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[This corrects the article DOI: 10.1371/journal.pgen.1004749.].
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Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.
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Enfermedad de Machado-Joseph , Enfermedades Neurodegenerativas , Ratones , Animales , Enfermedad de Machado-Joseph/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/genética , Ratones Transgénicos , Calcio/metabolismo , Ataxina-3/genética , Ataxina-3/metabolismo , Ratones Noqueados , Modelos Animales de Enfermedad , Progresión de la EnfermedadRESUMEN
BACKGROUND AND OBJECTIVE: Mitochondrial dysfunction has been implicated in several neurodegenerative diseases. Creatine administration increases concentration of the energy buffer phosphocreatine, exerting protective effects in the brain. We evaluate whether a creatine-enriched diet would be beneficial for a mouse model of spinocerebellar ataxia type 3, a genetically defined neurodegenerative disease for which no treatment is available. METHODS: We performed 2 independent preclinical trials using the CMVMJD135 mouse model (treating 2 groups of animals with different disease severity) and wild-type mice, to which 2% creatine was provided for 19 (preclinical trial 1) or 29 (preclinical trial 2) weeks, starting at a presymptomatic age. Motor behavior was evaluated at several time points from 5 to 34 weeks of age, and neuropathological studies were performed at the end of each trial. RESULTS: Creatine supplementation led to an overall improvement in the motor phenotype of CMVMJD135 mice in both trials, rescuing motor balance and coordination and also restored brain weight, mitigated astrogliosis, and preserved Calbindin-positive cells in the cerebellum. Moreover, a reduction of mutant ataxin-3 aggregates occurred despite maintained steady-state levels of the protein and the absence of autophagy activation. Creatine treatment also restored the expression of the mitochondrial mass marker Porin and reduced the expression of antioxidant enzymes Heme oxygenase 1 (HO1) and NAD(P)H Quinone Dehydrogenase 1 (NQO1), suggesting a beneficial effect at the level of mitochondria and oxidative stress. CONCLUSIONS: Creatine slows disease progression and improves motor dysfunction as well as ameliorates neuropathology of the CMVMJD135 animals, supporting this as a useful strategy to slow the progression of spinocerebellar ataxia type 3. © 2018 International Parkinson and Movement Disorder Society.
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Creatina/administración & dosificación , Dieta/métodos , Enfermedad de Machado-Joseph/dietoterapia , Enfermedad de Machado-Joseph/genética , Fármacos Neuroprotectores/administración & dosificación , Animales , Ataxina-3/genética , Ataxina-3/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Calbindinas/genética , Calbindinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Estudios de Seguimiento , Trastornos Neurológicos de la Marcha/dietoterapia , Trastornos Neurológicos de la Marcha/etiología , Gliosis/dietoterapia , Gliosis/genética , Enfermedad de Machado-Joseph/complicaciones , Enfermedad de Machado-Joseph/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Fuerza Muscular/efectos de los fármacos , Fuerza Muscular/genética , ARN Mensajero/metabolismoRESUMEN
DNA strand-breaks (SBs) with non-ligatable ends are generated by ionizing radiation, oxidative stress, various chemotherapeutic agents, and also as base excision repair (BER) intermediates. Several neurological diseases have already been identified as being due to a deficiency in DNA end-processing activities. Two common dirty ends, 3'-P and 5'-OH, are processed by mammalian polynucleotide kinase 3'-phosphatase (PNKP), a bifunctional enzyme with 3'-phosphatase and 5'-kinase activities. We have made the unexpected observation that PNKP stably associates with Ataxin-3 (ATXN3), a polyglutamine repeat-containing protein mutated in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease (MJD). This disease is one of the most common dominantly inherited ataxias worldwide; the defect in SCA3 is due to CAG repeat expansion (from the normal 14-41 to 55-82 repeats) in the ATXN3 coding region. However, how the expanded form gains its toxic function is still not clearly understood. Here we report that purified wild-type (WT) ATXN3 stimulates, and by contrast the mutant form specifically inhibits, PNKP's 3' phosphatase activity in vitro. ATXN3-deficient cells also show decreased PNKP activity. Furthermore, transgenic mice conditionally expressing the pathological form of human ATXN3 also showed decreased 3'-phosphatase activity of PNKP, mostly in the deep cerebellar nuclei, one of the most affected regions in MJD patients' brain. Finally, long amplicon quantitative PCR analysis of human MJD patients' brain samples showed a significant accumulation of DNA strand breaks. Our results thus indicate that the accumulation of DNA strand breaks due to functional deficiency of PNKP is etiologically linked to the pathogenesis of SCA3/MJD.
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Enzimas Reparadoras del ADN/genética , Enfermedad de Machado-Joseph/genética , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genética , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Represoras/genética , Expansión de Repetición de Trinucleótido/genética , Animales , Ataxina-3 , Línea Celular , Daño del ADN/genética , Reparación del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Humanos , Enfermedad de Machado-Joseph/enzimología , Enfermedad de Machado-Joseph/fisiopatología , Mamíferos , Ratones , Ratones Transgénicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Estrés Oxidativo/genética , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas Represoras/metabolismoRESUMEN
The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.
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Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Péptidos/metabolismo , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , Animales , Ataxina-3 , Diferenciación Celular , Células Cultivadas , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Integrina alfa5/metabolismo , Ratones , Células PC12 , Complejo de la Endopetidasa Proteasomal/metabolismo , Ratas , Ratas WistarRESUMEN
Polyglutamine diseases are a class of dominantly inherited neurodegenerative disorders for which there is no effective treatment. Here we provide evidence that activation of serotonergic signalling is beneficial in animal models of Machado-Joseph disease. We identified citalopram, a selective serotonin reuptake inhibitor, in a small molecule screen of FDA-approved drugs that rescued neuronal dysfunction and reduced aggregation using a Caenorhabditis elegans model of mutant ataxin 3-induced neurotoxicity. MOD-5, the C. elegans orthologue of the serotonin transporter and cellular target of citalopram, and the serotonin receptors SER-1 and SER-4 were strong genetic modifiers of ataxin 3 neurotoxicity and necessary for therapeutic efficacy. Moreover, chronic treatment of CMVMJD135 mice with citalopram significantly reduced ataxin 3 neuronal inclusions and astrogliosis, rescued diminished body weight and strikingly ameliorated motor symptoms. These results suggest that small molecule modulation of serotonergic signalling represents a promising therapeutic target for Machado-Joseph disease.
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Ataxina-3/efectos de los fármacos , Proteínas de Caenorhabditis elegans/efectos de los fármacos , Citalopram/farmacología , Gliosis/metabolismo , Cuerpos de Inclusión/efectos de los fármacos , Locomoción/efectos de los fármacos , Enfermedad de Machado-Joseph/metabolismo , Neuronas/efectos de los fármacos , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Serotonina/metabolismo , Animales , Ataxina-3/metabolismo , Conducta Animal/efectos de los fármacos , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Modelos Animales de Enfermedad , Cuerpos de Inclusión/metabolismo , Cuerpos de Inclusión/patología , Ratones , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/patología , Proteínas de Transporte de Serotonina en la Membrana Plasmática , Transmisión Sináptica/efectos de los fármacosRESUMEN
The accumulation of misfolded proteins in neurons, leading to the formation of cytoplasmic and nuclear aggregates, is a common theme in age-related neurodegenerative diseases, possibly due to disturbances of the proteostasis and insufficient activity of cellular protein clearance pathways. Lithium is a well-known autophagy inducer that exerts neuroprotective effects in different conditions and has been proposed as a promising therapeutic agent for several neurodegenerative diseases. We tested the efficacy of chronic lithium (10.4 mg/kg) treatment in a transgenic mouse model of Machado-Joseph disease, an inherited neurodegenerative disease, caused by an expansion of a polyglutamine tract within the protein ataxin-3. A battery of behavioral tests was used to assess disease progression. In spite of activating autophagy, as suggested by the increased levels of Beclin-1, Atg7, and LC3-II, and a reduction in the p62 protein levels, lithium administration showed no overall beneficial effects in this model concerning motor performance, showing a positive impact only in the reduction of tremors at 24 weeks of age. Our results do not support lithium chronic treatment as a promising strategy for the treatment of Machado-Joseph disease (MJD).
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Cloruro de Litio/farmacología , Enfermedad de Machado-Joseph/tratamiento farmacológico , Enfermedad de Machado-Joseph/fisiopatología , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fármacos Neuromusculares/farmacología , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Ataxina-3 , Autofagia/efectos de los fármacos , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Conducta Exploratoria/efectos de los fármacos , Conducta Exploratoria/fisiología , Humanos , Masculino , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Equilibrio Postural/efectos de los fármacos , Equilibrio Postural/fisiología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Resultado del Tratamiento , Temblor/tratamiento farmacológico , Temblor/fisiopatología , Pérdida de Peso/efectos de los fármacos , Pérdida de Peso/fisiologíaRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is an adult-onset neurodegenerative disease caused by a polyglutamine expansion in the ataxin-3 (ATXN3) gene. No effective treatment is available for this disorder, other than symptom-directed approaches. Bile acids have shown therapeutic efficacy in neurodegenerative disease models. Here, we pinpointed tauroursodeoxycholic acid (TUDCA) as an efficient therapeutic, improving the motor and neuropathological phenotype of SCA3 nematode and mouse models. Surprisingly, transcriptomic and functional in vivo data showed that TUDCA acts in neuronal tissue through the glucocorticoid receptor (GR), but independently of its canonical receptor, the farnesoid X receptor (FXR). TUDCA was predicted to bind to the GR, in a similar fashion to corticosteroid molecules. GR levels were decreased in disease-affected brain regions, likely due to increased protein degradation as a consequence of ATXN3 dysfunction being restored by TUDCA treatment. Analysis of a SCA3 clinical cohort showed intriguing correlations between the peripheral expression of GR and the predicted age at disease onset in presymptomatic subjects and FKBP5 expression with disease progression, suggesting this pathway as a potential source of biomarkers for future study. We have established a novel in vivo mechanism for the neuroprotective effects of TUDCA in SCA3 and propose this readily available drug for clinical trials in SCA3 patients.
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Enfermedad de Machado-Joseph , Enfermedades Neurodegenerativas , Ácido Tauroquenodesoxicólico , Ratones , Adulto , Animales , Humanos , Enfermedad de Machado-Joseph/tratamiento farmacológico , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/metabolismo , Receptores de Glucocorticoides/genética , Ratones TransgénicosRESUMEN
The risk of developing neurodegenerative diseases increases with age. Although many of the molecular pathways regulating proteotoxic stress and longevity are well characterized, their contribution to disease susceptibility remains unclear. In this study, we describe a new Caenorhabditis elegans model of Machado-Joseph disease pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron susceptibility to mutant ataxin-3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This reveals that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated pathogenesis. Aging influences the ATXN3 phenotypes which can be suppressed by the downregulation of the insulin/insulin growth factor-1-like signaling pathway and activation of heat shock factor-1.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Factores de Transcripción/metabolismo , Animales , Ataxina-3 , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Agregación Celular/genética , Agregación Celular/fisiología , Factores de Transcripción Forkhead , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Neuronas/patología , Péptidos/metabolismo , Factores de Transcripción/genéticaRESUMEN
Spinocerebellar ataxia type 3 (SCA3) is an adult-onset, progressive ataxia. SCA3 presents with ataxia before any gross neuropathology. A feature of many cerebellar ataxias is aberrant cerebellar output that contributes to motor dysfunction. We examined whether abnormal cerebellar output was present in the CMVMJD135 SCA3 mouse model and, if so, whether it correlated with the disease onset and progression. In vivo recordings showed that the activity of deep cerebellar nuclei neurons, the main output of the cerebellum, was altered. The aberrant activity correlated with the onset of ataxia. However, although the severity of ataxia increased with age, the severity of the aberrant cerebellar output was not progressive. The abnormal cerebellar output, however, was accompanied by non-progressive abnormal activity of their upstream synaptic inputs, the Purkinje cells. In vitro recordings indicated that alterations in intrinsic Purkinje cell pacemaking and in their synaptic inputs contributed to abnormal Purkinje cell activity. These findings implicate abnormal cerebellar physiology as an early, consistent contributor to pathophysiology in SCA3, and suggest that the aberrant cerebellar output could be an appropriate therapeutic target in SCA3.
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Ataxia Cerebelosa , Enfermedad de Machado-Joseph , Ataxias Espinocerebelosas , Animales , Ataxia/patología , Ataxia Cerebelosa/patología , Cerebelo/patología , Enfermedad de Machado-Joseph/patología , Ratones , Neuronas/patología , Células de Purkinje/patología , Ataxias Espinocerebelosas/patologíaRESUMEN
Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado-Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.
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Spinal cord injury (SCI) leads to dramatic impairments of motor, sensory, and autonomic functions of affected individuals. Following the primary injury, there is an increased release of glutamate that leads to excitotoxicity and further neuronal death. Therefore, modulating glutamate excitotoxicity seems to be a promising target to promote neuroprotection during the acute phase of the injury. In this study, we evaluated the therapeutic effect of a FDA approved antiepileptic drug (levetiracetam-LEV), known for binding to the synaptic vesicle protein SV2A in the brain and spinal cord. LEV therapy was tested in two models of SCI-one affecting the cervical and other the thoracic level of the spinal cord. The treatment was effective on both SCI models. Treated animals presented significant improvements on gross and fine motor functions. The histological assessment revealed a significant decrease of cavity size, as well as higher neuronal and oligodendrocyte survival on treated animals. Molecular analysis revealed that LEV acts by stabilizing the astrocytes allowing an effective uptake of the excess glutamate from the extracellular space. Overall, our results demonstrate that Levetiracetam may be a promising drug for acute management of SCI.
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The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)-or Machado-Joseph disease (MJD)-is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation-particularly in the cerebellum and basal ganglia-while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.
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INTRODUCTION: Six of the most frequent dominantly inherited spinocerebellar ataxias (SCAs) worldwide - SCA1, SCA2, SCA3, SCA6, SCA7, and SCA17 - are caused by an expansion of a polyglutamine (polyQ) tract in the corresponding proteins. While the identification of the causative mutation has advanced knowledge on the pathogenesis of polyQ SCAs, effective therapeutics able to mitigate the severe clinical manifestation of these highly incapacitating disorders are not yet available. AREAS COVERED: This review provides a comprehensive and critical perspective on well-established and emerging therapeutic targets for polyQ SCAs; it aims to inspire prospective drug discovery efforts. EXPERT OPINION: The landscape of polyQ SCAs therapeutic targets and strategies includes (1) the mutant genes and proteins themselves, (2) enhancement of endogenous protein quality control responses, (3) abnormal protein-protein interactions of the mutant proteins, (4) disturbed neuronal function, (5) mitochondrial function, energy availability and oxidative stress, and (6) glial dysfunction, growth factor or hormone imbalances. Challenges include gaining a clearer definition of therapeutic targets for the drugs in clinical development, the discovery of novel drug-like molecules for challenging key targets, and the attainment of a stronger translation of preclinical findings to the clinic.
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Terapia Molecular Dirigida , Péptidos/metabolismo , Ataxias Espinocerebelosas/tratamiento farmacológico , Animales , Desarrollo de Medicamentos , Descubrimiento de Drogas , Humanos , Estrés Oxidativo , Ataxias Espinocerebelosas/fisiopatologíaRESUMEN
Ataxin-3 is the protein involved in Machado-Joseph disease, a neurodegenerative disorder caused by a polyglutamine expansion. Ataxin-3 binds ubiquitylated proteins and acts as a deubiquitylating enzyme in vitro. It was previously proposed that ataxin-3, along with the VCP/p97 protein, escorts ubiquitylated substrates for proteasomal degradation, although other players of this escort complex were not identified yet. In this work, we show that the Caenorhabditis elegans ataxin-3 protein (ATX-3) interacts with both VCP/p97 worm homologs, CDC-48.1 and CDC-48.2 and we map the interaction domains. We describe a motility defect in both ATX-3 and CDC-48.1 mutants and, in addition, we identify a new protein interactor, UBXN-5, potentially an adaptor of the CDC-48-ATX-3 escort complex. CDC-48 binds to both ATX-3 and UBXN-5 in a non-competitive manner, suggesting the formation of a trimolecular complex. Both CDC-48 and ATX-3, but not UBXN-5, were able to bind K-48 polyubiquitin chains, the standard signal for proteasomal degradation. Additionally, we describe several common interactors of ATX-3 and UBXN-5, some of which can be in vivo targets of this complex.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adenosina Trifosfatasas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Adenosina Trifosfatasas/genética , Animales , Ataxina-3 , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Complejos Multiproteicos/genética , Proteínas del Tejido Nervioso/genética , Dominios y Motivos de Interacción de Proteínas/genética , Mapeo de Interacción de Proteínas , Proteína que Contiene ValosinaRESUMEN
MPTP-induced dopaminergic neurotoxicity involves major biochemical processes such as oxidative stress and impaired energy metabolism, leading to a significant reduction in the number of nigrostriatal dopaminergic neurons. Glutathione S-transferase pi (GSTpi) is a phase II detoxifying enzyme that provides protection of cells from injury by toxic chemicals and products of oxidative stress. In humans, polymorphisms of GSTP1 affect substrate selectivity and stability increasing the susceptibility to parkinsonism-inducing effects of environmental toxins. Given the ability of MPTP to increase the levels of reactive oxygen species and the link between altered redox potential and the expression and activity of GSTpi, we investigated the effect of MPTP on GSTpi cellular concentration in an in vivo model of Parkinson's disease. The present study demonstrates that GSTpi is actively expressed in both substantia nigra pars compacta and striatum of C57BL/6 mice brain, mostly in oligodendrocytes and astrocytes. After systemic administration of MPTP, GSTpi expression is significantly increased in glial cells in the vicinity of dopaminergic neurons cell bodies and fibers. The results suggest that GSTpi expression may be part of the mechanism underlying the ability of glial cells to elicit protection against the mechanisms involved in MPTP-induced neuronal death.
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Cuerpo Estriado , Dopamina/metabolismo , Gutatión-S-Transferasa pi/metabolismo , Intoxicación por MPTP/metabolismo , Mesencéfalo , Neuronas , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina/farmacología , Animales , Caspasa 3/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Dopaminérgicos/farmacología , Gutatión-S-Transferasa pi/genética , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Mesencéfalo/efectos de los fármacos , Mesencéfalo/patología , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/inducido químicamente , Degeneración Nerviosa/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tirosina 3-Monooxigenasa/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Imbalance of neuronal proteostasis associated with misfolding and aggregation of Tau protein is a common neurodegenerative feature in Alzheimer's disease (AD) and other Tauopathies. Consistent with suggestions that lifetime stress may be an important AD precipitating factor, we previously reported that environmental stress and high glucocorticoid (GC) levels induce accumulation of aggregated Tau; however, the molecular mechanisms for such process remain unclear. Herein, we monitor a novel interplay between RNA-binding proteins (RBPs) and autophagic machinery in the underlying mechanisms through which chronic stress and high GC levels impact on Tau proteostasis precipitating Tau aggregation. Using molecular, pharmacological and behavioral analysis, we demonstrate that chronic stress and high GC trigger mTOR-dependent inhibition of autophagy, leading to accumulation of Tau aggregates and cell death in P301L-Tau expressing mice and cells. In parallel, we found that environmental stress and GC disturb cellular homeostasis and trigger the insoluble accumulation of different RBPs, such as PABP, G3BP1, TIA-1, and FUS, shown to form stress granules (SGs) and Tau aggregation. Interestingly, an mTOR-driven pharmacological stimulation of autophagy attenuates the GC-driven accumulation of Tau and SG-related proteins as well as the related cell death, suggesting a critical interface between autophagy and the response of the SG-related protein in the neurodegenerative potential of chronic stress and GC. These studies provide novel insights into the RNA-protein intracellular signaling regulating the precipitating role of environmental stress and GC on Tau-driven brain pathology.
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Enfermedad de Alzheimer/metabolismo , Autofagia , Proteínas de Choque Térmico/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Animales , Ratones TransgénicosRESUMEN
It was recently shown that the conditioned media (CM) of Human Umbilical Cord Perivascular Cells (HUCPVCs), a mesenchymal progenitor population residing within the Wharton Jelly of the umbilical cord, was able to modulate in vitro the survival and viability of different neuronal and glial cells populations. In the present work, we aimed to assess if the secretome of HUCPVCs is able to 1) induce the differentiation of human telencephalon neural precursor cells (htNPCs) in vitro, and 2) modulate neural/glial proliferation, differentiation and survival in the dentate gyrus (DG) of adult rat hippocampus. For this purpose, two separate experimental setups were performed: 1) htNPCs were incubated with HUCPVCs-CM for 5 days after which neuronal differentiation was assessed and, 2) HUCPVCs, or their respective CM, were injected into the DG of young adult rats and their effects assessed 7 days later. Results revealed that the secretome of HUCPVCs was able to increase neuronal cell differentiation in vitro; indeed, higher densities of immature (DCX(+) cells) and mature neurons (MAP-2(+) cells) were observed when htNPCs were incubated with the HUCPVCs-CM. Additionally, when HUCPVCs and their CM were injected in the DG, results revealed that both cells or CM were able to increase the endogenous proliferation (BrdU(+) cells) 7 days after injection. It was also possible to observe an increased number of newborn neurons (DCX(+) cells), upon injection of HUCPVCs or their respective CM. Finally western blot analysis revealed that after CM or HUCPVCs transplantation, there was an increase of fibroblast growth factor-2 (FGF-2) and, to a lesser extent, of nerve growth factor (NGF) in the DG tissue. Concluding, our results have shown that the transplantation of HUCPVCs or the administration of their secretome were able to potentiate neuronal survival and differentiation in vitro and in vivo.
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Diferenciación Celular/efectos de los fármacos , Células-Madre Neurales/trasplante , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Giro Dentado/citología , Giro Dentado/efectos de los fármacos , Giro Dentado/crecimiento & desarrollo , Proteína Doblecortina , Humanos , Masculino , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/citología , Células-Madre Neurales/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Neuronas/citología , Ratas , Telencéfalo/citología , Telencéfalo/crecimiento & desarrollo , Cordón Umbilical/citología , Cordón Umbilical/crecimiento & desarrollo , Cordón Umbilical/metabolismoRESUMEN
Machado-Joseph disease (MJD) is an inherited neurodegenerative disease, caused by a CAG repeat expansion within the coding region of ATXN3 gene, and which currently lacks effective treatment. In this work we tested the therapeutic efficacy of chronic treatment with valproic acid (VPA) (200mg/kg), a compound with known neuroprotection activity, and previously shown to be effective in cell, fly and nematode models of MJD. We show that chronic VPA treatment in the CMVMJD135 mouse model had limited effects in the motor deficits of these mice, seen mostly at late stages in the motor swimming, beam walk, rotarod and spontaneous locomotor activity tests, and did not modify the ATXN3 inclusion load and astrogliosis in affected brain regions. However, VPA chronic treatment was able to increase GRP78 protein levels at 30 weeks of age, one of its known neuroprotective effects, confirming target engagement. In spite of limited results, the use of another dosage of VPA or of VPA in a combined therapy with molecules targeting other pathways, cannot be excluded as potential strategies for MJD therapeutics.
Asunto(s)
Ataxina-3/genética , Proteínas de Choque Térmico/biosíntesis , Enfermedad de Machado-Joseph/genética , Ácido Valproico/administración & dosificación , Animales , Astrocitos/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/patología , Modelos Animales de Enfermedad , Chaperón BiP del Retículo Endoplásmico , Proteínas de Choque Térmico/genética , Humanos , Enfermedad de Machado-Joseph/tratamiento farmacológico , Enfermedad de Machado-Joseph/patología , Ratones , Mutación , Expansión de Repetición de Trinucleótido/efectos de los fármacos , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.