis (ALS) is no exception. In particular, Ca²⁺ dysregulation has been shown to correlate with superoxide dismutase-1 (SOD1) aggregation in a cellular model of ALS. Here we present evidence that SOD1 aggregation is enhanced and modulated by Ca²⁺. We show that at physiological pH, Ca²⁺ induces conformational changes that increase SOD1 β-sheet content, as probed by far UV CD and attenuated total reflectance-FTIR, and enhances SOD1 hydrophobicity, as probed by ANS fluorescence emission. Moreover, dynamic light scattering analysis showed that Ca²⁺ boosts the onset of SOD1 aggregation. In agreement, Ca²⁺ decreases SOD1 critical concentration and nucleation time during aggregation kinetics, as evidenced by thioflavin T fluorescence emission. Attenuated total reflectance FTIR analysis showed that Ca²⁺ induced aggregates consisting preferentially of antiparallel β-sheets, thus suggesting a modulation effect on the aggregation pathway. Transmission electron microscopy and analysis with conformational anti-fibril and anti-oligomer antibodies showed that oligomers and amyloidogenic aggregates constitute the prevalent morphology of Ca²⁺-induced aggregates, thus indicating that Ca²⁺ diverts SOD1 aggregation from fibrils toward amorphous aggregates. Interestingly, the same heterogeneity of conformations is found in ALS-derived protein inclusions. We thus hypothesize that transient variations and dysregulation of cellular Ca²⁺ levels contribute to the formation of SOD1 aggregates in ALS patients. In this scenario, Ca²⁺ may be considered as a pathogenic effector in the formation of ALS proteinaceous inclusions.     

Alsin and SOD1(G93A) proteins regulate endosomal reactive oxygen species production by glial cells and proinflammatory pathways responsible for neurotoxicity

Recent studies have implicated enhanced Nox2-mediated reactive oxygen species (ROS) by microglia in the pathogenesis of motor neuron death observed in familial amyotrophic lateral sclerosis (ALS). In this context, ALS mutant forms of SOD1 enhance Rac1 activation, leading to increased Nox2-dependent microglial ROS production and neuron cell death in mice. It remains unclear if other genetic mutations that cause ALS also function through similar Nox-dependent pathways to enhance ROS-mediate motor neuron death. In the present study, we sought to understand whether alsin, which is mutated in an inherited juvenile form of ALS, functionally converges on Rac1-dependent pathways acted upon by SOD1(G93A) to regulate Nox-dependent ROS production. Our studies demonstrate that glial cell expression of SOD1(G93A) or wild type alsin induces ROS production, Rac1 activation, secretion of TNFα, and activation of NFκB, leading to decreased motor neuron survival in co-culture. Interestingly, coexpression of alsin, or shRNA against Nox2, with SOD1(G93A) in glial cells attenuated these proinflammatory indicators and protected motor neurons in co-culture, although shRNAs against Nox1 and Nox4 had little effect. SOD1(G93A) expression dramatically enhanced TNFα-mediated endosomal ROS in glial cells in a Rac1-dependent manner and alsin overexpression inhibited SOD1(G93A)-induced endosomal ROS and Rac1 activation. SOD1(G93A) expression enhanced recruitment of alsin to the endomembrane compartment in glial cells, suggesting that these two proteins act to modulate Nox2-dependent endosomal ROS and proinflammatory signals that modulate NFκB. These studies suggest that glial proinflammatory signals regulated by endosomal ROS are influenced by two gene products known to cause ALS.     

Misfolded Polyglutamine,Polyalanine, and Superoxide Dismutase 1 Aggregate via Distinct Pathways in the Cell

Protein aggregation into intracellular inclusions is a key feature of many neurodegenerative disorders. A common theme has emerged that inappropriate self-aggregation of misfolded or mutant polypeptide sequences is detrimental to cell health. Yet protein quality control mechanisms may also deliberately cluster them together into distinct inclusion subtypes, including the insoluble protein deposit (IPOD) and the juxtanuclear quality control (JUNQ). Here we investigated how the intrinsic oligomeric state of three model systems of disease-relevant mutant protein and peptide sequences relates to the IPOD and JUNQ patterns of aggregation using sedimentation velocity analysis. Two of the models (polyalanine (37A) and superoxide dismutase 1 (SOD1) mutants A4V and G85R) accumulated into the same JUNQ-like inclusion whereas the other, polyglutamine (72Q), formed spatially distinct IPOD-like inclusions. Using flow cytometry pulse shape analysis (PulSA) to separate cells with inclusions from those without revealed the SOD1 mutants and 37A to have abruptly altered oligomeric states with respect to the nonaggregating forms, regardless of whether cells had inclusions or not, whereas 72Q was almost exclusively monomeric until inclusions formed. We propose that mutations leading to JUNQ inclusions induce a constitutively “misfolded” state exposing hydrophobic side chains that attract and ultimately overextend protein quality capacity, which leads to aggregation into JUNQ inclusions. Poly(Q) is not misfolded in this same sense due to universal polar side chains, but is highly prone to forming amyloid fibrils that we propose invoke a different engagement mechanism with quality control.     

Fused in Sarcoma (FUS) Protein Lacking Nuclear Localization Signal (NLS) and Major RNA Binding Motifs Triggers Proteinopathy and Severe Motor Phenotype in Transgenic Mice

Dysfunction of two structurally and functionally related proteins, FUS and TAR DNA-binding protein of 43 kDa (TDP-43), implicated in crucial steps of cellular RNA metabolism can cause amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. The proteins are intrinsically aggregate-prone and form non-amyloid inclusions in the affected nervous tissues, but the role of these proteinaceous aggregates in disease onset and progression is still uncertain. To address this question, we designed a variant of FUS, FUS 1–359, which is predominantly cytoplasmic, highly aggregate-prone, and lacks a region responsible for RNA recognition and binding. Expression of FUS 1–359 in neurons of transgenic mice, at a level lower than that of endogenous FUS, triggers FUSopathy associated with severe damage of motor neurons and their axons, neuroinflammatory reaction, and eventual loss of selective motor neuron populations. These pathological changes cause abrupt development of a severe motor phenotype at the age of 2.5–4.5 months and death of affected animals within several days of onset. The pattern of pathology in transgenic FUS 1–359 mice recapitulates several key features of human ALS with the dynamics of the disease progression compressed in line with shorter mouse lifespan. Our data indicate that neuronal FUS aggregation is sufficient to cause ALS-like phenotype in transgenic mice.     

Targeted Depletion of TDP-43 Expression in the Spinal Cord Motor Neurons Leads to the Development of Amyotrophic Lateral Sclerosis-like Phenotypes in Mice

ALS, or amyotrophic lateral sclerosis, is a progressive and fatal motor neuron disease with no effective medicine. Importantly, the majority of the ALS cases are with TDP-43 proteinopathies characterized with TDP-43-positive, ubiquitin-positive inclusions (UBIs) in the cytosol. However, the role of the mismetabolism of TDP-43 in the pathogenesis of ALS with TDP-43 proteinopathies is unclear. Using the conditional mouse gene targeting approach, we show that mice with inactivation of the Tardbp gene in the spinal cord motor neurons (HB9:Cre-Tardbp(lx/-)) exhibit progressive and male-dominant development of ALS-related phenotypes including kyphosis, motor dysfunctions, muscle weakness/atrophy, motor neuron loss, and astrocytosis in the spinal cord. Significantly, ubiquitinated proteins accumulate in the TDP-43-depleted motor neurons of the spinal cords of HB9:Cre-Tardbp(lx/-) mice with the ALS phenotypes. This study not only establishes an important role of TDP-43 in the long term survival and functioning of the mammalian spinal cord motor neurons, but also establishes that loss of TDP-43 function could be one major cause for neurodegeneration in ALS with TDP-43 proteinopathies.