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Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

ABSTRACT: Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS). Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old) SOD1(G93A) mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrated the deregulated genes related to death, stress and mitochondrion, which allowed the identification of Cell cycle, ErbB signaling, Tryptophan metabolism and Rig-I-like receptor signaling as the most representative KEGG pathways. The protein-protein interaction networks based upon deregulated genes have identified the top hubs (TRAF2, H2AFX, E2F1, FOXO3, MSH2, NGFR, TGFBR1) and bottlenecks (TRAF2, E2F1, CDKN1B, TWIST1, FOXO3). Schwann cells were enriched from the sciatic nerve of presymptomatic mice using flow cytometry cell sorting. qPCR showed the up regulated (Ngfr, Cdnkn1b, E2f1, Traf2 and Erbb3, H2afx, Cdkn1a, Hspa1, Prdx, Mapk10) and down-regulated (Foxo3, Mtor) genes in the enriched Schwann cells. In conclusion, molecular analyses in the presymptomatic sciatic nerve demonstrated the involvement of death, oxidative stress, and mitochondrial pathways in the Schwann cell non-autonomous mechanisms in the early stages of ALS.

SUBMITTER: Alves CJ

PROVIDER: S-EPMC4555015 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

Alves Chrystian J CJ Maximino Jessica R JR Chadi Gerson G

Frontiers in cellular neuroscience 20150901

Schwann cells are the main source of paracrine support to motor neurons. Oxidative stress and mitochondrial dysfunction have been correlated to motor neuron death in Amyotrophic Lateral Sclerosis (ALS). Despite the involvement of Schwann cells in early neuromuscular disruption in ALS, detailed molecular events of a dying-back triggering are unknown. Sciatic nerves of presymptomatic (60-day-old) SOD1(G93A) mice were submitted to a high-density oligonucleotide microarray analysis. DAVID demonstrat ...[more]

PMID: 26339226

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Project description:Early molecular events related to cytoskeleton are poorly described in Amyotrophic Lateral Sclerosis (ALS), especially in the Schwann cell (SC), which offers strong trophic support to motor neurons. Database for Annotation, Visualization and Integrated Discovery (DAVID) tool identified cytoskeleton-related genes by employing the Cellular Component Ontology (CCO) in a large gene profiling of lumbar spinal cord and sciatic nerve of presymptomatic SOD1(G93A) mice. One and five CCO terms related to cytoskeleton were described from the spinal cord deregulated genes of 40 days (actin cytoskeleton) and 80 days (microtubule cytoskeleton, cytoskeleton part, actin cytoskeleton, neurofilament cytoskeleton, and cytoskeleton) old transgene mice, respectively. Also, four terms were depicted from the deregulated genes of sciatic nerve of 60 days old transgenes (actin cytoskeleton, cytoskeleton part, microtubule cytoskeleton and cytoskeleton). Kif1b was the unique deregulated gene in more than one studied region or presymptomatic age. The expression of Kif1b [quantitative polymerase chain reaction (qPCR)] elevated in the lumbar spinal cord (40 days old) and decreased in the sciatic nerve (60 days old) of presymptomatic ALS mice, results that were in line to microarray findings. Upregulation (24.8 fold) of Kif1b was seen in laser microdissected enriched immunolabeled motor neurons from the spinal cord of 40 days old presymptomatic SOD1(G93A) mice. Furthermore, Kif1b was dowregulated in the sciatic nerve Schwann cells of presymptomatic ALS mice (60 days old) that were enriched by means of cell microdissection (6.35 fold), cell sorting (3.53 fold), and primary culture (2.70 fold) technologies. The gene regulation of cytoskeleton molecules is an important occurrence in motor neurons and Schwann cells in presymptomatic stages of ALS and may be relevant in the dying back mechanisms of neuronal death. Furthermore, a differential regulation of Kif1b in the spinal cord and sciatic nerve cells emerged as key event in ALS.

Project description:Innate neuroimmune dysfunction is a pathobiological feature of amyotrophic lateral sclerosis (ALS). However, links, if any, between disease and adaptive immunity are poorly understood. Thus, the role of T cell immunity in disease was investigated in human G93A superoxide dismutase 1 (SOD1) transgenic (Tg) mice and subsequently in ALS patients.Quantitative and qualitative immune deficits in lymphoid cell and T cell function were seen in G93A-SOD1 Tg mice. Spleens of Tg animals showed reductions in size, weight, lymphocyte numbers, and morphological deficits at terminal stages of disease compared to their wild-type (Wt) littermates. Spleen sizes and weights of pre-symptomatic Tg mice were unchanged, but deficits were readily seen in T cell proliferation coincident with increased annexin-V associated apoptosis and necrosis of lymphocytes. These lymphoid deficits paralleled failure of Copolymer-1 (COP-1) immunization to affect longevity. In addition, among CD4(+) T cells in ALS patients, levels of CD45RA(+) (naïve) T cells were diminished, while CD45RO(+) (memory) T cells were increased compared to age-matched caregivers. In attempts to correct mutant SOD1 associated immune deficits, we reconstituted SOD1 Tg mice with unfractionated naïve lymphocytes or anti-CD3 activated CD4(+)CD25(+) T regulatory cells (Treg) or CD4(+)CD25(-) T effector cells (Teff) from Wt donor mice. While naive lymphocytes failed to enhance survival, both polyclonal-activated Treg and Teff subsets delayed loss of motor function and extended survival; however, only Treg delayed neurological symptom onset, whereas Teff increased latency between disease onset and entry into late stage.A profound and progressive immunodeficiency is operative in G93A-SOD1 mice and is linked to T cell dysfunction and the failure to elicit COP-1 neuroprotective immune responses. In preliminary studies T cell deficits were also observed in human ALS. These findings, taken together, suggest caution in ascribing vaccination outcomes when these animal models of human ALS are used for study. Nonetheless, the abilities to improve neurological function and life expectancy in G93A-SOD1 Tg mice by reconstitution with activated T cells do provide opportunities for therapeutic intervention.

Project description:IntroductionAmyotrophic Lateral Sclerosis (ALS) is a paralyzing, multifactorial neurodegenerative disease with limited therapeutics and no known cure. The study goal was to determine which pathophysiological treatment targets appear most beneficial.MethodsA big data approach was used to analyze high copy SOD1 G93A experimental data. The secondary data set comprised 227 published studies and 4,296 data points. Treatments were classified by pathophysiological target: apoptosis, axonal transport, cellular chemistry, energetics, neuron excitability, inflammation, oxidative stress, proteomics, or systemic function. Outcome assessment modalities included onset delay, health status (rotarod performance, body weight, grip strength), and survival duration. Pairwise statistical analysis (two-tailed t-test with Bonferroni correction) of normalized fold change (treatment/control) assessed significant differences in treatment efficacy. Cohen's d quantified pathophysiological treatment category effect size compared to "all" (e.g., all pathophysiological treatment categories combined).ResultsInflammation treatments were best at delaying onset (d = 0.42, p > 0.05). Oxidative stress treatments were significantly better for prolonging survival duration (d = 0.18, p < 0.05). Excitability treatments were significantly better for prolonging overall health status (d = 0.22, p < 0.05). However, the absolute best pathophysiological treatment category for prolonging health status varied with disease progression: oxidative stress was best for pre-onset health (d = 0.18, p > 0.05); excitability was best for prolonging function near onset (d = 0.34, p < 0.05); inflammation was best for prolonging post-onset function (d = 0.24, p > 0.05); and apoptosis was best for prolonging end-stage function (d = 0.49, p > 0.05). Finally, combination treatments simultaneously targeting multiple pathophysiological categories (e.g., polytherapy) performed significantly (p < 0.05) better than monotherapies at end-stage.DiscussionIn summary, the most effective pathophysiological treatments change as function of assessment modality and disease progression. Shifting pathophysiological treatment category efficacy with disease progression supports the homeostatic instability theory of ALS disease progression.

Project description:Amyotrophic Lateral Sclerosis (ALS) is an adult-onset and fast progression neurodegenerative disease that leads to the loss of motor neurons. Mechanisms of selective motor neuron loss in ALS are unknown. The early events occurring in the spinal cord that may contribute to motor neuron death are not described, neither astrocytes participation in the pre-symptomatic phases of the disease. In order to identify ALS early events, we performed a microarray analysis employing a whole mouse genome platform to evaluate the gene expression pattern of lumbar spinal cords of transgenic SOD1(G93A) mice and their littermate controls at pre-symptomatic ages of 40 and 80 days. Differentially expressed genes were identified by means of the Bioconductor packages Agi4×44Preprocess and limma. FunNet web based tool was used for analysis of over-represented pathways. Furthermore, immunolabeled astrocytes from 40 and 80 days old mice were submitted to laser microdissection and RNA was extracted for evaluation of a selected gene by qPCR. Statistical analysis has pointed to 492 differentially expressed genes (155 up and 337 down regulated) in 40 days and 1105 (433 up and 672 down) in 80 days old ALS mice. KEGG analysis demonstrated the over-represented pathways tight junction, antigen processing and presentation, oxidative phosphorylation, endocytosis, chemokine signaling pathway, ubiquitin mediated proteolysis and glutamatergic synapse at both pre-symptomatic ages. Ube2i gene expression was evaluated in astrocytes from both transgenic ages, being up regulated in 40 and 80 days astrocytes enriched samples. Our data points to important early molecular events occurring in pre-symptomatic phases of ALS in mouse model. Early SUMOylation process linked to astrocytes might account to non-autonomous cell toxicity in ALS. Further studies on the signaling pathways presented here may provide new insights to better understand the events triggering motor neuron death in this devastating disorder.

Project description:A preclinical strategy to broaden the search of potentially effective treatments in amyotrophic lateral sclerosis (ALS) relies on identifying factors controlling motor neuron (MN) excitability. These partners might be part of still unknown pathogenic pathways and/or useful for the design of new interventions to affect disease progression. In this framework, the bioactive membrane-derived phospholipid lysophosphatidic acid (LPA) affects MN excitability through LPA receptor 1 (LPA1 ). Furthermore, LPA1 knockdown is neuroprotective in transgenic ALS SOD1-G93A mice. On this basis, we raised the hypothesis that the major LPA-synthesizing ectoenzyme, autotaxin (ATX), regulates MN excitability and is a potential target to modulate disease development in ALS mice. We show here that PF-8380, a specific ATX inhibitor, reduced intrinsic membrane excitability (IME) of hypoglossal MNs in brainstem slices, supporting that baseline ATX activity regulates MN IME. PF-8380-induced alterations were prevented by a small-interfering RNA directed against mRNA for lpa1 . These outcomes support that impact of ATX-originated lysophospholipids on MN IME engages, at least, the G-protein-coupled receptor LPA1 . Interestingly, mRNAatx levels increased in the spinal cord of pre-symptomatic (1-2 months old) SOD1-G93A mice, thus preceding MN loss. The rise in transcripts levels also occurred in cultured spinal cord MNs from SOD1-G93A embryos, suggesting that mRNAatx upregulation in MNs is an etiopathogenic event in the ALS cell model. Remarkably, chronic administration in the drinking water of the orally bioavailable ATX inhibitor PF-8380 delayed MN loss, motor deterioration and prolonged life span in ALS mice. Treatment also led to a reduction in LPA1 -immunoreactive patches in transgenic animals mostly in MNs. These outcomes support that neuroprotective effects of interfering with ATX in SOD1-G93A mice rely, at least in part, on LPA1 knockdown in MNs. Therefore, we propose ATX as a potential target and/or a biomarker in ALS and highlight ATX inhibitors as reasonable tools with therapeutic usefulness for this lethal pathology.

Dataset Information

Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

Publications

Dysregulated expression of death, stress and mitochondrion related genes in the sciatic nerve of presymptomatic SOD1(G93A) mouse model of Amyotrophic Lateral Sclerosis.

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