RESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Syncytin-1 (Syn), an envelope glycoprotein encoded by the env gene of the human endogenous retrovirus-W family, has been resorted to be highly expressed in biopsies from the muscles from ALS patients; however, the specific regulatory role of Syn during ALS progression remains uncovered. In this study, C57BL/6 mice were injected with adeno-associated virus-overexpressing Syn, with or without Fasudil administration. The Syn expression was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry analysis. The histological change of anterior tibial muscles was determined by hematoxylin-eosin staining. Qualitative ultrastructural analysis of electron micrographs obtained from lumbar spinal cords was carried out. Serum inflammatory cytokines were assessed by enzyme linked immunosorbent assay (ELISA) assay and motor function was recorded using Basso, Beattie, and Bresnahan (BBB) scoring, climbing test and treadmill running test. Immunofluorescence and western blot assays were conducted to examine microglial- and motor neurons-related proteins. Syn overexpression significantly caused systemic inflammatory response, muscle tissue lesions, and motor dysfunction in mice. Meanwhile, Syn overexpression promoted the impairment of motor neuron, evidenced by the damaged structure of the neurons and reduced expression of microtubule-associated protein 2, HB9, neuronal nuclei and neuron-specific enolase in Syn-induced mice. In addition, Syn overexpression greatly promoted the expression of CD16/CD32 and inducible nitric oxide synthase (M1 phenotype markers), and reduced the expression of CD206 and arginase 1 (M2 phenotype markers). Importantly, the above changes caused by Syn overexpression were partly abolished by Fasudil administration. This study provides evidence that Syn-activated microglia plays a pivotal role during the progression of ALS.
Asunto(s)
1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Ratones Endogámicos C57BL , Microglía , Neuronas Motoras , Animales , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Ratones , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Productos del Gen env , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Gestacionales/metabolismo , Masculino , Citocinas/metabolismo , Modelos Animales de Enfermedad , Actividad Motora/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacosRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by progressive motoneuron degeneration, and effective clinical treatments are lacking. In this study, we evaluated whether intranasal delivery of mesenchymal stem cell-derived small extracellular vesicles (sEVs) is a strategy for ALS therapy using SOD1G93A mice. In vivo tracing showed that intranasally-delivered sEVs entered the central nervous system and were extensively taken up by spinal neurons and some microglia. SOD1G93A mice that intranasally received sEV administration showed significant improvements in motor performances and survival time. After sEV administration, pathological changes, including spinal motoneuron death and synaptic denervation, axon demyelination, neuromuscular junction degeneration and electrophysiological defects, and mitochondrial vacuolization were remarkably alleviated. sEV administration attenuated the elevation of proinflammatory cytokines and glial responses. Proteomics and transcriptomics analysis revealed upregulation of the complement and coagulation cascade and NF-ĸB signaling pathway in SOD1G93A mouse spinal cords, which was significantly inhibited by sEV administration. The changes were further confirmed by detecting C1q and NF-ĸB expression using Western blots. In conclusion, intranasal administration of sEVs effectively delays the progression of ALS by inhibiting neuroinflammation and overactivation of the complement and coagulation cascades and NF-ĸB signaling pathway and is a potential option for ALS therapy.
Asunto(s)
Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , FN-kappa B , Transducción de Señal , Animales , Masculino , Ratones , Administración Intranasal , Esclerosis Amiotrófica Lateral/metabolismo , Coagulación Sanguínea , Modelos Animales de Enfermedad , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas Motoras/metabolismo , FN-kappa B/metabolismo , Médula Espinal/metabolismo , Médula Espinal/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
The majority of severe early-onset and juvenile cases of amyotrophic lateral sclerosis (ALS) are caused by mutations in the FUS gene, resulting in rapid disease progression. Mutant FUS accumulates within stress granules (SGs), thereby affecting the dynamics of these ribonucleoprotein complexes. Here, we define the interactome of the severe mutant FUSP525L variant in human induced pluripotent stem cell (iPSC)-derived motor neurons. We find increased interaction of FUSP525L with the PARP1 enzyme, promoting poly-ADP-ribosylation (PARylation) and binding of FUS to histone H1.2. Inhibiting PARylation or reducing H1.2 levels alleviates mutant FUS aggregation, SG alterations, and apoptosis in human motor neurons. Conversely, elevated H1.2 levels exacerbate FUS-ALS phenotypes, driven by the internally disordered terminal domains of H1.2. In C. elegans models, knockdown of H1.2 and PARP1 orthologs also decreases FUSP525L aggregation and neurodegeneration, whereas H1.2 overexpression worsens ALS-related changes. Our findings indicate a link between PARylation, H1.2, and FUS with potential therapeutic implications.
Asunto(s)
Esclerosis Amiotrófica Lateral , Caenorhabditis elegans , Histonas , Mutación , Poli(ADP-Ribosa) Polimerasa-1 , Proteína FUS de Unión a ARN , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Humanos , Histonas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Animales , Mutación/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Poli ADP Ribosilación , Células Madre Pluripotentes Inducidas/metabolismo , Unión ProteicaRESUMEN
Given the increasing trend of aging population in the world, neurodegenerative diseases (NDDs), a common type of diseases that mostly occur in the elderly, have attracted much more attention. It has been shown that tumor necrosis factor receptor-associated factor 6 (TRAF6) is involved in the regulation of neuroinflammation, an important pathological feature of NDDs, and affects the occurrence and development of NDDs. Most importantly, the regulatory effect of TRAF6 is related to its ubiquitination. Therefore, in the present paper, the molecular structure, biological function, and ubiquitination mechanism of TRAF6, and its relationship with some common NDDs, including Alzheimer's disease, Parkinson's disease, multiple sclerosis, and amyotrophic lateral sclerosis, were analyzed and summarized. The possible molecular mechanisms by which TRAF6 regulates the occurrence of NDDs were also elucidated, providing a theoretical basis for exploring the etiology and treatment of NDDs.
Asunto(s)
Enfermedades Neurodegenerativas , Factor 6 Asociado a Receptor de TNF , Humanos , Enfermedades Neurodegenerativas/metabolismo , Enfermedades Neurodegenerativas/etiología , Factor 6 Asociado a Receptor de TNF/metabolismo , Factor 6 Asociado a Receptor de TNF/genética , Factor 6 Asociado a Receptor de TNF/fisiología , Ubiquitinación , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/fisiopatología , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/fisiopatología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/etiología , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/fisiopatología , Esclerosis Múltiple/etiologíaRESUMEN
Intermediate-length repeat expansions in ATAXIN-2 (ATXN2) are the strongest genetic risk factor for amyotrophic lateral sclerosis (ALS). At the molecular level, ATXN2 intermediate expansions enhance TDP-43 toxicity and pathology. However, whether this triggers ALS pathogenesis at the cellular and functional level remains unknown. Here, we combine patient-derived and mouse models to dissect the effects of ATXN2 intermediate expansions in an ALS background. iPSC-derived motor neurons from ATXN2-ALS patients show altered stress granules, neurite damage and abnormal electrophysiological properties compared to healthy control and other familial ALS mutations. In TDP-43Tg-ALS mice, ATXN2-Q33 causes reduced motor function, NMJ alterations, neuron degeneration and altered in vitro stress granule dynamics. Furthermore, gene expression changes related to mitochondrial function and inflammatory response are detected and confirmed at the cellular level in mice and human neuron and organoid models. Together, these results define pathogenic defects underlying ATXN2-ALS and provide a framework for future research into ATXN2-dependent pathogenesis and therapy.
Asunto(s)
Esclerosis Amiotrófica Lateral , Ataxina-2 , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas , Ratones Transgénicos , Neuronas Motoras , Péptidos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Ataxina-2/genética , Ataxina-2/metabolismo , Humanos , Animales , Péptidos/metabolismo , Péptidos/genética , Ratones , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fenotipo , Masculino , Femenino , Mitocondrias/metabolismo , Neuritas/metabolismoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disorder with minimally effective treatment options. An important hurdle in ALS drug development is the non-invasive therapeutic access to the motor cortex currently limited by the presence of the blood-brain barrier (BBB). Focused ultrasound and microbubble (FUS+ MB) treatment is an emerging technology that was successfully used in ALS patients to temporarily open the cortical BBB. However, FUS+ MB-mediated drug delivery across ALS patients' BBB has not yet been reported. Similarly, the effects of FUS+ MB on human ALS BBB cells remain unexplored. METHODS: Here we established the first FUS+ MB-compatible, fully-human ALS patient-cell-derived BBB model based on induced brain endothelial-like cells (iBECs) to study anti-TDP-43 antibody delivery and FUS+ MB bioeffects in vitro. RESULTS: Generated ALS iBECs recapitulated disease-specific hallmarks of BBB pathology, including reduced BBB integrity and permeability, and TDP-43 proteinopathy. The results also identified differences between sporadic ALS and familial (C9orf72 expansion carrying) ALS iBECs reflecting patient heterogeneity associated with disease subgroups. Studies in these models revealed successful ALS iBEC monolayer opening in vitro with no adverse cellular effects of FUS+ MB as reflected by lactate dehydrogenase (LDH) release viability assay and the lack of visible monolayer damage or morphology change in FUS+ MB treated cells. This was accompanied by the molecular bioeffects of FUS+ MB in ALS iBECs including changes in expression of tight and adherens junction markers, and drug transporter and inflammatory mediators, with sporadic and C9orf72 ALS iBECs generating transient specific responses. Additionally, we demonstrated an effective increase in the delivery of anti-TDP-43 antibody with FUS+ MB in C9orf72 (2.7-fold) and sporadic (1.9-fold) ALS iBECs providing the first proof-of-concept evidence that FUS+ MB can be used to enhance the permeability of large molecule therapeutics across the BBB in a human ALS in vitro model. CONCLUSIONS: Together, this study describes the first characterisation of cellular and molecular responses of ALS iBECs to FUS+ MB and provides a fully-human platform for FUS+ MB-mediated drug delivery screening on an ALS BBB in vitro model.
Asunto(s)
Esclerosis Amiotrófica Lateral , Barrera Hematoencefálica , Proteínas de Unión al ADN , Microburbujas , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Barrera Hematoencefálica/metabolismo , Barrera Hematoencefálica/efectos de los fármacos , Humanos , Proteínas de Unión al ADN/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Células Endoteliales/metabolismo , Anticuerpos/administración & dosificación , Ondas Ultrasónicas , Células CultivadasRESUMEN
Spinal motor neurons (MNs) represent a highly vulnerable cellular population, which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study, we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/- hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover, analysis of the SYT13+/- transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures, displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes, which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family, SYT13, is sufficient to trigger a series of abnormal alterations leading to MN sufferance, thus revealing novel insights into the selective vulnerability of this cell population.
Asunto(s)
Esclerosis Amiotrófica Lateral , Neuronas Motoras , Sinaptotagminas , Proteína p53 Supresora de Tumor , Humanos , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Sinaptotagminas/metabolismo , Sinaptotagminas/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Heterocigoto , Fenotipo , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Diferenciación Celular/genética , Técnicas de Inactivación de GenesRESUMEN
Post-translational modifications (PTMs) affecting proteins during or after their synthesis play a crucial role in their localization and function. The modification of these PTMs under pathophysiological conditions, i.e., their appearance, disappearance, or variation in quantity caused by a pathological environment or a mutation, corresponds to post-translational variants (PTVs). These PTVs can be directly or indirectly involved in the pathophysiology of diseases. Here, we present the PTMs and PTVs of four major amyotrophic lateral sclerosis (ALS) proteins, SOD1, TDP-43, FUS, and TBK1. These modifications involve acetylation, phosphorylation, methylation, ubiquitination, SUMOylation, and enzymatic cleavage. We list the PTM positions known to be mutated in ALS patients and discuss the roles of PTVs in the pathophysiological processes of ALS. In-depth knowledge of the PTMs and PTVs of ALS proteins is needed to better understand their role in the disease. We believe it is also crucial for developing new therapies that may be more effective in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Procesamiento Proteico-Postraduccional , Proteína FUS de Unión a ARN , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Humanos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Mutación , Animales , Fosforilación , AcetilaciónRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder distinguished by gradual depletion of motor neurons. RNA binding motif protein 5 (RBM5), an abundantly expressed RNA-binding protein, plays a critical role in the process of cellular death. However, little is known about the role of RBM5 in the pathogenesis of ALS. Here, we found that RBM5 was upregulated in ALS hSOD1G93A-NSC34 cell models and hSOD1G93A mice due to a reduction of miR-141-5p. The upregulation of RBM5 increased the apoptosis of motor neurons by inhibiting Rac1-mediated neuroprotection. In contrast, genetic knockdown of RBM5 rescued motor neurons from hSOD1G93A-induced degeneration by activating Rac1 signaling. The neuroprotective effect of RBM5-knockdown was significantly inhibited by the Rac1 inhibitor, NSC23766. These findings suggest that RBM5 could potentially serve as a therapeutic target in ALS by activating the Rac1 signalling.
Asunto(s)
Esclerosis Amiotrófica Lateral , Apoptosis , Neuronas Motoras , Proteínas Proto-Oncogénicas c-akt , Proteínas de Unión al ARN , Transducción de Señal , Proteína de Unión al GTP rac1 , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rac1/genética , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Apoptosis/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genética , Ratones , Humanos , Transducción de Señal/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratones Transgénicos , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/genética , Masculino , Proteínas de Unión al ADN , Proteínas de Ciclo Celular , Proteínas Supresoras de TumorRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , Células Madre Mesenquimatosas , Superóxido Dismutasa-1 , Animales , Vesículas Extracelulares/metabolismo , Células Madre Mesenquimatosas/metabolismo , Ratones , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Esclerosis Amiotrófica Lateral/patología , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Tejido Adiposo/metabolismo , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismoRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron (MN) degeneration, leading to neuromuscular junction (NMJ) dismantling and severe muscle atrophy. The nuclear receptor interaction protein (NRIP) functions as a multifunctional protein. It directly interacts with calmodulin or α-actinin 2, serving as a calcium sensor for muscle contraction and maintaining sarcomere integrity. Additionally, NRIP binds with the acetylcholine receptor (AChR) for NMJ stabilization. Loss of NRIP in muscles results in progressive motor neuron degeneration with abnormal NMJ architecture, resembling ALS phenotypes. Therefore, we hypothesize that NRIP could be a therapeutic factor for ALS. METHODS: We used SOD1 G93A mice, expressing human SOD1 with the ALS-linked G93A mutation, as an ALS model. An adeno-associated virus vector encoding the human NRIP gene (AAV-NRIP) was generated and injected into the muscles of SOD1 G93A mice at 60 days of age, before disease onset. Pathological and behavioral changes were measured to evaluate the therapeutic effects of AAV-NRIP on the disease progression of SOD1 G93A mice. RESULTS: SOD1 G93A mice exhibited lower NRIP expression than wild-type mice in both the spinal cord and skeletal muscle tissues. Forced NRIP expression through AAV-NRIP intramuscular injection was observed in skeletal muscles and retrogradely transduced into the spinal cord. AAV-NRIP gene therapy enhanced movement distance and rearing frequencies in SOD1 G93A mice. Moreover, AAV-NRIP increased myofiber size and slow myosin expression, ameliorated NMJ degeneration and axon terminal denervation at NMJ, and increased the number of α-motor neurons (α-MNs) and compound muscle action potential (CMAP) in SOD1 G93A mice. CONCLUSIONS: AAV-NRIP gene therapy ameliorates muscle atrophy, motor neuron degeneration, and axon terminal denervation at NMJ, leading to increased NMJ transmission and improved motor functions in SOD1 G93A mice. Collectively, AAV-NRIP could be a potential therapeutic drug for ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Ratones Transgénicos , Neuronas Motoras , Atrofia Muscular , Animales , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/terapia , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Terapia Genética/métodos , Atrofia Muscular/genética , Atrofia Muscular/terapia , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Dependovirus/genética , Ratones , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Unión Neuromuscular/metabolismo , Unión Neuromuscular/patología , Vectores Genéticos/administración & dosificación , Degeneración Nerviosa/genética , Degeneración Nerviosa/terapia , Masculino , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the dysfunction and death of motor neurons through multifactorial mechanisms that remain unclear. ALS has been recognized as a multisystemic disease, and the potential role of skeletal muscle in disease progression has been investigated. Reactive aldehydes formed as secondary lipid peroxidation products in the redox processes react with biomolecules, such as DNA, proteins, and amino acids, resulting in cytotoxic effects. 4-Hydroxy-2-nonenal (HNE) levels are elevated in the spinal cord motor neurons of ALS patients, and HNE-modified proteins have been identified in the spinal cord tissue of an ALS transgenic mice model, suggesting that reactive aldehydes can contribute to motor neuron degeneration in ALS. One biological pathway of aldehyde detoxification involves conjugation with glutathione (GSH) or carnosine (Car). Here, the detection and quantification of Car, GSH, GSSG (glutathione disulfide), and the corresponding adducts with HNE, Car-HNE, and GS-HNE, were performed in muscle and liver tissues of a hSOD1G93A ALS rat model by reverse-phase high-performance liquid chromatography coupled to electrospray ion trap tandem mass spectrometry in the selected reaction monitoring mode. A significant increase in the levels of GS-HNE and Car-HNE was observed in the muscle tissue of the end-stage ALS animals. Therefore, analyzing variations in the levels of these adducts in ALS animal tissue is crucial from a toxicological perspective and can contribute to the development of new therapeutic strategies.
Asunto(s)
Aldehídos , Esclerosis Amiotrófica Lateral , Carnosina , Modelos Animales de Enfermedad , Glutatión , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Aldehídos/metabolismo , Aldehídos/química , Carnosina/metabolismo , Glutatión/metabolismo , Ratas , Músculo Esquelético/metabolismo , Humanos , Superóxido Dismutasa/metabolismo , Masculino , Cromatografía Líquida de Alta Presión , Ratas Transgénicas , Superóxido Dismutasa-1/metabolismo , Ratas Sprague-DawleyRESUMEN
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive loss of motor neurons. The limited efficacy of recent therapies in clinical development may be linked to lack of drug penetration to the affected motor neurons due to the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB). METHODS: In this work, the safety and efficacy of repeated short transient opening of the BSCB by low intensity pulsed ultrasound (US, sonication) was studied in females of an ALS mouse model (B6.Cg-Tg(SOD1∗G93A)1Gur/J). The BSCB was disrupted using a 1 MHz ultrasound transducer coupled to the spinal cord, with and without injection of insulin-like growth factor 1 (IGF1), a neurotrophic factor that has previously shown efficacy in ALS models. FINDINGS: Results in wild-type (WT) animals demonstrated that the BSCB can be safely disrupted and IGF1 concentrations significantly enhanced after a single session of transient BSCB disruption (176 ± 32 µg/g vs. 0.16 ± 0.008 µg/g, p < 0.0001). Five repeated weekly US sessions performed in female ALS mice demonstrated a survival advantage in mice treated with IGF1 and US (US IGF1) compared to treatment with IGF1 alone (176 vs. 166 days, p = 0.0038). Surprisingly, this survival advantage was also present in mice treated with US alone vs. untreated mice (178.5 vs. 166.5 days, p = 0.0061). Muscle strength did not show difference among the groups. Analysis of glial cell immunoreactivity and microglial transcriptome showing reduced cell proliferation pathways, in addition to lymphocyte infiltration, suggested that the beneficial effect of US or US IGF1 could act through immune cell modulation. INTERPRETATION: These results show the first step towards a possible beneficial impact of transient BSCB opening for ALS therapy and suggest implication of immune cells. FUNDING: Fondation pour la Recherche Médicale (FRM). Investissements d'avenirANR-10-IAIHU-06, Société Française de Neurochirurgie (SFNC), Fond d'étude et de Recherche du Corps Medical (FERCM), Aide à la Recherche des Maladies du Cerveau (ARMC), SLA Fondation Recherche (SLAFR), French Ministry for High Education and Research (MENR), Carthera, Laboratoire de Recherche en Technologies Chirurgicales Avancées (LRTCA).
Asunto(s)
Esclerosis Amiotrófica Lateral , Barrera Hematoencefálica , Modelos Animales de Enfermedad , Factor I del Crecimiento Similar a la Insulina , Médula Espinal , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/terapia , Femenino , Ratones , Médula Espinal/metabolismo , Barrera Hematoencefálica/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones Transgénicos , Humanos , Neuronas Motoras/metabolismo , Ondas UltrasónicasRESUMEN
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motoneurons (MN) degeneration. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by aggregation of mutant proteins, among which the RNA binding protein FUS. Here we show that, in neuronal cells and in iPSC-derived MN expressing mutant FUS, such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished. Interestingly, stress granules formed in ALS conditions showed a distinctive transcriptome with respect to control cells, which reverted to similar to control after m6A downregulation. Notably, cells expressing mutant FUS were characterized by higher m6A levels suggesting a possible link between m6A homeostasis and pathological aggregates. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, an inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Proteína FUS de Unión a ARN , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Células Madre Pluripotentes Inducidas/metabolismo , Gránulos Citoplasmáticos/metabolismo , Fibroblastos/metabolismo , Adenosina/metabolismo , Adenosina/análogos & derivados , Metiltransferasas/metabolismo , Metiltransferasas/genética , Mutación , Cuerpos de Inclusión/metabolismo , Gránulos de Estrés/metabolismo , TranscriptomaRESUMEN
Matrin-3 (MATR3) was initially discovered as a component of the nuclear matrix about thirty years ago. Since then, accumulating studies have provided evidence that MATR3 not only plays a structural role in the nucleus, but that it is also an active protein involved in regulating gene expression at multiple levels, including chromatin organization, DNA transcription, RNA metabolism, and protein translation in the nucleus and cytoplasm. Furthermore, MATR3 may play a critical role in various cellular processes, including DNA damage response, cell proliferation, differentiation, and survival. In addition to the revelation of its biological role, recent studies have reported MATR3's involvement in the context of various diseases, including neurodegenerative and neurodevelopmental diseases, as well as cancer. Moreover, sequencing studies of patients revealed a handful of disease-associated mutations in MATR3 linked to amyotrophic lateral sclerosis (ALS), which further elevated the gene's importance as a topic of study. In this review, we synthesize the current knowledge regarding the diverse functions of MATR3 in DNA- and RNA-related processes, as well as its involvement in various diseases, with a particular emphasis on ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Regulación de la Expresión Génica , Proteínas Asociadas a Matriz Nuclear , Matriz Nuclear , Humanos , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteínas Asociadas a Matriz Nuclear/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Matriz Nuclear/metabolismo , Animales , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease targeting the brain and spinal cord. Non-neuronal cells, including macrophages, may contribute to the disruption of motor neurons (MNs), neuromuscular junction dismantling and clinical signs of ALS. Understanding the modality and the effect of MNs-macrophage communication is pivotal. Here, we focus on extracellular vesicle (EVS)-mediated communication and, in particular, we analyze the response of macrophages. NSC-34 cells transfected with mutant SOD1 (G93A, A4V, G85R, G37R) and differentiated towards MN-like cells, and Raw 264.7 macrophages are the cellular models of the study. mSOD1 NSC-34 cells release a high number of vesicles, both large-lEVs (300 nm diameter) and small-sEVs (90 nm diameter), containing inflammation-modulating molecules, and are efficiently taken up by macrophages. RT-PCR analysis of inflammation mediators demonstrated that the conditioned medium of mSOD1 NSC-34 cells polarizes Raw 264.7 macrophages towards both pro-inflammatory and anti-inflammatory phenotypes. sEVs act on macrophages in a time-dependent manner: an anti-inflammatory response mediated by TGFß firstly starts (12 h); successively, the response shifts towards a pro-inflammation IL-1ß-mediated (48 h). The response of macrophages is strictly dependent on the SOD1 mutation type. The results suggest that EVs impact physiological and behavioral macrophage processes and are of potential relevance to MN degeneration.
Asunto(s)
Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , Macrófagos , Neuronas Motoras , Superóxido Dismutasa-1 , Animales , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/genética , Ratones , Células RAW 264.7 , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Macrófagos/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Neuronas Motoras/metabolismo , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Mutación , Transfección , HumanosRESUMEN
RNA binding proteins have emerged as central players in the mechanisms of many neurodegenerative diseases. In particular, a proteinopathy of fused in sarcoma (FUS) is present in some instances of familial Amyotrophic lateral sclerosis (ALS) and about 10% of sporadic Frontotemporal lobar degeneration (FTLD). Here we establish that focal injection of sonicated human FUS fibrils into brains of mice in which ALS-linked mutant or wild-type human FUS replaces endogenous mouse FUS is sufficient to induce focal cytoplasmic mislocalization and aggregation of mutant and wild-type FUS which with time spreads to distal regions of the brain. Human FUS fibril-induced FUS aggregation in the mouse brain of humanized FUS mice is accelerated by an ALS-causing FUS mutant relative to wild-type human FUS. Injection of sonicated human FUS fibrils does not induce FUS aggregation and subsequent spreading after injection into naïve mouse brains containing only mouse FUS, indicating a species barrier to human FUS aggregation and its prion-like spread. Fibril-induced human FUS aggregates recapitulate pathological features of FTLD including increased detergent insolubility of FUS and TAF15 and amyloid-like, cytoplasmic deposits of FUS that accumulate ubiquitin and p62, but not TDP-43. Finally, injection of sonicated FUS fibrils is shown to exacerbate age-dependent cognitive and behavioral deficits from mutant human FUS expression. Thus, focal seeded aggregation of FUS and further propagation through prion-like spread elicits FUS-proteinopathy and FTLD-like disease progression.
Asunto(s)
Progresión de la Enfermedad , Demencia Frontotemporal , Ratones Transgénicos , Proteína FUS de Unión a ARN , Animales , Humanos , Ratones , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Demencia Frontotemporal/patología , Demencia Frontotemporal/metabolismo , Demencia Frontotemporal/genética , Agregación Patológica de Proteínas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína FUS de Unión a ARN/genéticaRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral , Vesículas Extracelulares , Células Madre Mesenquimatosas , Microglía , Superóxido Dismutasa-1 , Esclerosis Amiotrófica Lateral/terapia , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Vesículas Extracelulares/metabolismo , Microglía/metabolismo , Células Madre Mesenquimatosas/metabolismo , Humanos , Superóxido Dismutasa-1/metabolismo , Superóxido Dismutasa-1/genética , Especies Reactivas de Oxígeno/metabolismo , Línea Celular , Tejido Adiposo/citología , Tejido Adiposo/metabolismoRESUMEN
A CAG repeat sequence in the ATXN2 gene encodes a polyglutamine (polyQ) tract within the ataxin-2 (ATXN2) protein, showcasing a complex landscape of functions that have been progressively unveiled over recent decades. Despite significant progresses in the field, a comprehensive overview of the mechanisms governed by ATXN2 remains elusive. This multifaceted protein emerges as a key player in RNA metabolism, stress granules dynamics, endocytosis, calcium signaling, and the regulation of the circadian rhythm. The CAG overexpansion within the ATXN2 gene produces a protein with an extended poly(Q) tract, inducing consequential alterations in conformational dynamics which confer a toxic gain and/or partial loss of function. Although overexpanded ATXN2 is predominantly linked to spinocerebellar ataxia type 2 (SCA2), intermediate expansions are also implicated in amyotrophic lateral sclerosis (ALS) and parkinsonism. While the molecular intricacies await full elucidation, SCA2 presents ATXN2-associated pathological features, encompassing autophagy impairment, RNA-mediated toxicity, heightened oxidative stress, and disruption of calcium homeostasis. Presently, SCA2 remains incurable, with patients reliant on symptomatic and supportive treatments. In the pursuit of therapeutic solutions, various studies have explored avenues ranging from pharmacological drugs to advanced therapies, including cell or gene-based approaches. These endeavours aim to address the root causes or counteract distinct pathological features of SCA2. This review is intended to provide an updated compendium of ATXN2 functions, delineate the associated pathological mechanisms, and present current perspectives on the development of innovative therapeutic strategies.
Asunto(s)
Ataxina-2 , Péptidos , Humanos , Ataxina-2/metabolismo , Ataxina-2/genética , Péptidos/metabolismo , Péptidos/genética , Animales , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Ataxias Espinocerebelosas/metabolismo , Ataxias Espinocerebelosas/genética , Ataxias Espinocerebelosas/patologíaRESUMEN
Cytoplasmic mislocalization and aggregation of TDP-43 protein are hallmarks of amyotrophic lateral sclerosis (ALS) and are observed in the vast majority of both familial and sporadic cases. How these two interconnected processes are regulated on a molecular level, however, remains enigmatic. Genome-wide screens for modifiers of the ALS-associated genes TDP-43 and FUS have identified the phospholipase D (Pld) pathway as a key regulator of ALS-related phenotypes in the fruit fly Drosophila melanogaster [M. W. Kankel et al., Genetics 215, 747-766 (2020)]. Here, we report the results of our search for downstream targets of the enzymatic product of Pld, phosphatidic acid. We identify two conserved negative regulators of the cAMP/PKA signaling pathway, the phosphodiesterase dunce and the inhibitory subunit PKA-R2, as modifiers of pathogenic phenotypes resulting from overexpression of the Drosophila TDP-43 ortholog TBPH. We show that knockdown of either of these genes results in a mitigation of both TBPH aggregation and mislocalization in larval motor neuron cell bodies, as well as an amelioration of adult-onset motor defects and shortened lifespan induced by TBPH. We determine that PKA kinase activity is downstream of both TBPH and Pld and that overexpression of the PKA target CrebA can rescue TBPH mislocalization. These findings suggest a model whereby increasing cAMP/PKA signaling can ameliorate the molecular and functional effects of pathological TDP-43.