SOD1-positive aggregate accumulation in the CNS predicts slower disease progression and increased longevity in a mutant SOD1 mouse model of ALS.
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ABSTRACT:
SUBMITTER: Gill C
PROVIDER: S-EPMC6491559 | biostudies-literature | 2019 Apr
REPOSITORIES: biostudies-literature
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Project description:Amyotrophic lateral sclerosis (ALS) is a fatal motoneuron (MN) disease characterized by protein misfolding and aggregation leading to cellular degeneration. So far neither biomarker, nor effective treatment has been found. ATP signaling and P2X4 receptors (P2X4) are upregulated in various neurodegenerative diseases. Here we show that several ALS-related misfolded proteins including mutants of SOD1 or TDP-43 lead to a significant increase in surface P2X4 receptor density and function in vitro. In addition, we demonstrate in the spinal the cord of SOD1-G93A (SOD1) mice that misfolded SOD1-G93A proteins directly interact with endocytic adaptor protein-2 (AP2); thus, acting as negative competitors for the interaction between AP2 and P2X4, impairing constitutive P2X4 endocytosis. The higher P2X4 surface density was particularly observed in peripheral macrophages of SOD1 mice before the onset and during the progression of ALS symptoms positioning P2X4 as a potential early biomarker for ALS. P2X4 expression was also upregulated in spinal microglia of SOD1 mice during ALS and affect microglial inflammatory responses. Importantly, we report using double transgenic SOD1 mice expressing internalization-defective P2X4mCherryIN knock-in gene or invalidated for the P2X4 gene that P2X4 is instrumental for motor symptoms, ALS progression and survival. This study highlights the role of P2X4 in the pathophysiology of ALS and thus its potential for the development of biomarkers and treatments. We also decipher the molecular mechanism by which misfolded proteins related to ALS impact P2X4 trafficking at early pathological stage in cells expressing-P2X4.
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Project description:Point mutations in the gene encoding copper-zinc superoxide dismutase (SOD1) impart a gain-of-function to this protein that underlies 20-25% of all familial amyotrophic lateral sclerosis (FALS) cases. However, the specific mechanism of mutant SOD1 toxicity has remained elusive. Using the complementary techniques of atomic force microscopy (AFM), electrophysiology, and cell and molecular biology, here we examine the structure and activity of A4VSOD1, a mutant SOD1. AFM of A4VSOD1 reconstituted in lipid membrane shows discrete tetrameric pore-like structure with outer and inner diameters 12.2 and 3.0nm respectively. Electrophysiological recordings show distinct ionic conductances across bilayer for A4VSOD1 and none for wildtype SOD1. Mouse neuroblastoma cells exposed to A4VSOD1 undergo membrane depolarization and increases in intracellular calcium. These results provide compelling new evidence that a mutant SOD1 is capable of disrupting cellular homeostasis via an unregulated ion channel mechanism. Such a "toxic channel" mechanism presents a new therapeutic direction for ALS research.
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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.
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Project description:Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.
| S-EPMC4355022 | biostudies-literature
Project description:Varied stresses to cells can lead to a repression in translation by triggering phosphorylation of eukaryotic translation initiator factor 2? (eIF2?), which is central to a process known as the integrated stress response (ISR). PKR-like ER-localized eIF2 kinase (PERK), one of the kinases that phosphorylates eIF2? and coordinates the ISR, is activated by stress occurring from the accumulation of misfolded or unfolded proteins in the endoplasmic reticulum (ER). Mutant Cu/Zn superoxide dismutase (mtSOD1) is thought to cause familial amyotrophic lateral sclerosis (FALS) because it misfolds and aggregates. Published studies have suggested that ER stress is involved in FALS pathogenesis since mtSOD1 accumulates inside the ER and activates PERK leading to phosphorylated eIF2? (p-eIF2?). We previously used a genetic approach to show that haploinsufficiency of PERK significantly accelerates disease onset and shortens survival of G85R mtSOD1 FALS transgenic mice. We now show that G85R mice that express reduced levels of active GADD34, which normally dephosphorylates p-eIF2? and allows recovery from the global suppression of protein synthesis, markedly ameliorates disease. These studies emphasize the importance of the ISR, and specifically the PERK pathway, in the pathogenesis of mtSOD1-induced FALS and as a target for treatment. Furthermore, the ISR may be an appropriate therapeutic target for sporadic ALS and other neurodegenerative diseases since misfolded proteins have been implicated in these disorders.
| S-EPMC3990163 | biostudies-literature
Project description:Amyotrophic lateral sclerosis (ALS) is caused by the progressive degeneration of motor neurons. Mutations in the Cu/Zn superoxide dismutase (SOD1) are found in approximately 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although the molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in nonneuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to nontransgenic cells, SOD1(G93A)-expressing astrocytes induced apoptosis of cocultured motor neurons. This prompted us to investigate whether the effect on motor neuron survival was related to a change in the gene expression profile. Through high-density oligonucleotide microarrays, we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix synthesis, response to stress, and steroid and lipid metabolism. The most up-regulated gene was decorin (Dcn), a small multifunctional extracellular proteoglycan. Down-regulated genes included the insulin-like growth factor-1 receptor (Igf-1r) and the RNA binding protein ROD1. Rod1 was also found down-regulated in purified motor neurons expressing SOD1(G93A). Changes in the expression of Dcn, Igf-1r, and Rod1 were found in the spinal cord of asymptomatic animals, suggesting these changes occur before overt neuronal degeneration and potentially influence astrocyte-motor neuron interaction in the course of the disease. The astrocyte-specific gene expression profile might contribute to the identification of possible candidates for cell type-specific therapies in ALS.
| S-EPMC4048747 | biostudies-literature
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Mutations in Cu/Zn superoxide dismutase (SOD1) are responsible for approximately 20 % of the familial ALS cases. ALS-causing SOD1 mutants display a gain-of-toxicity phenotype, but the nature of this toxicity is still not fully understood. The Ras GTPase-activating protein-binding protein G3BP1 plays a critical role in stress granule dynamics. Alterations in the dynamics of stress granules have been reported in several other forms of ALS unrelated to SOD1. To our surprise, the mutant G93A SOD1 transgenic mice exhibited pathological cytoplasmic inclusions that co-localized with G3BP1-positive granules in spinal cord motor neurons. The co-localization was also observed in fibroblast cells derived from familial ALS patient carrying SOD1 mutation L144F. Mutant SOD1, unlike wild-type SOD1, interacted with G3BP1 in an RNA-independent manner. Moreover, the interaction is specific for G3BP1 since mutant SOD1 showed little interaction with four other RNA-binding proteins implicated in ALS. The RNA-binding RRM domain of G3BP1 and two particular phenylalanine residues (F380 and F382) are critical for this interaction. Mutant SOD1 delayed the formation of G3BP1- and TIA1-positive stress granules in response to hyperosmolar shock and arsenite treatment in N2A cells. In summary, the aberrant mutant SOD1-G3BP1 interaction affects stress granule dynamics, suggesting a potential link between pathogenic SOD1 mutations and RNA metabolism alterations in ALS.
| S-EPMC5023729 | biostudies-literature
Project description:Background:SOD1 mutations are the most common cause of amyotrophic lateral sclerosis (ALS) in non-Caucasian patients. Detailed natural history profiles of SOD1-mutant patients will be beneficial for the strategy and interpretation of future SOD1-targeted clinical practice. Methods:Mutational distribution, age at onset (AAO), site of onset, diagnostic delay, disease progression (rate of ALSFRS-R decrease, ?FS) and survival were analysed. Further comparisons between heredity of disease, gender, and mutations were performed. Results:Sixty-six cases with 43 SOD1 mutations were included and analysed, with p.His47Arg as the leading mutation and seven novel variants identified. The mean (SD) AAO was 43.92?years (9.24) for all subjects, with a significant difference between patients carrying mutations in exon 2 (n?=?24,46.83, 8.31) and exon 4 (n?=?18, 37.75, 7.67) (p?=?0.002). The median (IQR) diagnostic delay from symptom onset was 14.50 (6.00-36.50) months for all SOD1-mutant patients, 9.50 (4.75-24.25) months for males and 24.00 (9.50-47.50) months for females, revealing a gender difference (p?=?0.009). Similar advantages in median (IQR) ?FS [male: female, 0.55 (0.24-0.94) vs 0.19 (0.06-0.90), p?=?0.041] and mean (95% CI) survival [57.4 (38.90-75.90) months vs 125.6 (99.80-151.50) months, p?=?0.006] were also observed in females, both of which existed in sporadic ALS only when stratified by familiar or sporadic ALS. Conclusions:The results highlight a distinct mutational distribution and natural history spectrum in ALS patients carrying SOD1 mutations in China. A prominent mild disease progression was observed in female patients, which had rarely been reported in the previous literature. This finding, together with the detailed analysis of natural history among each mutation, can have important implications for future genetic counselling and SOD1-targeted clinical trials.
| S-EPMC6325854 | biostudies-literature
Project description:Increasing evidence suggests that propagation of the motor neuron disease amyotrophic lateral sclerosis (ALS) involves the pathogenic aggregation of disease-associated proteins that spread in a prion-like manner. We have identified two aggregate strains of human superoxide dismutase 1 (hSOD1) that arise in the CNS of transgenic mouse models of SOD1-mediated ALS. Both strains transmit template-directed aggregation and premature fatal paralysis when inoculated into the spinal cord of adult hSOD1 transgenic mice. This spread of pathogenic aggregation could be a potential target for immunotherapeutic intervention. Here we generated mouse monoclonal antibodies (mAbs) directed to exposed epitopes in hSOD1 aggregate strains and identified an aggregate selective mAb that targets the aa 143-153 C-terminal extremity of hSOD1 (αSOD1143-153). Both pre-incubation of seeds with αSOD1143-153 prior to inoculation, and weekly intraperitoneal (i.p.) administration attenuated transmission of pathogenic aggregation and prolonged the survival of seed-inoculated hSOD1G85R Tg mice. In contrast, administration of a mAb targeting aa 65-72 (αSOD165-72), which exhibits high affinity towards monomeric disordered hSOD1, had an adverse effect and aggravated seed induced premature ALS-like disease. Although the mAbs reached similar concentrations in CSF, only αSOD1143-153 was found in association with aggregated hSOD1 in spinal cord homogenates. Our results suggest that an aggregate-selective immunotherapeutic approach may suppress seeded transmission of pathogenic aggregation in ALS. However, long-term administration of αSOD1143-153 was unable to prolong the lifespan of non-inoculated hSOD1G85R Tg mice. Thus, spontaneously initiated hSOD1 aggregation in spinal motor neurons may be poorly accessible to therapeutic antibodies.
| S-EPMC7488686 | biostudies-literature
Project description:Mutations in superoxide dismutase 1 (SOD1) are associated with familial cases of amyotrophic lateral sclerosis (fALS). Studies in transgenic mice have suggested that wild-type (WT) SOD1 can modulate the toxicity of mutant SOD1. In the present study, we demonstrate that the effects of WT SOD1 on the age at which transgenic mice expressing mutant human SOD1 (hSOD1) develop paralysis are influenced by the nature of the ALS mutation and the expression levels of WT hSOD1. We show that regardless of whether WT SOD1 changes the course of disease, both WT and mutant hSOD1 accumulate as detergent-insoluble aggregates in symptomatic mice expressing both proteins. However, using a panel of fluorescently tagged variants of SOD1 in a cell model of mutant SOD1 aggregation, we demonstrate that the interactions between mutant and WT SOD1 in aggregate formation are not simply a co-assembly of mutant and WT proteins. Overall, these data demonstrate that the product of the normal SOD1 allele in fALS has potential to influence the toxicity of mutant SOD1 and that complex interactions with the mutant protein may influence the formation of aggregates and inclusion bodies generated by mutant SOD1.
| S-EPMC2989888 | biostudies-literature