Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 5 de 5
Filtrar
1.
Neurobiol Dis ; 134: 104614, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31605779

RESUMO

The G2019S substitution in the kinase domain of LRRK2 (LRRK2G2019S) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2WT or LV-ΔLRRK2G2019S, the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2WT and ΔLRRK2G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10.


Assuntos
Neurônios Dopaminérgicos/patologia , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Degeneração Neural/genética , Doença de Parkinson , Domínios Proteicos/genética , Animais , Técnicas de Transferência de Genes , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus , Masculino , Mutação , Degeneração Neural/patologia , Parte Compacta da Substância Negra , Ratos , Ratos Sprague-Dawley
2.
Front Immunol ; 10: 154, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30787931

RESUMO

NKG2D is an activating receptor expressed on the surface of immune cells including subsets of T lymphocytes. NKG2D binds multiple ligands (NKG2DL) whose expression are differentially triggered in a cell type and stress specific manner. The NKG2D-NKG2DL interaction has been involved in autoimmune disorders but its role in animal models of multiple sclerosis (MS) remains incompletely resolved. Here we show that NKG2D and its ligand MULT1 contribute to the pathobiology of experimental autoimmune encephalomyelitis (EAE). MULT1 protein levels are increased in the central nervous system (CNS) at EAE disease peak; soluble MULT1 is elevated in the cerebrospinal fluid of both active and passive EAE. We establish that such soluble MULT1 enhances effector functions (e.g., IFNγ production) of activated CD8 T lymphocytes from wild type but not from NKG2D-deficient (Klrk1-/-) mice in vitro. The adoptive transfer of activated T lymphocytes from wild type donors induced a significantly reduced EAE disease in Klrk1-/- compared to wild type (Klrk1+/+) recipients. Characterization of T lymphocytes infiltrating the CNS of recipient mice shows that donor (CD45.1) rather than endogenous (CD45.2) CD4 T cells are the main producers of key cytokines (IFNγ, GM-CSF). In contrast, infiltrating CD8 T lymphocytes include mainly endogenous (CD45.2) cells exhibiting effector properties (NKG2D, granzyme B and IFNγ). Our data support the notion that endogenous CD8 T cells contribute to passive EAE pathobiology in a NKG2D-dependent manner. Collectively, our results point to the deleterious role of NKG2D and its MULT1 in the pathobiology of a MS mouse model.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Proteínas de Membrana/imunologia , Esclerose Múltipla/imunologia , Subfamília K de Receptores Semelhantes a Lectina de Células NK/imunologia , Animais , Encéfalo/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Modelos Animais de Doenças , Progressão da Doença , Camundongos Endogâmicos C57BL , Camundongos Knockout , Subfamília K de Receptores Semelhantes a Lectina de Células NK/genética , Medula Espinal/imunologia
3.
Acta Neuropathol Commun ; 4(1): 43, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27121871

RESUMO

Approximately 20 % of familial Amyotrophic Lateral Sclerosis (ALS) is caused by mutations in superoxide dismutase (SOD1), which leads to misfolding of the SOD1 protein, resulting in a toxic gain of function. Several conformation-restricted antibodies have been generated that specifically recognize misfolded SOD1 protein, and have been used as therapeutics in pre-clinical models. Misfolded SOD1 selectively associates with spinal cord mitochondria in SOD1 rodent models. Using the SOD1(G93A) rat model, we find that SOD1 conformational specific antibodies AMF7-63 and DSE2-3H1 labeled a fibrillar network concentrated in the anterior horn; while A5C3, B8H10, C4F6 and D3H5 labeled motor neurons as well as puncta in the neuropil. There is a time-dependent accumulation of misfolded SOD1 at the surface of spinal cord mitochondria with AMF7-63-labeled mitochondria having increased volume in contrast to a mitochondrial subset labeled with B8H10. In spinal cord homogenates and isolated mitochondria, AMF7-63, DSE2-3H1 and B8H10 detect misfolded SOD1 aggregates. SOD1 that lacks its metal cofactors has an increased affinity for naïve mitochondria and misfolded SOD1 antibodies B8H10 and DSE2-3H1 readily detect demetalated mutant and wild-type SOD1. Together, these data suggest that multiple non-native species of misfolded SOD1 may exist, some of which are associated with mitochondrial damage. Conformational antibodies are invaluable tools to identify and characterize the variation in misfolded SOD1 species with regards to biochemical characteristics and toxicity. This information is highly relevant to the further development of these reagents as therapeutics.


Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Mitocôndrias/metabolismo , Dobramento de Proteína , Superóxido Dismutase-1/metabolismo , Superóxido Dismutase/metabolismo , Esclerose Lateral Amiotrófica/patologia , Animais , Anticorpos/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Metais/metabolismo , Mitocôndrias/patologia , Agregação Patológica de Proteínas/metabolismo , Agregação Patológica de Proteínas/patologia , Deficiências na Proteostase/metabolismo , Deficiências na Proteostase/patologia , Ratos Transgênicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/genética
4.
J Neuroimmune Pharmacol ; 10(4): 528-46, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25946987

RESUMO

Multiple sclerosis (MS) is the prototypic inflammatory disease of the central nervous system (CNS) characterized by multifocal areas of demyelination, axonal damage, activation of glial cells, and immune cell infiltration. Despite intensive years of research, the etiology of this neurological disorder remains elusive. Nevertheless, the abundance of immune cells such as T lymphocytes and their products in CNS lesions of MS patients supports the notion that MS is an immune-mediated disorder. An important body of evidence gathered from MS animal models such as experimental autoimmune encephalomyelitis (EAE), points to the central contribution of CD4 T lymphocytes in disease pathogenesis. Both Th1 (producing interferon-γ) and Th17 (producing interleukin 17) CD4 T lymphocytes targeting CNS self-antigens have been implicated in MS and EAE pathobiology. Moreover, several publications suggest that CD8 T lymphocytes also participate in the development of MS lesions. The migration of activated T lymphocytes from the periphery into the CNS has been identified as a crucial step in the formation of MS lesions. Several factors promote such T cell extravasation including: molecules (e.g., cell adhesion molecules) implicated in the T cell-blood brain barrier interaction, and chemokines produced by neural cells. Finally, once in the CNS, T lymphocytes need to be reactivated by local antigen presenting cells prior to enter the parenchyma where they can initiate damage. Further investigations will be necessary to elucidate the impact of environmental factors (e.g., gut microbiota) and CNS intrinsic properties (e.g., microglial activation) on this inflammatory neurological disease.


Assuntos
Autoimunidade/imunologia , Sistema Nervoso Central/imunologia , Encefalomielite Autoimune Experimental/imunologia , Esclerose Múltipla/imunologia , Linfócitos T/imunologia , Animais , Humanos
5.
J Neurosci Methods ; 247: 23-31, 2015 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-25819540

RESUMO

BACKGROUND: Flow cytometry is an efficient and powerful technique to characterize and quantify numerous cells. However, the strengths of this technique have not been widely harnessed in neurosciences due to the critical step of CNS tissue preparation into a single cell suspension. Previous reports assessed either neural cells or infiltrating leukocytes but simultaneous detection has not been extensively implemented. We optimized CNS tissue preparation for flow cytometry analysis. NEW METHOD: We subjected CNS tissue from individual adult mice to different digestion protocols and Percoll™ methods. We quantified and characterized by flow cytometry neural cells (neurons, oligodendrocytes, microglia) and leukocytes (macrophages, T lymphocytes). RESULTS: The one step Percoll™ method significantly increased cell yield compared to the gradient Percoll™ method. The collagenase D+DNase I digestion led to the maximal cell number recovery while preserving cell marker (O4, NeuN, CD45, CD11b, CD3, CD4, CD8) integrity compared to papain, trypsin digestion, and no digestion. The combination of collagenase D+DNase I digestion and one step Percoll™ method was optimal for the recovery and analysis of cells from the CNS of naïve and experimental autoimmune encephalomyelitis (multiple sclerosis model) mice. COMPARISON WITH EXISTING METHOD(S): Although flow cytometry does not reveal CNS localization, this technique allows concurrent quantification of multiple parameters. In contrast to other protocols, our novel method simultaneously analyzes neural and immune cells in individual mice in healthy and pathological conditions. CONCLUSIONS: We strongly believe that the field of neurosciences will benefit from an optimal use of flow cytometry to elucidate physiological and pathological processes.


Assuntos
Sistema Nervoso Central/citologia , Citometria de Fluxo/métodos , Leucócitos/citologia , Neurônios/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA