Dataset Information

Impaired Cu-Zn Superoxide Dismutase (SOD1) and Calcineurin (Cn) Interaction in ALS: A Presumed Consequence for TDP-43 and Zinc Aggregation in Tg SOD1G93A Rodent Spinal Cord Tissue.

ABSTRACT: Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)- enriched phosphorylated TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1G93A protein does not properly bind zinc (Zn) in animal models; hence, mutant SOD1G93A-Cn interaction weakens. Consequently, unstable Cn fails to dephosphorylate TDP-43 that yields hyperphosphorylated TDP-43 aggregates. Our previous studies had suggested that Cn and SOD1 interaction was necessary to keep Cn enzyme functional. We have observed low Cn level, increased Zn concentrations, and increased TDP-43 protein levels in cervical, thoracic, lumbar, and sacral regions of the spinal cord tissue homogenates. This study further supports our previously published work indicating that Cn stability depends on functional Cn-SOD1 interaction because Zn is crucial for maintaining the Cn stability. Less active Cn did not efficiently dephosphorylate TDP-43; hence TDP-43 aggregations appeared in the spinal cord tissue.

SUBMITTER: Kim JM

PROVIDER: S-EPMC6345727 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature

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Impaired Cu-Zn Superoxide Dismutase (SOD1) and Calcineurin (Cn) Interaction in ALS: A Presumed Consequence for TDP-43 and Zinc Aggregation in Tg SOD1<sup>G93A</sup> Rodent Spinal Cord Tissue.

Kim Jolene M JM Billington Elizabeth E Reyes Ada A Notarianni Tara T Sage Jessica J Agbas Emre E Taylor Michael M Monast Ian I Stanford John A JA Agbas Abdulbaki A

Neurochemical research 20180103 1

Impaired interactions between Calcineurin (Cn) and (Cu/Zn) superoxide dismutase (SOD1) are suspected to be responsible for the formation of hyperphosphorylated protein aggregation in amyotrophic lateral sclerosis (ALS). Serine (Ser)- enriched phosphorylated TDP-43 protein aggregation appears in the spinal cord of ALS animal models, and may be linked to the reduced phosphatase activity of Cn. The mutant overexpressed SOD1<sup>G93A</sup> protein does not properly bind zinc (Zn) in animal models; h ...[more]

PMID: 29299811

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Project description:Mutations in TDP-43 (an RNA binding protein) are known to cause amyotrophic lateral sclerosis (ALS). Previously, our group and several other studies showed that TDP-43 binds to several RNA targets in the mammalian CNS. ALS causing mutations in the C-terminal region of TDP-43 that is involved in splicing regulation may lead to aberrant splicing of several RNA transcripts. The main aim of this study is to identify the effect of an ALS causing mutation on the splicing regulation of previously known targets of TDP-43. Mice overexpressing a mutant human TDP-43 (hTDP-43) gene were obtained from The Jackson Laboratory (strain name - B6;CB-Tg(Prnp-TARDBP*A315T) 95Balo/J; Stock no:010700). Mutant hTDP43 was expressed under the control of a prion protein promoter that drives the expression mainly in the mouse CNS and the male transgenic mice developed symptoms around 12 weeks of age while the female mice develop symptoms approximately 20 weeks of age. Therefore, Tg animals that are 50 days old were considered pre-symptomatic and 100 days old (Tg) animals were considered to be in the post-symptomatic stages of the disease for exon array experiments. Age and sex matched pre and post-symptomatic transgenic animals and their wild type littermates were euthanised by cervical dislocation and tissues (brain and spinal cord) were harvested immediately for total RNA isolation experiments (n=3 per group). 200 ng of total RNA from transgenic TDP-43 mice and wild type animals was converted to cDNA and then amplified using the Applause WT-Amp Plus ST kit (NuGEN). Amplified cDNA was then fragmented and biotin labeled using the Encore Biotin Module kit (NuGEN) according to the manufacturer's instruction manual. Labelled cDNA was then hybridised onto Affymetrix GeneChip Exon 1.0 ST arrays in a hybridisation oven at 45 degrees C for 20 hours at 60rpm. After hybridization, washing and staining of the arrays were carried out using Affymetrix fluidics station 450 followed by scanning using GeneChip scanner. GeneChip Command Console Software (AGCC) controlled both the fluidics station and the scanner. CEL files generated by AGCC were uploaded onto Partek software and the data analysis was carried out using the exon array analysis workflow. Exon 1.0 ST arrays contain many more probes, which are classified into three major types based on their source. They are Core, extended and full probe set annotation. Core annotation refers the probe sets that are the most reliable of the three and is derived based on evidences from Refseq and GenBank. Extended annotations refer to probe sets that are generated based on EST sequences, ENSEMBL gene collections and other databases including those used for generating core probe sets. The full annotation refers to probe sets that are purely based on computational predictions. Core probe set annotation, unlike the extended or full annotation excludes the speculative probes reducing the incidence of false positives and was employed for exon array analysis. Alt-splice Anova, a statistical tool available in Partek was used for identifying novel alternative splicing events.

Project description:Multiple neurotoxic proteinopathies co-exist within vulnerable neuronal populations in all major neurodegenerative diseases. Interactions between these pathologies may modulate disease progression, suggesting they may constitute targets for disease-modifying treatments aiming to slow or halt neurodegeneration. Pairwise interactions between superoxide dismutase 1 (SOD1), TAR DNA-binding protein 43 (TDP-43) and ubiquitin-binding protein 62/sequestosome 1 (p62) proteinopathies have been reported in multiple transgenic cellular and animal models of amyotrophic lateral sclerosis (ALS), however corresponding examination of these relationships in patient tissues is lacking. Further, the coalescence of all three proteinopathies has not been studied in vitro or in vivo to date. These data are essential to guide therapeutic development and enhance the translation of relevant therapies into the clinic. Our group recently profiled SOD1 proteinopathy in post-mortem spinal cord tissues from familial and sporadic ALS cases, demonstrating an abundance of structurally-disordered (dis)SOD1 conformers which become mislocalized within these vulnerable neurons compared with those of aged controls. To explore any relationships between this, and other, ALS-linked proteinopathies, we profiled TDP-43 and p62 within spinal cord motor neurons of the same post-mortem tissue cohort using multiplexed immunofluorescence and immunohistochemistry. We identified distinct patterns of SOD1, TDP43 and p62 co-deposition and subcellular mislocalization between motor neurons of familial and sporadic ALS cases, which we primarily attribute to SOD1 gene status. Our data demonstrate co-deposition of p62 with mutant and wild-type disSOD1 and phosphorylated TDP-43 in familial and sporadic ALS spinal cord motor neurons, consistent with attempts by p62 to mitigate SOD1 and TDP-43 deposition. Wild-type SOD1 and TDP-43 co-deposition was also frequently observed in ALS cases lacking SOD1 mutations. Finally, alterations to the subcellular localization of the three proteins were tightly correlated, suggesting close relationships between the regulatory mechanisms governing the subcellular compartmentalization of these proteins. Our study is the first to profile spatial relationships between SOD1, TDP-43 and p62 pathologies in post-mortem spinal cord motor neurons of ALS patients, previously only studied in vitro. Our findings suggest interactions between these three key ALS-linked proteins are likely to modulate the formation of their respective proteinopathies, and perhaps the rate of motor neuron degeneration, in ALS patients.

Dataset Information

Impaired Cu-Zn Superoxide Dismutase (SOD1) and Calcineurin (Cn) Interaction in ALS: A Presumed Consequence for TDP-43 and Zinc Aggregation in Tg SOD1G93A Rodent Spinal Cord Tissue.

Publications

Impaired Cu-Zn Superoxide Dismutase (SOD1) and Calcineurin (Cn) Interaction in ALS: A Presumed Consequence for TDP-43 and Zinc Aggregation in Tg SOD1<sup>G93A</sup> Rodent Spinal Cord Tissue.

Similar Datasets

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