Lysosomal and phagocytic activity is increased in astrocytes during disease progression in the SOD1 (G93A) mouse model of amyotrophic lateral sclerosis.
ABSTRACT: Astrocytes are key players in the progression of amyotrophic lateral sclerosis (ALS). Previously, gene expression profiling of astrocytes from the pre-symptomatic stage of the SOD1(G93A) model of ALS has revealed reduced lactate metabolism and altered trophic support. Here, we have performed microarray analysis of symptomatic and late-stage disease astrocytes isolated by laser capture microdissection (LCM) from the lumbar spinal cord of the SOD1(G93A) mouse to complete the picture of astrocyte behavior throughout the disease course. Astrocytes at symptomatic and late-stage disease show a distinct up-regulation of transcripts defining a reactive phenotype, such as those involved in the lysosome and phagocytic pathways. Functional analysis of hexosaminidase B enzyme activity in the spinal cord and of astrocyte phagocytic ability has demonstrated a significant increase in lysosomal enzyme activity and phagocytic activity in SOD1(G93A) vs. littermate controls, validating the findings of the microarray study. In addition to the increased reactivity seen at both stages, astrocytes from late-stage disease showed decreased expression of many transcripts involved in cholesterol homeostasis. Staining for the master regulator of cholesterol synthesis, SREBP2, has revealed an increased localization to the cytoplasm of astrocytes and motor neurons in late-stage SOD1(G93A) spinal cord, indicating that down-regulation of transcripts may be due to an excess of cholesterol in the CNS during late-stage disease possibly due to phagocytosis of neuronal debris. Our data reveal that SOD1(G93A) astrocytes are characterized more by a loss of supportive function than a toxic phenotype during ALS disease progression and future studies should focus upon restorative therapies.
Project description:Adipose stromal cells (ASC) secrete various trophic factors that assist in the protection of neurons in a variety of neuronal death models. In this study, we tested the effects of human ASC conditional medium (ASC-CM) in human amyotrophic lateral sclerosis (ALS) transgenic mouse model expressing mutant superoxide dismutase (SOD1(G93A)). Treating symptomatic SOD1(G93A) mice with ASC-CM significantly increased post-onset survival time and lifespan. Moreover, SOD1(G93A) mice given ASC-CM treatment showed high motor neuron counts, less activation of microglia and astrocytes at an early symptomatic stage in the spinal cords under immunohistochemical analysis. SOD1(G93A) mice treated with ASC-CM for 7 days showed reduced levels of phosphorylated p38 (pp38) in the spinal cord, a mitogen-activated protein kinase that is involved in both inflammation and neuronal death. Additionally, the levels of α-II spectrin in spinal cords were also inhibited in SOD1(G93A) mice treated with ASC-CM for 3 days. Interestingly, nerve growth factor (NGF), a neurotrophic factor found in ASC-CM, played a significant role in the protection of neurodegeneration inSOD1(G93A) mouse. These results indicate that ASC-CM has the potential to develop into a novel and effective therapeutic treatment for ALS.
Project description:Despite some advances in the understanding of amyotrophic lateral sclerosis (ALS) pathogenesis, significant achievements in treating this disease are still lacking. Mesenchymal stromal (stem) cells (MSCs) have been shown to be effective in several models of neurological disease. To determine the effects of the intravenous injection of MSCs in an ALS mouse model during the symptomatic stage of disease, MSCs (1 × 10?) were intravenously injected in mice expressing human superoxide dismutase 1 (SOD1) carrying the G93A mutation (SOD1/G93A) presenting with experimental ALS. Survival, motor abilities, histology, oxidative stress markers and [³H]D-aspartate release in the spinal cord were investigated. MSC injection in SOD1/G93A mice improved survival and motor functions compared with saline-injected controls. Injected MSCs scantly home to the central nervous system and poorly engraft. We observed a reduced accumulation of ubiquitin agglomerates and of activated astrocytes and microglia in the spinal cord of MSC-treated SOD1/G93A mice, with no changes in the number of choline acetyltransferase- and glutamate transporter type 1-positive cells. MSC administration turned around the upregulation of metallothionein mRNA expression and of the activity of the antioxidant enzyme glutathione S-transferase, both associated with disease progression. Last, we observed that MSCs reverted both spontaneous and stimulus-evoked neuronal release of [³H]D-aspartate, a marker of endogenous glutamate, which is upregulated in SOD1/G93A mice. These findings suggest that intravenous administration of MSCs significantly improves the clinical outcome and pathological scores of mutant SOD1/G93A mice, thus providing the rationale for their exploitation for the treatment of ALS.
Project description:VGF mRNA is widely expressed in areas of the nervous system known to degenerate in Amyotrophic Lateral Sclerosis (ALS), including cerebral cortex, brainstem and spinal cord. Despite certain VGF alterations are reported in animal models, little information is available with respect to the ALS patients. We addressed VGF peptide changes in fibroblast cell cultures and in plasma obtained from ALS patients, in parallel with spinal cord and plasma samples from the G93A-SOD1 mouse model. Antisera specific for the C-terminal end of the human and mouse VGF proteins, respectively, were used in immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), while gel chromatography and HPLC/ESI-MS/MS were used to identify the VGF peptides present. Immunoreactive VGF C-terminus peptides were reduced in both fibroblast and plasma samples from ALS patients in an advanced stage of the disease. In the G93A-SOD1 mice, the same VGF peptides were also decreased in plasma in the late-symptomatic stage, while showing an earlier down-regulation in the spinal cord. In immunohistochemistry, a large number of gray matter structures were VGF C-terminus immunoreactive in control mice (including nerve terminals, axons and a few perikarya identified as motoneurons), with a striking reduction already in the pre-symptomatic stage. Through gel chromatography and spectrometry analysis, we identified one form likely to be the VGF precursor as well as peptides containing the NAPP- sequence in all tissues studied, while in the mice and fibroblasts, we revealed also AQEE- and TLQP- peptides. Taken together, selective VGF fragment depletion may participate in disease onset and/or progression of ALS.
Project description:The aim of the present study is to combine LCM and microarray analysis to study how astrocytes in the spinal cord of transgenic SOD1 G93A mice and their non-transgenic (NTg) littermates respond to stimuli determined by the presence of the human mutant protein throughout the evolution of the disease by looking at the symptomatic and late-stage disease time points. Astrocytes have been isolated from the spinal cord of G93A mice and non transgenic littermates at different time points and the transcription expression profile of the isolated astrocytes has been analysed
Project description:Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting predominantly motor neurons. Recent studies suggest that the disease progression of ALS is non-cell-autonomous, although the interaction between neurons and glial cells in different disease stages is not entirely clear. Here, we demonstrate that the interferon (IFN) signaling pathway is activated in human SOD1(G93A) transgenic mice, a rodent model of ALS. IFN-stimulated genes (ISGs) increased in the spinal cord of SOD1(G93A) mice at a presymptomatic age. In addition, the up-regulated ISGs, and most likely their transcriptional activators, were found specifically in astrocytes surrounding motor neurons, suggesting that IFN signaling in astrocytes was triggered by specific pathologic changes in motor neurons. Furthermore, induction of ISGs in cultured astrocytes was highly sensitive to IFN, especially Type I IFN. ISGs in astrocytes were activated specifically by endoplasmic reticulum stress-induced neurodegeneration in vitro, implicating a similar process in the presymptomatic stage of SOD1 mutant mice. Finally, reduction or deletion of IFN? receptor 1 inhibited IFN signaling and increased the life-span of SOD1(G93A) mice. Thus, the activation of IFN signaling pathways represents an early "dialogue" between motor neurons and astrocytes in response to pathological changes in ALS.
Project description:Cellular abnormalities in amyotrophic lateral sclerosis (ALS) are not limited to motor neurons. Astrocyte dysfunction also occurs in human ALS and transgenic rodents expressing mutant human SOD1 protein (SOD1(G93A)). Here we investigated focal enrichment of normal astrocytes using transplantation of lineage-restricted astrocyte precursors, called glial-restricted precursors (GRPs). We transplanted GRPs around cervical spinal cord respiratory motor neuron pools, the principal cells whose dysfunction precipitates death in ALS. GRPs survived in diseased tissue, differentiated efficiently into astrocytes and reduced microgliosis in the cervical spinal cords of SOD1(G93A) rats. GRPs also extended survival and disease duration, attenuated motor neuron loss and slowed declines in forelimb motor and respiratory physiological functions. Neuroprotection was mediated in part by the primary astrocyte glutamate transporter GLT1. These findings indicate the feasibility and efficacy of transplantation-based astrocyte replacement and show that targeted multisegmental cell delivery to the cervical spinal cord is a promising therapeutic strategy for slowing focal motor neuron loss associated with ALS.
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive fatal neurodegenerative disease characterised by loss of motor neurons that currently has no cure. Omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have many health benefits including neuroprotective and myoprotective potential. We tested the hypothesis that a high level of dietary EPA could exert beneficial effects in ALS. The dietary exposure to EPA (300 mg/kg/day) in a well-established mouse model of ALS expressing the G93A superoxide dismutase 1 (SOD1) mutation was initiated at a pre-symptomatic or symptomatic stage, and the disease progression was monitored until the end stage. Daily dietary EPA exposure initiated at the disease onset did not significantly alter disease presentation and progression. In contrast, EPA treatment initiated at the pre-symptomatic stage induced a significantly shorter lifespan. In a separate group of animals sacrificed before the end stage, the tissue analysis showed that the vacuolisation detected in G93A-SOD1 mice was significantly increased by exposure to EPA. Although EPA did not alter motor neurone loss, EPA reversed the significant increase in activated microglia and the astrocytic activation seen in G93A-SOD1 mice. The microglia in the spinal cord of G93A-SOD1 mice treated with EPA showed a significant increase in 4-hydroxy-2-hexenal, a highly toxic aldehydic oxidation product of omega-3 fatty acids. These data show that dietary EPA supplementation in ALS has the potential to worsen the condition and accelerate the disease progression. This suggests that great caution should be exerted when considering dietary omega-3 fatty acid supplements in ALS patients.
Project description:<h4>Background</h4>Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1(G93A) rats leads to a moderate therapeutical effect as evidenced by local ?-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1(G93A) rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1(G93A) animals.<h4>Methods/principal findings</h4>Presymptomatic SOD1(G93A) rats (60-65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1(G93A) rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1(G93A) rats protected ?-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to ?-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1(G93A) rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50-65%) loss of large caliber descending motor axons.<h4>Conclusions/significance</h4>These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.
Project description:Motoneuron loss and reactive astrocytosis are pathological hallmarks of amyotrophic lateral sclerosis (ALS), a paralytic neurodegenerative disease that can be triggered by mutations in Cu-Zn superoxide dismutase (SOD1). Dysfunctional astrocytes contribute to ALS pathogenesis, inducing motoneuron damage and accelerating disease progression. However, it is unknown whether ALS progression is associated with the appearance of a specific astrocytic phenotype with neurotoxic potential. Here, we report the isolation of astrocytes with aberrant phenotype (referred as "AbA cells") from primary spinal cord cultures of symptomatic rats expressing the SOD1(G93A) mutation. Isolation was based on AbA cells' marked proliferative capacity and lack of replicative senescence, which allowed oligoclonal cell expansion for 1 y. AbA cells displayed astrocytic markers including glial fibrillary acidic protein, S100? protein, glutamine synthase, and connexin 43 but lacked glutamate transporter 1 and the glial progenitor marker NG2 glycoprotein. Notably, AbA cells secreted soluble factors that induced motoneuron death with a 10-fold higher potency than neonatal SOD1(G93A) astrocytes. AbA-like aberrant astrocytes expressing S100? and connexin 43 but lacking NG2 were identified in nearby motoneurons, and their number increased sharply after disease onset. Thus, AbA cells appear to be an as-yet unknown astrocyte population arising during ALS progression with unprecedented proliferative and neurotoxic capacity and may be potential cellular targets for slowing ALS progression.