RESUMO
Long considered to fluctuate between pro- and anti-inflammatory states, it has now become evident that microglia occupy a variegated phenotypic landscape with relevance to aging and neurodegeneration. However, whether specific microglial subsets converge in or contribute to both processes that eventually affect brain function is less clear. To investigate this, we analyzed microglial heterogeneity in a tauopathy mouse model (K18-seeded P301L) and an accelerated aging model (Senescence-Accelerated Mouse-Prone 8, SAMP8) using cellular indexing of transcriptomes and epitopes by sequencing. We found that widespread tau pathology in K18-seeded P301L mice caused a significant change in the number and morphology of microglia, but only a mild overrepresentation of disease-associated microglia. At the cell population-level, we observed a marked upregulation of the calprotectin-encoding genes S100a8 and S100a9. In 9-month-old SAMP8 mice, we identified a unique microglial subpopulation that showed partial similarity with the disease-associated microglia phenotype and was additionally characterized by a high expression of the same calprotectin gene set. Immunostaining for S100A8 revealed that this population was enriched in the hippocampus, correlating with the cognitive impairment observed in this model. However, incomplete colocalization between their residence and markers of neuronal loss suggests regional specificity. Importantly, S100A8-positive microglia were also retrieved in brain biopsies of human AD and tauopathy patients as well as in a biopsy of an aged individual without reported pathology. Thus, the emergence of S100A8-positive microglia portrays a conspicuous commonality between accelerated aging and tauopathy progression, which may have relevance for ensuing brain dysfunction.
Assuntos
Envelhecimento , Encéfalo , Calgranulina A , Microglia , Animais , Microglia/metabolismo , Camundongos , Encéfalo/metabolismo , Encéfalo/patologia , Calgranulina A/metabolismo , Calgranulina A/genética , Envelhecimento/metabolismo , Proteínas tau/metabolismo , Proteínas tau/genética , Humanos , Modelos Animais de Doenças , Tauopatias/metabolismo , Tauopatias/patologia , Masculino , Camundongos TransgênicosRESUMO
Alzheimer disease (AD) is the most common neurodegenerative disease, and with no efficient curative treatment available, its medical, social, and economic burdens are expected to dramatically increase. AD is historically characterized by amyloid ß (Aß) plaques and tau neurofibrillary tangles, but over the last 25 years chronic immune activation has been identified as an important factor contributing to AD pathogenesis. In this article, we review recent and important advances in our understanding of the significance of immune activation in the development of AD. We describe how brain-resident macrophages, the microglia, are able to detect Aß species and be activated, as well as the consequences of activated microglia in AD pathogenesis. We discuss transcriptional changes of microglia in AD, their unique heterogeneity in humans, and emerging strategies to study human microglia. Finally, we expose, beyond Aß and microglia, the role of peripheral signals and different cell types in immune activation.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Microglia , Doença de Alzheimer/imunologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/metabolismo , Humanos , Animais , Microglia/imunologia , Microglia/metabolismo , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/imunologia , Encéfalo/imunologia , Encéfalo/metabolismo , Encéfalo/patologia , Macrófagos/imunologia , Macrófagos/metabolismoRESUMO
Neuroinflammation is an important component of many neurodegenerative diseases, whether as a primary cause or a secondary outcome. For that reason, either as diagnostic tools or to monitor progression and/or pharmacological interventions, there is a need for robust biomarkers of neuroinflammation in the brain. Mitochondrial TSPO (18 kDa Translocator protein) is one of few available biomarkers of neuroinflammation for which there are clinically available PET imaging agents. In this study, we further characterised neuroinflammation in a mouse model of prion-induced chronic neurodegeneration (ME7) including a pharmacological intervention via a CSF1R inhibitor. This was achieved by autoradiographic binding of the second-generation TSPO tracer, [3H]PBR28, along with a more comprehensive examination of the cellular contributors to the TSPO signal changes by immunohistochemistry. We observed regional increases of TSPO in the ME7 mouse brains, particularly in the hippocampus, cortex and thalamus. This increased TSPO signal was detected in the cells of microglia/macrophage lineage as well as in astrocytes, endothelial cells and neurons. Importantly, we show that the selective CSF1R inhibitor, JNJ-40346527 (JNJ527), attenuated the disease-dependent increase in TSPO signal, particularly in the dentate gyrus of the hippocampus, where JNJ527 attenuated the number of Iba1+ microglia and neurons, but not GFAP+ astrocytes or endothelial cells. These findings suggest that [3H]PBR28 quantitative autoradiography in combination with immunohistochemistry are important translational tools for detecting and quantifying neuroinflammation, and its treatments, in neurodegenerative disease. Furthermore, we demonstrate that although TSPO overexpression in the ME7 brains was driven by various cell types, the therapeutic effect of the CSF1R inhibitor was primarily to modulate TSPO expression in microglia and neurons, which identifies an important route of biological action of this particular CSF1R inhibitor and provides an example of a cell-specific effect of this type of therapeutic agent on the neuroinflammatory process.
Assuntos
Doenças Neurodegenerativas , Doenças Priônicas , Camundongos , Animais , Microglia/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças Neuroinflamatórias , Células Endoteliais/metabolismo , Receptores de GABA/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Macrófagos/metabolismo , Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Neurônios/metabolismo , Doenças Priônicas/metabolismo , Biomarcadores/metabolismoRESUMO
Microglia are critically involved in complex neurological disorders with a strong genetic component, such as Alzheimer's disease, Parkinson's disease and frontotemporal dementia. Although mouse microglia can recapitulate aspects of human microglia physiology, they do not fully capture the human genetic aspects of disease and do not reproduce all human cell states. Primary cultures of human microglia or microglia derived from human induced pluripotent stem cells (PSCs) are difficult to maintain in brain-relevant cell states in vitro. Here we describe MIGRATE (microglia in vitro generation refined for advanced transplantation experiments, which provides a combined in vitro differentiation and in vivo xenotransplantation protocol to study human microglia in the context of the mouse brain. This article details an accurate, step-by-step workflow that includes in vitro microglia differentiation from human PSCs, transplantation into the mouse brain and quantitative analysis of engraftment. Compared to current differentiation and xenotransplantation protocols, we present an optimized, faster and more efficient approach that yields up to 80% chimerism. To quantitatively assess engraftment efficiency by flow cytometry, access to specialized flow cytometry is required. Alternatively, the percentage of chimerism can be estimated by standard immunohistochemical analysis. The MIGRATE protocol takes ~40 d to complete, from culturing PSCs to engraftment efficiency assessment.
Assuntos
Transplante de Células-Tronco Mesenquimais/métodos , Microglia/citologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Encéfalo/fisiologia , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Microglia/metabolismo , Microglia/fisiologia , Células-Tronco Pluripotentes/citologia , GravidezRESUMO
BACKGROUND: The simultaneous contribution of several etiopathogenic disturbances makes amyotrophic lateral sclerosis (ALS) a fatal and challenging disease. Here, we studied two different cell therapy protocols to protect both central and peripheral nervous system in a murine model of ALS. METHODS: Since ALS begins with a distal axonopathy, in a first assay, we performed injection of bone marrow cells into two hindlimb muscles of transgenic SOD1G93A mice. In a second study, we combined intramuscular and intraspinal injection of bone marrow cells. Fluorescence-activated cell sorting was used to assess the survival of the transplanted cells into the injected tissues. The mice were assessed from 8 to 16 weeks of age by means of locomotion and electrophysiological tests. After follow-up, the spinal cord was processed for analysis of motoneuron survival and glial cell reactivity. RESULTS: We found that, after intramuscular injection, bone marrow cells were able to engraft within the muscle. However, bone marrow cell intramuscular injection failed to promote a general therapeutic effect. In the second approach, we found that bone marrow cells had limited survival in the spinal cord, but this strategy significantly improved motor outcomes. Moreover, we also found that the dual cell therapy tended to preserve spinal motoneurons at late stages of the disease and to reduce microgliosis, although this did not prolong mice survival. CONCLUSION: Overall, our findings suggest that targeting more than one affected area of the motor system at once with bone marrow cell therapy results in a valuable therapeutic intervention for ALS.
Assuntos
Células da Medula Óssea/metabolismo , Transplante de Medula Óssea/métodos , Superóxido Dismutase-1/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Injeções Intramusculares , Injeções Espinhais , Camundongos , Camundongos TransgênicosRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by progressive degeneration of motoneurons, which is preceded by loss of neuromuscular connections in a "dying back" process. Neuregulin-1 (Nrg1) is a neurotrophic factor essential for the development and maintenance of neuromuscular junctions, and Nrg1 receptor ErbB4 loss-of-function mutations have been reported as causative for ALS. Our main goal was to investigate the role of Nrg1 type I (Nrg1-I) in SOD1(G93A) mice muscles. We overexpressed Nrg1-I by means of an adeno-associated viral (AAV) vector, and investigated its effect by means of neurophysiological techniques assessing neuromuscular function, as well as molecular approaches (RT-PCR, western blot, immunohistochemistry, ELISA) to determine the mechanisms underlying Nrg1-I action. AAV-Nrg1-I intramuscular administration promoted motor axon collateral sprouting by acting on terminal Schwann cells, preventing denervation of the injected muscles through Akt and ERK1/2 pathways. We further used a model of muscle partial denervation by transecting the L4 spinal nerve. AAV-Nrg1-I intramuscular injection enhanced muscle reinnervation by collateral sprouting, whereas administration of lapatinib (ErbB receptor inhibitor) completely blocked it. We demonstrated that Nrg1-I plays a crucial role in the collateral reinnervation process, opening a new window for developing novel ALS therapies for functional recovery rather than preservation.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Axônios/metabolismo , Neuregulina-1/metabolismo , Junção Neuromuscular/metabolismo , Quinazolinas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Lapatinib , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Denervação Muscular/métodos , Neurogênese/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismoRESUMO
Inflammation is a common neuropathological feature in several neurological disorders, including amyotrophic lateral sclerosis (ALS). We have studied the contribution of CSF1R signalling to inflammation in ALS, as a pathway previously reported to control the expansion and activation of microglial cells. We found that microglial cell proliferation in the spinal cord of SOD1(G93A) transgenic mice correlates with the expression of CSF1R and its ligand CSF1. Administration of GW2580, a selective CSF1R inhibitor, reduced microglial cell proliferation in SOD1(G93A) mice, indicating the importance of CSF1-CSF1R signalling in microgliosis in ALS. Moreover, GW2580 treatment slowed disease progression, attenuated motoneuron cell death and extended survival of SOD1(G93A) mice. Electrophysiological assessment revealed that GW2580 treatment protected skeletal muscle from denervation prior to its effects on microglial cells. We found that macrophages invaded the peripheral nerve of ALS mice before CSF1R-induced microgliosis occurred. Interestingly, treatment with GW2580 attenuated the influx of macrophages into the nerve, which was partly caused by the monocytopenia induced by CSF1R inhibition. Overall, our findings provide evidence that CSF1R signalling regulates inflammation in the central and peripheral nervous system in ALS, supporting therapeutic targeting of CSF1R in this disease.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Macrófagos/metabolismo , Microglia/metabolismo , Nervos Periféricos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Esclerose Lateral Amiotrófica/genética , Animais , Anisóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Progressão da Doença , Gliose/genética , Gliose/metabolismo , Inflamação/genética , Inflamação/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/patologia , Neurônios Motores/metabolismo , Pirimidinas/farmacologia , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/antagonistas & inibidores , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
BACKGROUND: Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease characterized by the loss of motoneurons (MNs) in the spinal cord, brainstem and motor cortex, causing progressive paralysis and death. Nowadays, there is no effective therapy and most patients die 2-5 years after diagnosis. Sigma-1R is a transmembrane protein highly expressed in the CNS and specially enriched in MNs. Mutations on the Sigma-1R leading to frontotemporal lobar degeneration-ALS were recently described in human patients. We previously reported the therapeutic role of the selective sigma-1R agonist 2-(4-morpholi-nethyl)1-phenylcyclohexanecarboxylate (PRE-084) in SOD1G93A ALS mice, that promoted spinal MN preservation and extended animal survival by controlling NMDA receptor calcium influx. Resveratrol (RSV, trans-3,4',5-trihydroxystilbene) is a natural polyphenol with promising neuroprotective effects. We recently found that RSV administration to SOD1G93A mice preserves spinal MN function and increases mice survival. These beneficial effects were associated to activation of Sirtuin 1 (Sirt1) and AMP-activated protein kinase (AMPK) pathways, leading to the modulation of autophagy and an increase of mitochondrial biogenesis. The main goal of this work was to assess the effect of combined RSV and PRE-084 administration in SOD1G93A ALS mice. METHODS: We determined the locomotor performance of the animals by rotarod test and evaluated spinal motoneuron function using electrophysiological tests. RESULTS: RSV plus PRE-084 treatment from 8 weeks of age significantly improved locomotor performance and spinal MN function, accompanied by a significant reduction of MN degeneration and an extension of mice lifespan. In agreement with our previous findings, there was an induction of PKC-specific phosphorylation of the NMDA-NR1 subunit and an increased expression and activation of Sirt1 and AMPK in the ventral spinal cord of treated SOD1G93A animals. CONCLUSIONS: Although combined PRE and RSV treatment significantly ameliorated SOD1G93A mice, it did not show a synergistic effect compared to RSV-only and PRE-084-only treated groups.
Assuntos
Esclerose Lateral Amiotrófica/genética , Morfolinas/uso terapêutico , Receptores sigma/agonistas , Estilbenos/uso terapêutico , Superóxido Dismutase/genética , Adenilato Quinase/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Sinergismo Farmacológico , Camundongos , Camundongos Transgênicos , Morfolinas/farmacologia , Condução Nervosa , Fosforilação , Proteína Quinase C/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Resveratrol , Teste de Desempenho do Rota-Rod , Sirtuína 1/metabolismo , Estilbenos/farmacologia , Receptor Sigma-1RESUMO
Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease that causes progressive paralysis and death due to degeneration of motoneurons in spinal cord, brainstem and motor cortex. Nowadays, there is no effective therapy and patients die 2-5 years after diagnosis. Resveratrol (trans-3,4',5-trihydroxystilbene) is a natural polyphenol found in grapes, with promising neuroprotective effects since it induces expression and activation of several neuroprotective pathways involving Sirtuin1 and AMPK. The objective of this work was to assess the effect of resveratrol administration on SOD1(G93A) ALS mice. We determined the onset of symptoms by rotarod test and evaluated upper and lower motoneuron function using electrophysiological tests. We assessed the survival of the animals and determined the number of spinal motoneurons. Finally, we further investigated resveratrol mechanism of action by means of western blot and immunohistochemical analysis. Resveratrol treatment from 8 weeks of age significantly delayed disease onset and preserved lower and upper motoneuron function in female and male animals. Moreover, resveratrol significantly extended SOD1(G93A) mice lifespan and promoted survival of spinal motoneurons. Delayed resveratrol administration from 12 weeks of age also improved spinal motoneuron function preservation and survival. Further experiments revealed that resveratrol protective effects were associated with increased expression and activation of Sirtuin 1 and AMPK in the ventral spinal cord. Both mediators promoted normalization of the autophagic flux and, more importantly, increased mitochondrial biogenesis in the SOD1(G93A) spinal cord. Taken together, our findings suggest that resveratrol may represent a promising therapy for ALS.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estilbenos/farmacologia , Superóxido Dismutase/genética , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fármacos Neuroprotetores/uso terapêutico , Resveratrol , Sirtuínas/metabolismo , Estilbenos/uso terapêutico , Superóxido Dismutase-1RESUMO
Dysferlinopathies are caused by mutations in the DYSF gene. Dysferlin is a protein mainly expressed in the skeletal muscle and monocytes. Cell therapy constitutes a promising tool for the treatment of muscular dystrophies. The aim of our study was to evaluate the effect of bone marrow transplantation (BMT) using the A/J Dysf(prmd) mouse model of dysferlinopathy. For that purpose, we studied dysferlin expression by western blot and/or immunohistochemistry in transplanted mice and controls. Computerized analyses of locomotion and electrophysiological techniques were also performed to test the functional improvement. We observed dysferlin expression in splenocytes, but not in the skeletal muscle of the transplanted mice. However, the locomotion test, electromyography studies, and muscle histology showed an improvement in all transplanted mice that was more significant in the animals transplanted with dysferlinâº/⺠cells. In conclusion, although BMT restores dysferlin expression in monocytes, but not in skeletal muscle, muscle function was partially recovered. We propose that the slight improvement observed in the functional studies could be related with factors, such as the hepatocyte growth factor, released after BMT that prevented muscle degeneration.