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An in vitro model for Lewy body-like hyaline inclusion/astrocytic hyaline inclusion: induction by ER stress with an ALS-linked SOD1 mutation.

ABSTRACT: Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15-25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction.

SUBMITTER: Yamagishi S

PROVIDER: S-EPMC2000355 | biostudies-literature | 2007 Oct

REPOSITORIES: biostudies-literature

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An in vitro model for Lewy body-like hyaline inclusion/astrocytic hyaline inclusion: induction by ER stress with an ALS-linked SOD1 mutation.

Yamagishi Satoru S Koyama Yoshihisa Y Katayama Taiichi T Taniguchi Manabu M Hitomi Junichi J Kato Masaaki M Aoki Masashi M Itoyama Yasuto Y Kato Shinsuke S Tohyama Masaya M

PloS one 20071010 10

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI clo ...[more]

PMID: 17925878

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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 about 20% of patients with familial ALS. Mutant SOD1 causes motor neuron death through an acquired toxic property. Although, molecular mechanism underlying this toxic gain-of-function remains unknown, evidence support the role of mutant SOD1 expression in non-neuronal cells in shaping motor neuron degeneration. We have previously found that in contrast to non-transgenic, SOD1G93A-expressing astrocytes induced apoptosis of co-cultured 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 oligonucletide microarrays we found changes in the expression of genes involved in transcription, signaling, cell proliferation, extracellular matrix construction, response to stress and steroid and lipid metabolism. Decorin, a small multifunctional proteoglycan, was the most up-regulated gene. Down-regulated genes included the insulin-like growth factor-1 receptor and the RNA binding protein ROD1. We also analyzed the expression of selected genes in purified motor neurons expressing SOD1G93A and in spinal cord of asymptomatic and early symptomatic ALS-rodent model. The expression of mutated SOD1 in astrocytes cause gene expression changes with potential consequences for its interaction with motor neurons. The astrocyte-specific gene expression profile contributes to the identification of possible candidates for cell type-specific therapies in ALS Keywords: Cell type comparison Astrocytes were plated at a density of 2x104 cells/cm2 and maintained as described in Cassina P, et al.J Neurosci Res. 2002;67(1):21-9. Confluent astrocytes monolayers were changed to supplemented L15 medium (Vargas et al., 2006) for 24h before RNA isolation. Total RNA was isolated with RNeasy kit/RNase-Free DNase Set (Qiagen, CA, USA). RNA quality was assessed with the A260/280 ratio and the 2100 Bioanalyzer (Agilent Technologies, CA, USA) to ensure integrity of the samples used for microarray analysis. Double-stranded cDNA was synthesized from 5 μg of total RNA and used for microarray analysis with the Rat Genome 230 2.0 array (Affymetrix, CA, USA). A total of six arrays divided in three control and three transgenic samples were used. Labeling was performed with one-cycle target labeling assay according to Affymetrix, including the eukaryotic poly-A RNA control and the eukaryotic hybridization control kit. Hybridization, washing and scanning were carried out as described in the Affymetrix GeneChip expression analysis technical manual at the Oregon State University’s Center for Genome Research and Biocomputing. Image processing was done using Affymetrix GCOS 1.4 software. The quality of hybridization and overall chip performance was determined by visual inspection of the raw scanned data and the GCOS-generated report file. Microarray data (.CEL files) was normalized using GC-RMA probe-level analysis in ArrayAssist 4.0 (Stratagene, CA, USA). Following variance stabilization and Log transformation, fold chance versus p-value was calculated using nontransgenic (NonTG) samples as base values.

Project description:Oxidative stress (OS) is regarded as the dominant theory for aging. While compelling correlative data have been generated to support the OS theory, a direct cause-and-effect relationship between the accumulation of oxidation-mediated damage and aging has not been firmly established. Superoxide dismutase 1 (SOD1) is a primary antioxidant in all cells. It is, however, susceptible to oxidation due to OS and gains toxic properties to cells. This study investigates the role of oxidized SOD1 derived from amyotrophic lateral sclerosis (ALS) linked SOD1 mutations in cell senescence and aging. Herein, we have shown that the cell line NSC34 expressing the G93A mutation of human SOD1 (hSOD1G93A) entered premature senescence as evidenced by a decreased number of the 5-ethynyl-2'-deoxyuridine (EdU)-positive cells. There was an upregulation of cellular senescence markers compared to cells expressing the wild-type human SOD1 (hSOD1WT). Transgenic mice carrying the hSOD1G93A gene showed aging phenotypes at an early age (135 days) with high levels of P53 and P16 but low levels of SIRT1 and SIRT6 compared with age-matched hSOD1WT transgenic mice. Notably, the levels of oxidized SOD1 were significantly elevated in both the senescent NSC34 cells and 135-day hSOD1G93A mice. Selective removal of oxidized SOD1 by our CT4-directed autophagy significantly decelerated aging, indicating that oxidized SOD1 is a causal factor of aging. Intriguingly, mitochondria malfunctioned in both senescent NSC34 cells and middle-aged hSODG93A transgenic mice. They exhibited increased production of mitochondrial-derived vesicles (MDVs) in response to mild OS in mutant humanSOD1 (hSOD1) transgenic mice at a younger age; however, the mitochondrial response gradually declined with aging. In conclusion, our data show that oxidized SOD1 derived from ALS-linked SOD1 mutants is a causal factor for cellular senescence and aging. Compromised mitochondrial responsiveness to OS may serve as an indicator of premature aging.

Dataset Information

An in vitro model for Lewy body-like hyaline inclusion/astrocytic hyaline inclusion: induction by ER stress with an ALS-linked SOD1 mutation.

Publications

An in vitro model for Lewy body-like hyaline inclusion/astrocytic hyaline inclusion: induction by ER stress with an ALS-linked SOD1 mutation.

Similar Datasets

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