Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Song, Y. Synaptic actions of amyotrophic-lateral-sclerosis-associated G85R-SOD1 in the squid giant synapse. Eneuro, (2020): ENEURO.0369-19.2020, doi: 10.1523/ENEURO.0369-19.2020.Altered synaptic function is thought to play a role in many neurodegenerative diseases, but little is known about the underlying mechanisms for synaptic dysfunction. The squid giant synapse (SGS) is a classical model for studying synaptic electrophysiology and ultrastructure, as well as molecular mechanisms of neurotransmission. Here, we conduct a multidisciplinary study of synaptic actions of misfolded human G85R-SOD1 causing familial Amyotrophic Lateral Sclerosis (fALS). G85R-SOD1, but not WT-SOD1, inhibited synaptic transmission, altered presynaptic ultrastructure, and reduced both the size of the Readily Releasable Pool (RRP) of synaptic vesicles and mobility from the Reserved Pool (RP) to the RRP. Unexpectedly, intermittent high frequency stimulation (iHFS) blocked inhibitory effects of G85R-SOD1 on synaptic transmission, suggesting aberrant Ca2+ signaling may underlie G85R-SOD1 toxicity. Ratiometric Ca2+ imaging showed significantly increased presynaptic Ca2+ induced by G85R-SOD1 that preceded synaptic dysfunction. Chelating Ca2+ using EGTA prevented synaptic inhibition by G85R-SOD1, confirming the role of aberrant Ca2+ in mediating G85R-SOD1 toxicity. These results extended earlier findings in mammalian motor neurons and advanced our understanding by providing possible molecular mechanisms and therapeutic targets for synaptic dysfunctions in ALS as well as a unique model for further studies.Grass Foundation, HHMI, MGH Jack Satter Foundation, Harvard University ALS and Alzheimer's Endowed Research Fund, Harvard Brain Science Initiative

Familial ALS-superoxide dismutases associate with mitochondria and shift their redox potentials

Recent studies suggest that the toxicity of familial amyotrophic lateral sclerosis mutant Cu, Zn superoxide dismutase (SOD1) arises from its selective recruitment to mitochondria. Here we demonstrate that each of 12 different familial ALS-mutant SOD1s with widely differing biophysical properties are associated with mitochondria of motoneuronal cells to a much greater extent than wild-type SOD1, and that this effect may depend on the oxidation of Cys residues. We demonstrate further that mutant SOD1 proteins associated with the mitochondria tend to form cross-linked oligomers and that their presence causes a shift in the redox state of these organelles and results in impairment of respiratory complexes. The observation that such a diverse set of mutant SOD1 proteins behave so similarly in mitochondria of motoneuronal cells and so differently from wild-type SOD1 suggests that this behavior may explain the toxicity of ALS-mutant SOD1 proteins, which causes motor neurons to die

Single copy/knock-in models of ALS SOD1 in C. elegans suggest loss and gain of function have different contributions to cholinergic and glutamatergic neurodegeneration

Mutations in Cu/Zn superoxide dismutase 1 (SOD1) lead to Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease that disproportionately affects glutamatergic and cholinergic motor neurons. Previous work with SOD1 overexpression models supports a role for SOD1 toxic gain of function in ALS pathogenesis. However, the impact of SOD1 loss of function in ALS cannot be directly examined in overexpression models. In addition, overexpression may obscure the contribution of SOD1 loss of function in the degeneration of different neuronal populations. Here, we report the first single-copy, ALS knock-in models in C. elegans generated by transposon- or CRISPR/Cas9- mediated genome editing of the endogenous sod-1 gene. Introduction of ALS patient amino acid changes A4V, H71Y, L84V, G85R or G93A into the C. elegans sod-1 gene yielded single-copy/knock-in ALS SOD1 models. These differ from previously reported overexpression models in multiple assays. In single-copy/knock-in models, we observed differential impact of sod-1 ALS alleles on glutamatergic and cholinergic neurodegeneration. A4V, H71Y, G85R, and G93A animals showed increased SOD1 protein accumulation and oxidative stress induced degeneration, consistent with a toxic gain of function in cholinergic motor neurons. By contrast, H71Y, L84V, and G85R lead to glutamatergic neuron degeneration due to sod-1 loss of function after oxidative stress. However, dopaminergic and serotonergic neuronal populations were spared in single-copy ALS models, suggesting a neuronal-subtype specificity previously not reported in invertebrate ALS SOD1 models. Combined, these results suggest that knock-in models may reproduce the neurotransmitter-type specificity of ALS and that both SOD1 loss and gain of toxic function differentially contribute to ALS pathogenesis in different neuronal populations.Peer reviewe

Viral delivery of antioxidant genes as a therapeutic strategy in experimental models of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder with no effective treatment to date. Despite its multi-factorial aetiology, oxidative stress is hypothesized to be one of the key pathogenic mechanisms. It is thus proposed that manipulation of the expression of antioxidant genes that are downregulated in the presence of mutant SOD1 may serve as a therapeutic strategy for motor neuronal protection. Lentiviral vectors expressing either PRDX3 or NRF2 genes were tested in the motor neuronal-like NSC34 cell line, and in the ALS tissue culture model, NSC34 cells expressing the human SOD1(G93A) mutation. The NSC34 SOD1(G93A) cells overexpressing either PRDX3 or NRF2 showed a significant decrease in endogenous oxidation stress levels by 40 and 50% respectively compared with controls, whereas cell survival was increased by 30% in both cases. The neuroprotective potential of those two genes was further investigated in vivo in the SOD1(G93A) ALS mouse model, by administering intramuscular injections of adenoassociated virus serotype 6 (AAV6) expressing either of the target genes at a presymptomatic stage. Despite the absence of a significant effect in survival, disease onset or progression, which can be explained by the inefficient viral delivery, the promising in vitro data suggest that a more widespread CNS delivery is needed

Peroxynitrite activates the NLRP3 inflammasome cascade in SOD1(G93A) mouse model of amyotrophic lateral sclerosis

Neuroinflammation, characterized by the appearance of reactive microglial and astroglial cells, is one of the several pathogenic mechanisms of amyotrophic lateral sclerosis (ALS), a fast-progressing and fatal neurodegenerative disease. Cerebrospinal fluid and spinal cord of ALS patients and SOD1 mutant mice show high concentrations of IL-1β. This interleukin, expressed as an inactive precursor, undergoes a proteolytic maturation by caspase1, whose activation, in turn, depends on inflammasomes. Whether and how inflammasome is activated in ALS models is still to be clarified. The mechanism of inflammasome activation was studied in murine microglial cells overexpressing hSOD1(G93A) and verified in the spinal cord of hSOD1(G93A) mice. Murine microglial hSOD1(G93A) cells express all the inflammasome components and LPS activates caspase1 leading to an increase in the secretion of IL-1β. By activating NF-κB, LPS increases ROS and NO levels that spontaneously react to form peroxynitrite, thus leading to protein nitration. Reduction in peroxynitrite levels results in a decrease in caspase1 activity. Protein nitration and caspase1 activity are concomitantly increased in the spinal cord of pre-symptomatic SOD1(G93A) mice. Oxidative/nitrosative stress induces peroxynitrite formation that may be a key trigger of caspase1/inflammasome activation. Peroxynitrite formation may play a critical role in inflammasome activation and might be exploited as potential therapeutic target for ALS

Evaluating the role of the Hippo pathway in the onset and disease progression of the SOD1 mouse model of amyotrophic lateral sclerosis

The Hippo pathway is a cell signaling pathway involved in organ size regulation and tumorigenesis in mammals. This pathway regulates the activity of Yes-associated protein (YAP), a transcriptional coactivator which binds to the transcription factor TEAD to promote expression of genes controlling growth and proliferation of tissues, as well as inhibition of apoptosis. The Hippo pathway has recently been implicated as a pathogenic mechanism in neurodegenerative disorders. Specifically, mammalian sterile 20 (Ste20)-like kinase 1 (MST1), a protein kinase in the Hippo pathway, has been found to promote neuronal death under conditions of oxidative stress. Moreover, homozygous deletion of MST1 in a mouse model of Amyotrophic Lateral Sclerosis (ALS) significantly delayed onset of neurodegenerative symptoms. We examined the expression levels of key Hippo pathway components in cortex, lumbar spinal cord, and gastrocnemius muscle samples of male and female G39A SOD1 mice using western blots. Our results revealed a significant increase in phosphorylated MST1 (pMST1) in lumbar spinal cord of presymptomatic transgenic animals, and found this increase to be sex and gene copy number dependent. These results suggest that the Hippo pathway is dysregulated in the SOD1 mouse model and that MST1 may play a critical role in pathogenesis and disease progression in ALS

Folding of Cu, Zn superoxide dismutase and Familial Amyotrophic Lateral Sclerosis

Cu,Zn superoxide dismutase (SOD1) has been implicated in the familial form of the neurodegenerative disease Amyotrophic Lateral Sclerosis (ALS). It has been suggested that mutant mediated SOD1 misfolding/aggregation is an integral part of the pathology of ALS. We study the folding thermodynamics and kinetics of SOD1 using a hybrid molecular dynamics approach. We reproduce the experimentally observed SOD1 folding thermodynamics and find that the residues which contribute the most to SOD1 thermal stability are also crucial for apparent two-state folding kinetics. Surprisingly, we find that these residues are located on the surface of the protein and not in the hydrophobic core. Mutations in some of the identified residues are found in patients with the disease. We argue that the identified residues may play an important role in aggregation. To further characterize the folding of SOD1, we study the role of cysteine residues in folding and find that non-native disulfide bond formation may significantly alter SOD1 folding dynamics and aggregation propensity.Comment: 16 pages, 5 figure

The chaperone protein clusterin may serve as a cerebrospinal fluid biomarker for chronic spinal cord disorders in the dog

Chronic spinal cord dysfunction occurs in dogs as a consequence of diverse aetiologies, including long-standing spinal cord compression and insidious neurodegenerative conditions. One such neurodegenerative condition is canine degenerative myelopathy (DM), which clinically is a challenge to differentiate from other chronic spinal cord conditions. Although the clinical diagnosis of DM can be strengthened by the identification of the Sod1 mutations that are observed in affected dogs, genetic analysis alone is insufficient to provide a definitive diagnosis. There is a requirement to identify biomarkers that can differentiate conditions with a similar clinical presentation, thus facilitating patient diagnostic and management strategies. A comparison of the cerebrospinal fluid (CSF) protein gel electrophoresis profile between idiopathic epilepsy (IE) and DM identified a protein band that was more prominent in DM. This band was subsequently found to contain a multifunctional protein clusterin (apolipoprotein J) that is protective against endoplasmic reticulum (ER) stress-mediated apoptosis, oxidative stress, and also serves as an extracellular chaperone influencing protein aggregation. Western blot analysis of CSF clusterin confirmed elevated levels in DM compared to IE (p < 0.05). Analysis of spinal cord tissue from DM and control material found that clusterin expression was evident in neurons and that the clusterin mRNA levels from tissue extracts were elevated in DM compared to the control. The plasma clusterin levels was comparable between these groups. However, a comparison of clusterin CSF levels in a number of neurological conditions found that clusterin was elevated in both DM and chronic intervertebral disc disease (cIVDD) but not in meningoencephalitis and IE. These findings indicate that clusterin may potentially serve as a marker for chronic spinal cord disease in the dog; however, additional markers are required to differentiate DM from a concurrent condition such as cIVDD