ABSTRACT
BACKGROUND AIMS: Adipose mesenchymal stem cells (ASCs) represent a promising therapeutic approach in inflammatory neurological disorders, including multiple sclerosis (MS). Recent lines of evidence indicate that most biological activities of ASCs are mediated by the delivery of soluble factors enclosed in extracellular vesicles (EVs). Indeed, we have previously demonstrated that small EVs derived from ASCs (ASC-EVs) ameliorate experimental autoimmune encephalomyelitis (EAE), a murine model of MS. The precise mechanisms and molecular/cellular target of EVs during EAE are still unknown. METHODS: To investigate the homing of ASC-EVs, we intravenously injected small EVs loaded with ultra-small superparamagnetic iron oxide nanoparticles (USPIO) at disease onset in EAE-induced C57Bl/6J mice. Histochemical analysis and transmission electron microscopy were carried out 48 h after EV treatment. Moreover, to assess the cellular target of EVs, flow cytometry on cells extracted ex vivo from EAE mouse lymph nodes was performed. RESULTS: Histochemical and ultrastructural analysis showed the presence of labeled EVs in lymph nodes but not in lungs and spinal cord of EAE injected mice. Moreover, we identified the cellular target of EVs in EAE lymph nodes by flow cytometry: ASC-EVs were preferentially located in macrophages, with a consistent amount also noted in dendritic cells and CD4+ T lymphocytes. CONCLUSIONS: This represents the first direct evidence of the privileged localization of ASC-EVs in draining lymph nodes of EAE after systemic injection. These data provide prominent information on the distribution, uptake and retention of ASC-EVs, which may help in the development of EV-based therapy in MS.
Subject(s)
Encephalomyelitis, Autoimmune, Experimental , Extracellular Vesicles , Mesenchymal Stem Cells , Multiple Sclerosis , Mice , Animals , Encephalomyelitis, Autoimmune, Experimental/therapy , Encephalomyelitis, Autoimmune, Experimental/pathology , Multiple Sclerosis/therapy , Multiple Sclerosis/pathology , Lymph Nodes , Mice, Inbred C57BLABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive degeneration of upper and lower motor neurons (MNs) in the brain and spinal cord, leading to progressive paralysis and death. Increasing evidence indicates that neuroinflammation plays an important role in ALS's pathogenesis and disease progression. Neuroinflammatory responses, primarily driven by activated microglia and astrocytes, and followed by infiltrating peripheral immune cells, contribute to exacerbate/accelerate MN death. In particular, the role of the microglia in ALS remains unclear, partly due to the lack of experimental models that can fully recapitulate the complexity of ALS's pathology. In this study, we developed and characterized a microglial cell line, SIM-A9-expressing human mutant protein Cu+/Zn+ superoxide dismutase_1 (SIM-A9hSOD1(G93A)), as a suitable model in vitro mimicking the microglia activity in ALS. The expression of hSOD1(G93A) in SIM-A9 cells induced a change in their metabolic activity, causing polarization into a pro-inflammatory phenotype and enhancing reactive oxygen species production, which is known to activate cell death processes and apoptosis. Afterward, we used our microglial model as an experimental set-up to investigate the therapeutic action of extracellular vesicles isolated from adipose mesenchymal stem cells (ASC-EVs). ASC-EVs represent a promising therapeutic treatment for ALS due to their neuroprotective and immunomodulatory properties. Here, we demonstrated that treatment with ASC-EVs is able to modulate activated ALS microglia, reducing their metabolic activity and polarizing their phenotype toward an anti-inflammatory one through a mechanism of reduction of reactive oxygen species.
Subject(s)
Amyotrophic Lateral Sclerosis , Extracellular Vesicles , Mesenchymal Stem Cells , Microglia , Superoxide Dismutase-1 , Amyotrophic Lateral Sclerosis/therapy , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Extracellular Vesicles/metabolism , Microglia/metabolism , Mesenchymal Stem Cells/metabolism , Humans , Superoxide Dismutase-1/metabolism , Superoxide Dismutase-1/genetics , Reactive Oxygen Species/metabolism , Cell Line , Adipose Tissue/cytology , Adipose Tissue/metabolismABSTRACT
Down syndrome is a common genetic disorder caused by partial or complete triplication of chromosome 21. This syndrome shows an overall and progressive impairment of olfactory function, detected early in adulthood. The olfactory neuronal cells are located in the nasal olfactory mucosa and represent the first sensory neurons of the olfactory pathway. Herein, we applied the olfactory swabbing procedure to allow a gentle collection of olfactory epithelial cells in seven individuals with Down syndrome and in ten euploid controls. The aim of this research was to investigate the peripheral gene expression pattern in olfactory epithelial cells through RNAseq analysis. Validated tests (Sniffin' Sticks Extended test) were used to assess olfactory function. Olfactory scores were correlated with RNAseq results and cognitive scores (Vineland II and Leiter scales). All Down syndrome individuals showed both olfactory deficit and intellectual disability. Down syndrome individuals and euploid controls exhibited clear expression differences in genes located in and outside the chromosome 21. In addition, a significant correlation was found between olfactory test scores and gene expression, while a non-significant correlation emerged between olfactory and cognitive scores. This first preliminary step gives new insights into the Down syndrome olfactory system research, starting from the olfactory neuroepithelium, the first cellular step on the olfactory way.
Subject(s)
Down Syndrome , Olfaction Disorders , Humans , Pilot Projects , Olfaction Disorders/etiology , Odorants , Smell/physiologyABSTRACT
Neurodegenerative diseases are fatal disorders of the central nervous system (CNS) which currently lack effective treatments. The application of mesenchymal stem cells (MSCs) represents a new promising approach for treating these incurable disorders. Growing evidence suggest that the therapeutic effects of MSCs are due to the secretion of neurotrophic molecules through extracellular vesicles. The extracellular vesicles produced by MSCs (MSC-EVs) have valuable innate properties deriving from parental cells and could be exploited as cell-free treatments for many neurological diseases. In particular, thanks to their small size, they are able to overcome biological barriers and reach lesion sites inside the CNS. They have a considerable pharmacokinetic and safety profile, avoiding the critical issues related to the fate of cells following transplantation. This review discusses the therapeutic potential of MSC-EVs in the treatment of neurodegenerative diseases, focusing on the strategies to further enhance their beneficial effects such as tracking methods, bioengineering applications, with particular attention to intranasal delivery as a feasible strategy to deliver MSC-EVs directly to the CNS in an effective and minimally invasive way. Current progresses and limiting issues to the extent of the use of MSC-EVs treatment for human neurodegenerative diseases will be also revised.
Subject(s)
Extracellular Vesicles , Mesenchymal Stem Cell Transplantation , Mesenchymal Stem Cells , Neurodegenerative Diseases , Humans , Neurodegenerative Diseases/therapy , Central Nervous System , Mesenchymal Stem Cells/physiologyABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disorder. There is no cure and current treatments fail to slow the progression of the disease. Epigenetic modulation in the acetylation state of NF-kB RelA and the histone 3 (H3) protein, involved in the development of neurodegeneration, is a drugable target for the class-I histone deacetylases (HDAC) inhibitors, entinostat or valproate, and the AMP-activated kinase (AMPK)-sirtuin 1 pathway activator, resveratrol. In this study, we demonstrated that the combination of valproate and resveratrol can restore the normal acetylation state of RelA in the SOD1(G93A) murine model of ALS, in order to obtain the neuroprotective form of NF-kB. We also investigated the sexually dimorphic development of the disease, as well as the sex-sensibility to the treatment administered. We showed that the combined drugs, which rescued AMPK activation, RelA and the histone 3 acetylation state, reduced the motor deficit and the disease pathology associated with motor neuron loss and microglial reactivity, Brain-Derived Neurotrophic Factor (BDNF) and B-cell lymphoma-extra large (Bcl-xL) level decline. Specifically, vehicle-administered males showed earlier onset and slower progression of the disease when compared to females. The treatment, administered at 50 days of life, postponed the time of onset in the male by 22 days, but not in a significant way in females. Nevertheless, in females, the drugs significantly reduced symptom severity of the later phase of the disease and prolonged the mice's survival. Only minor beneficial effects were produced in the latter stage in males. Overall, this study shows a beneficial and sexually dimorphic response to valproate and resveratrol treatment in ALS mice.
Subject(s)
Amyotrophic Lateral Sclerosis , AMP-Activated Protein Kinases/metabolism , Amyotrophic Lateral Sclerosis/drug therapy , Amyotrophic Lateral Sclerosis/pathology , Animals , Disease Models, Animal , Female , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/therapeutic use , Histones/metabolism , Male , Mice , Mice, Transgenic , NF-kappa B/metabolism , Resveratrol/pharmacology , Resveratrol/therapeutic use , Sirtuin 1/metabolism , Superoxide Dismutase/metabolism , Valproic Acid/pharmacology , Valproic Acid/therapeutic useABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive degeneration of motoneurons. To date, there is no effective treatment available. Exosomes are extracellular vesicles that play important roles in intercellular communication, recapitulating the effect of origin cells. In this study, we tested the potential neuroprotective effect of exosomes isolated from adipose-derived stem cells (ASC-exosomes) on the in vivo model most widely used to study ALS, the human SOD1 gene with a G93A mutation (SOD1(G93A)) mouse. Moreover, we compared the effect of two different routes of exosomes administration, intravenous and intranasal. The effect of exosomes administration on disease progression was monitored by motor tests and analysis of lumbar motoneurons and glial cells, neuromuscular junction, and muscle. Our results demonstrated that repeated administration of ASC-exosomes improved the motor performance; protected lumbar motoneurons, the neuromuscular junction, and muscle; and decreased the glial cells activation in treated SOD1(G93A) mice. Moreover, exosomes have the ability to home to lesioned ALS regions of the animal brain. These data contribute by providing additional knowledge for the promising use of ASC-exosomes as a therapy in human ALS.
Subject(s)
Amyotrophic Lateral Sclerosis/therapy , Exosomes/transplantation , Mesenchymal Stem Cell Transplantation/methods , Adipose Tissue/cytology , Amyotrophic Lateral Sclerosis/genetics , Animals , Cells, Cultured , Mice , Mice, Inbred C57BL , Motor Neurons/metabolism , Motor Neurons/physiology , Movement , Mutation, Missense , Neuromuscular Junction/metabolism , Neuromuscular Junction/physiology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolismABSTRACT
PURPOSE: The first part of the experiment identifies and validates MRI biomarkers distinctive of the disease progression in the transgenic superoxide dismutase gene (SOD1(G93A)) animal model. The second part assesses the efficacy of a mesenchymal stem cell-based therapy through the MRI biomarkers previously defined. METHODS: The first part identifies MRI differences between SOD1(G93A) and healthy mice. The second part of the experiment follows the disease evolution of stem cell-treated and non-stem-cell treated SOD1(G93A) mice. The analysis focused on voxel-based morphometry and T2 mapping on the brain tissues, and T2-weighted imaging and diffusion tensor imaging (DTI) on the hind limbs. RESULTS: Comparing diseased mice to healthy control revealed gray matter alterations in the brainstem area, accompanied by increased T2 relaxation time. Differences in muscle volume, muscle signal intensity, fractional anisotropy, axial diffusivity, and radial diffusivity were measured in the hind limbs. In the comparison between stem cell-treated mice and nontreated ones, differences in muscle volume, muscle signal intensity, and DTI-derived maps were found. CONCLUSION: MRI-derived biomarkers can be used to identify differences between stem cell-treated and nontreated SOD1(G93A) mice. Magn Reson Med 79:459-469, 2018. © 2017 International Society for Magnetic Resonance in Medicine.
Subject(s)
Image Processing, Computer-Assisted , Magnetic Resonance Imaging , Stem Cell Transplantation , Stem Cells , Superoxide Dismutase-1/genetics , Animals , Anisotropy , Behavior, Animal , Biomarkers/metabolism , Brain/metabolism , Brain Stem/metabolism , Diffusion Tensor Imaging , Disease Models, Animal , Disease Progression , Female , Humans , Male , Mice , Phenotype , Promoter Regions, Genetic , Superoxide Dismutase/genetics , TransgenesABSTRACT
Malignant gliomas, the most frequent primary brain tumors, are characterized by a dismal prognosis. Reliable biomarkers complementary to neuroradiology in the differential diagnosis of gliomas and monitoring for post-surgical progression are unmet needs. Altered expression of several microRNAs in tumour tissues from patients with gliomas compared to normal brain tissue have been described, thus supporting the rationale of using microRNA-based biomarkers. Although different circulating microRNAs were proposed in association with gliomas, they have not been introduced into clinical practice so far. Blood samples were collected from patients with high and low grade gliomas, both before and after surgical resection, and the expression of miR-21, miR-222 and miR-124-3p was measured in exosomes isolated from serum. The expression levels of miR-21, miR-222 and miR-124-3p in serum exosomes of patients with high grade gliomas were significantly higher than those of low grade gliomas and healthy controls and were sharply decreased in samples obtained after surgery. The analysis of miR-21, miR-222 and miR-124-3p in serum exosomes of patients affected by gliomas can provide a minimally invasive and innovative tool to help the differential diagnosis of gliomas at their onset in the brain and predict glioma grading and non glial metastases before surgery.
Subject(s)
Brain Neoplasms/blood , Brain Neoplasms/diagnosis , Exosomes/metabolism , Glioma/blood , Glioma/diagnosis , MicroRNAs/blood , Adult , Aged , Biomarkers, Tumor , Brain Neoplasms/surgery , Diagnosis, Differential , Female , Glioma/surgery , Humans , Male , Middle Aged , Sensitivity and SpecificityABSTRACT
Therapeutic strategies for the fatal neurodegenerative disease amyotrophic lateral sclerosis (ALS) have not yet provided satisfactory results. Interest in stem cells for the treatment of neurodegenerative diseases is increasing and their beneficial action seems to be due to a paracrine effect via the release of exosomes, main mediators of cell-cell communication. Here we wished to assess, in vitro, the efficacy of a novel non-cell therapeutic approach based on the use of exosomes derived from murine adipose-derived stromal cells on motoneuron-like NSC-34 cells expressing ALS mutations, and used as in vitro models of disease. In particular, we set out to investigate the effect of exosomes on NSC-34 naïve cells and NSC-34 cells overexpressing human SOD1(G93A) or SOD1(G37R) or SOD1(A4V) mutants, exposed to oxidative stress. The data presented here indicate for the first time that exosomes (0.2 µg/ml) are able to protect NSC-34 cells from oxidative damage, which is one of the main mechanism of damage in ALS, increasing cell viability. These data highlight a promising role of exosomes derived from stem cells for potential therapeutic applications in motoneuron disease.
Subject(s)
Adipose Tissue/cytology , Amyotrophic Lateral Sclerosis/pathology , Exosomes/metabolism , Models, Biological , Neuroprotective Agents , Stem Cells/cytology , Stromal Cells/cytology , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/metabolism , Animals , Apoptosis/drug effects , Cell Survival , Cells, Cultured , Humans , Hydrogen Peroxide/pharmacology , Mice , Mice, Inbred C57BL , Mutation , Oxidative Stress/geneticsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving motor neuron (MN) loss in the motor cortex, brainstem and spinal cord leading to progressive paralysis and death. Due to the pathogenetic complexity, there are no effective therapies available. In this context the use of mesenchymal stem cells and their vesicular counterpart is an emerging therapeutic strategy to counteract neurodegeneration. The extracellular vesicles derived from adipose stem cells (ASC-EVs) recapitulate and ameliorate the neuroprotective effect of stem cells and, thanks to their small dimensions, makes their use suitable to develop novel therapeutic approaches for neurodegenerative diseases as ALS. Here we investigate a therapeutic regimen of ASC-EVs injection in SOD1(G93A) mice, the most widely used murine model of ALS. Repeated intranasal administrations of high doses of ASC-EVs were able to ameliorate motor performance of injected SOD1(G93A) mice at the early stage of the disease and produce a significant improvement at the end-stage in the lumbar MNs rescue. Moreover, ASC-EVs preserve the structure of neuromuscular junction without counteracting the muscle atrophy. The results indicate that the intranasal ASC-EVs administration acts in central nervous system sites rather than at peripheral level in SOD1(G93A) mice. These considerations allow us to identify future applications of ASC-EVs that involve different targets simultaneously to maximize the clinical and neuropathological outcomes in ALS in vivo models.
Subject(s)
Amyotrophic Lateral Sclerosis , Extracellular Vesicles , Mesenchymal Stem Cells , Superoxide Dismutase-1 , Animals , Extracellular Vesicles/metabolism , Mesenchymal Stem Cells/metabolism , Mice , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/therapy , Amyotrophic Lateral Sclerosis/pathology , Superoxide Dismutase-1/genetics , Superoxide Dismutase-1/metabolism , Mice, Transgenic , Disease Models, Animal , Adipose Tissue/metabolism , Motor Neurons/metabolism , Neuromuscular Junction/metabolismABSTRACT
BACKGROUND: Spinal Muscular Atrophy (SMA) is an autosomal-recessive neuromuscular disease affecting children. It is caused by the mutation or deletion of the survival motor neuron 1 (SMN1) gene resulting in lower motor neuron (MN) degeneration followed by motor impairment, progressive skeletal muscle paralysis and respiratory failure. In addition to the already existing therapies, a possible combinatorial strategy could be represented by the use of adipose-derived mesenchymal stem cells (ASCs) that can be obtained easily and in large amounts from adipose tissue. Their efficacy seems to be correlated to their paracrine activity and the production of soluble factors released through extracellular vesicles (EVs). EVs are important mediators of intercellular communication with a diameter between 30 and 100 nm. Their use in other neurodegenerative disorders showed a neuroprotective effect thanks to the release of their content, especially proteins, miRNAs and mRNAs. METHODS: In this study, we evaluated the effect of EVs isolated from ASCs (ASC-EVs) in the SMNΔ7 mice, a severe SMA model. With this purpose, we performed two administrations of ASC-EVs (0.5 µg) in SMA pups via intracerebroventricular injections at post-natal day 3 (P3) and P6. We then assessed the treatment efficacy by behavioural test from P2 to P10 and histological analyses at P10. RESULTS: The results showed positive effects of ASC-EVs on the disease progression, with improved motor performance and a significant delay in spinal MN degeneration of treated animals. ASC-EVs could also reduce the apoptotic activation (cleaved Caspase-3) and modulate the neuroinflammation with an observed decreased glial activation in lumbar spinal cord, while at peripheral level ASC-EVs could only partially limit the muscular atrophy and fiber denervation. CONCLUSIONS: Our results could encourage the use of ASC-EVs as a therapeutic combinatorial treatment for SMA, bypassing the controversial use of stem cells.
Subject(s)
Extracellular Vesicles , Muscular Atrophy, Spinal , Humans , Child , Mice , Animals , Disease Models, Animal , Muscular Atrophy, Spinal/genetics , Muscular Atrophy, Spinal/therapy , Muscular Atrophy, Spinal/pathology , Motor Neurons , Stem Cells/metabolism , Extracellular Vesicles/metabolismABSTRACT
Background: Cladribine has been introduced as a high-efficacy drug for treating relapsing-remitting multiple sclerosis (RRMS). Initial cohort studies showed early disease activity in the first year after drug initiation. Biomarkers that can predict early disease activity are needed. Aim: To estimate cerebrospinal fluid (CSF) markers of clinical and radiological responses after initiation of cladribine. Methods: Forty-two RRMS patients (30F/12M) treated with cladribine were included in a longitudinal prospective study. All patients underwent a CSF examination at treatment initiation, clinical follow-up including Expanded Disability Status Scale (EDSS) assessment, and a 3T MRI scan after 6,12 and 24 months, including the evaluation of white matter (WM) and cortical lesions (CLs). CSF levels of 67 inflammatory markers were assessed with immune-assay multiplex techniques. The 'no evidence of disease activity' (NEDA-3) status was assessed after two years and defined by no relapses, no disability worsening measured by EDSS and no MRI activity, including CLs. Results: Three patients were lost at follow-up. At the end of follow-up, 19 (48%) patients remained free from disease activity. IFNgamma, Chitinase3like1, IL32, Osteopontin, IL12(p40), IL34, IL28A, sTNFR2, IL20 and CCL2 showed the best association with disease activity. When added in a multivariate regression model including age, sex, and baseline EDSS, Chitinase 3 like1 (p = 0.049) significantly increased in those patients with disease activity. Finally, ROC analysis with Chitinase3like1 added to a model with EDSS, sex, age previous relapses, WM lesion number, CLs, number of Gad enhancing lesions and spinal cord lesions provided an AUC of 0.76 (95%CI 0.60-0.91). Conclusions: CSF Chitinase 3 like1 might provide prognostic information for predicting disease activity in the first years after initiation of cladribine. The drug's effect on chronic macrophage and microglia activation deserves further evaluation.
Subject(s)
Chitinase-3-Like Protein 1 , Cladribine , Multiple Sclerosis, Relapsing-Remitting , Humans , Cladribine/therapeutic use , Multiple Sclerosis, Relapsing-Remitting/diagnostic imaging , Multiple Sclerosis, Relapsing-Remitting/drug therapy , Prospective Studies , Chitinase-3-Like Protein 1/cerebrospinal fluidABSTRACT
BACKGROUND AND OBJECTIVES: To evaluate CSF inflammatory markers with accumulation of cortical damage as well as disease activity in patients with early relapsing-remitting MS (RRMS). METHODS: CSF levels of osteopontin (OPN) and 66 inflammatory markers were assessed using an immune-assay multiplex technique in 107 patients with RRMS (82 F/25 M, mean age 35.7 ± 11.8 years). All patients underwent regular clinical assessment and yearly 3T MRI scans for 2 years while 39 patients had a 4-year follow-up. White matter lesion number and volume, cortical lesions (CLs) and volume, and global cortical thickness (CTh) were evaluated together with the 'no evidence of disease activity' (NEDA-3) status, defined by no relapses, no disability worsening, and no MRI activity, including CLs. RESULTS: The random forest algorithm selected OPN, CXCL13, TWEAK, TNF, IL19, sCD30, sTNFR1, IL35, IL16, and sCD163 as significantly associated with changes in global CTh. OPN and CXCL13 were most related to accumulation of atrophy after 2 and 4 years. In a multivariate linear regression model on CSF markers, OPN (p < 0.001), CXCL13 (p = 0.001), and sTNFR1 (p = 0.024) were increased in those patients with accumulating atrophy (adjusted R-squared 0.615). The 10 markers were added in a model that included all clinical, demographic, and MRI variables: OPN (p = 0.002) and IL19 (p = 0.022) levels were confirmed to be significantly increased in patients developing more CTh change over the follow-up (adjusted R-squared 0.619). CXCL13 and OPN also revealed the best association with NEDA-3 after 2 years, with OPN significantly linked to disability accumulation (OR 2.468 [1.46-5.034], p = 0.004) at the multivariate logistic regression model. DISCUSSION: These data confirm and expand our knowledge on the prognostic role of the CSF inflammatory profile in predicting changes in cortical pathology and disease activity in early MS. The data emphasize a crucial role of OPN.
Subject(s)
Atrophy , Cerebral Cortex , Multiple Sclerosis, Relapsing-Remitting , Osteopontin , Humans , Osteopontin/cerebrospinal fluid , Female , Male , Adult , Multiple Sclerosis, Relapsing-Remitting/cerebrospinal fluid , Multiple Sclerosis, Relapsing-Remitting/pathology , Multiple Sclerosis, Relapsing-Remitting/diagnostic imaging , Atrophy/pathology , Middle Aged , Cerebral Cortex/pathology , Cerebral Cortex/diagnostic imaging , Magnetic Resonance Imaging , Biomarkers/cerebrospinal fluid , Follow-Up Studies , Young Adult , Disease ProgressionABSTRACT
Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.
Subject(s)
Amyotrophic Lateral Sclerosis/pathology , Anabolic Agents/pharmacology , Nandrolone/pharmacology , Neuromuscular Junction/ultrastructure , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/genetics , Animals , Disease Models, Animal , Humans , Male , Mice , Mice, Transgenic , Mitochondria/ultrastructure , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Muscle Fibers, Skeletal/physiology , Mutation , Neuromuscular Junction/drug effects , Neuromuscular Junction/genetics , Neuromuscular Junction/physiopathology , Presynaptic Terminals/physiology , Presynaptic Terminals/ultrastructure , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Synaptic Vesicles/ultrastructureABSTRACT
Anabolic/androgenic steroids (AAS) are drugs that enhance muscle mass, and are often illegally utilized in athletes to improve their performances. Recent data suggest that the increased risk for amyotrophic lateral sclerosis (ALS) in male soccer and football players could be linked to AAS abuse. ALS is a motor neuron disease mainly occurring in sporadic (sALS) forms, but some familial forms (fALS) exist and have been linked to mutations in different genes. Some of these, in their wild type (wt) form, have been proposed as risk factors for sALS, i.e. superoxide dismutase 1 (SOD1) gene, whose mutations are causative of about 20% of fALS. Notably, SOD1 toxicity might occur both in motor neurons and in muscle cells. Using gastrocnemius muscles of mice overexpressing human mutant SOD1 (mutSOD1) at different disease stages, we found that the expression of a selected set of genes associated to muscle atrophy, MyoD, myogenin, atrogin-1, and transforming growth factor (TGF)ß1, is up-regulated already at the presymptomatic stage. Atrogin-1 gene expression was increased also in mice overexpressing human wtSOD1. Similar alterations were found in axotomized mouse muscles and in cultured ALS myoblast models. In these ALS models, we then evaluated the pharmacological effects of the synthetic AAS nandrolone on the expression of the genes modified in ALS muscle. Nandrolone administration had no effects on MyoD, myogenin, and atrogin-1 expression, but it significantly increased TGFß1 expression at disease onset. Altogether, these data suggest that, in fALS, muscle gene expression is altered at early stages, and AAS may exacerbate some of the alterations induced by SOD1 possibly acting as a contributing factor also in sALS.
Subject(s)
Amyotrophic Lateral Sclerosis/genetics , Gene Expression/drug effects , Mutation , Nandrolone/pharmacology , Superoxide Dismutase/genetics , Amyotrophic Lateral Sclerosis/enzymology , Amyotrophic Lateral Sclerosis/metabolism , Anabolic Agents/pharmacology , Androgens/pharmacology , Animals , Cells, Cultured , Disease Models, Animal , Humans , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Motor Neurons/drug effects , Motor Neurons/metabolism , Muscle Proteins/genetics , Muscle Proteins/metabolism , Muscle, Skeletal/drug effects , Muscle, Skeletal/metabolism , Muscular Atrophy/genetics , Muscular Atrophy/metabolism , MyoD Protein/biosynthesis , MyoD Protein/genetics , MyoD Protein/metabolism , Myoblasts/drug effects , Myoblasts/metabolism , Myogenin/biosynthesis , Myogenin/genetics , Myogenin/metabolism , SKP Cullin F-Box Protein Ligases/genetics , SKP Cullin F-Box Protein Ligases/metabolism , Superoxide Dismutase/biosynthesis , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Transforming Growth Factor beta/biosynthesis , Transforming Growth Factor beta/genetics , Transforming Growth Factor beta/metabolism , Up-Regulation/drug effectsABSTRACT
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease associated with motor neuron degeneration, progressive paralysis and finally death. Despite the research efforts, currently there is no cure for ALS. In recent years, multiple epigenetic mechanisms have been associated with neurodegenerative diseases. A pathological role for histone hypoacetylation and the abnormal NF-κB/RelA activation involving deacetylation of lysines, with the exclusion of lysine 310, has been established in ALS. Recent findings indicate that the pathological acetylation state of NF-κB/RelA and histone 3 (H3) occurring in the SOD1(G93A) murine model of ALS can be corrected by the synergistic combination of low doses of the AMP-activated kinase (AMPK)-sirtuin 1 pathway activator resveratrol and the histone deacetylase (HDAC) inhibitors MS-275 (entinostat) or valproate. The combination of the epigenetic drugs, by rescuing RelA and the H3 acetylation state, promotes a beneficial and sexually dimorphic effect on disease onset, survival and motor neurons degeneration. In this mini review, we discuss the potential of the epigenetic combination of resveratrol with HDAC inhibitors in the ALS treatment.
ABSTRACT
Lower motoneuron abnormalities have been extensively documented in the murine model of familial amyotrophic lateral sclerosis, whereas information on corticospinal neurons in these mice is very limited. We investigated 1) mRNA levels of inflammation-related molecules in the deep layers in which corticospinal neurons reside, 2) corticospinal neurons labeled from tracer injections in the corticospinal tract at the cervical level, 3) axonal damage revealed by beta-amyloid precursor protein accumulation, and 4) glial cell activation in the sensorimotor cortex of presymptomatic and end-stage superoxide dismutase (SOD)-1 (G93A) mice. We demonstrated induction of inflammatory gene transcripts in the deep layers, early and progressive shrinkage of corticospinal cell bodies and activation of surrounding astrocytes and microglia with upregulation of major histocompatibility complex class I antigen. Accumulation of beta-amyloid precursor protein in proximal axonal swellings indicating axonal injury was also evident at the terminal stage in the motor cortex and internal capsule. Glial and axon changes were not observed elsewhere in the cortex. These data reveal that the entire motor circuit is affected in this murine amyotrophic lateral sclerosis model as it is in human amyotrophic lateral sclerosis. Sensorimotor cortical inflammation and progressive corticospinal cell body and fiber damage may reflect transsynaptic signaling of damage from lower motoneurons.
Subject(s)
Amyotrophic Lateral Sclerosis , Axons/pathology , Gene Expression/genetics , Neurons/pathology , Somatosensory Cortex/pathology , Amyloid beta-Protein Precursor/metabolism , Amyotrophic Lateral Sclerosis/genetics , Amyotrophic Lateral Sclerosis/pathology , Amyotrophic Lateral Sclerosis/physiopathology , Animals , CD11b Antigen/metabolism , Cytokines/genetics , Cytokines/metabolism , Disease Models, Animal , Glial Fibrillary Acidic Protein/metabolism , Humans , Mice , Mice, Transgenic , Neuroglia/pathology , Pyramidal Tracts/pathology , Stilbamidines/metabolism , Superoxide Dismutase/genetics , Superoxide Dismutase-1ABSTRACT
The Lgi1 protein is involved in the pathogenesis of autosomal dominant lateral temporal epilepsy because mutations in the leucine-rich, glioma-inactivated 1 (LGI1) gene have been found in affected subjects and families; however, the function of Lgi1 is still unclear. Definition of the fine intracellular distribution of Lgi1 in normal cortical tissue would add in understanding its role and envisage pathogenetic mechanisms. Therefore, we analyzed normal rat cortex for Lgi1 by combining biochemistry, immunohistochemistry and immunoelectron microscopy. Our results demonstrate that Lgi1 is a cytoplasmic protein distributed both in the soma and in the processes of neurons: it occurs on the rough endoplasmic reticulum, the site of synthesis, in the Golgi complex, where it undergoes glycosylation, and in close proximity to neurotubules and neurofilaments, especially in the axons, but it is scarce at synapses and the neurilemma. Lgi1 association with axonal cytoskeletal structures would imply Lgi1 is either transported along axons by motor proteins, or playing some role as a carrier in the axonal Xux, or it could be involved in the regulation of cytoskeletal organization.
Subject(s)
Cerebral Cortex/cytology , Neurons/metabolism , Proteins/analysis , Proteins/metabolism , Animals , Immunohistochemistry , Intercellular Signaling Peptides and Proteins , Male , Neurons/cytology , Proteins/genetics , Rats , Rats, WistarABSTRACT
Testosterone and physical exercise administration have been shown to affect hippocampal morphology in adult rodents. In aged animals, similar data are only available after physical exercise. In this work we used ultrastructural quantitative morphometry to investigate the effect of testosterone administration on the hippocampal synapses of old mice, either alone or in combination with aerobic physical exercise. The inner molecular layer of the hippocampal dentate gyrus (IMLDG) and the molecular stratum of Ammon's horn 1 neurons (SMCA1) were investigated in 27-month-old male Balb/c mice randomly allocated to one of four experimental conditions (five mice each): sedentary control (C), testosterone administration (10â¯mg/kg once a week, TA), treadmill training (30â¯min a day, five days a week for 4â¯weeks at belt speed 8â¯m/min, 0% incline, TT) and testosterone administration plus treadmill training (TTTA). At the end of a four-week period, hippocampi were excised, fixed, and processed by ethanol phosphotungstic acid procedure to contrast synapses. The following variables were measured in electron micrographs: number of synapses/µm3 of tissue (Nv), total area of contact zones/µm3 of tissue (Sv), average area of the synaptic contact zone (S), and percentage of perforated synapses (%PS). ANOVA showed a statistically significant main effect of experimental condition for Nv and Sv in IMLDG, and for Sv in SMCA1 (pâ¯≤â¯0.003). The S and %PS were similar within group in ANOVA. Post-hoc analysis revealed a significant (pâ¯<â¯0.05) increase of Sv vs. C in SMCA1 and IMLDG after TT and TA, respectively. In IMLDG, Nv was significantly increased vs. C and TT after both TA and TTTA. Overall, results showed that testosterone increases synaptic density in IMLDG of old mice independently of physical exercise or changes in synaptic size. Instead, synaptic density in SMCA1 was only sensitive to physical exercise. These findings show that exogenous testosterone administration exerts a positive effect of on synapses in selected areas of the old mouse hippocampus.