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1.
Cell Rep Med ; 5(5): 101546, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38703766

RESUMO

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.


Assuntos
Esclerose Lateral Amiotrófica , Fatores Inibidores da Migração de Macrófagos , Neurônios Motores , Superóxido Dismutase-1 , Fatores Inibidores da Migração de Macrófagos/metabolismo , Fatores Inibidores da Migração de Macrófagos/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/terapia , Esclerose Lateral Amiotrófica/patologia , Animais , Humanos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Camundongos , Células-Tronco Pluripotentes Induzidas/metabolismo , Oxirredutases Intramoleculares/metabolismo , Oxirredutases Intramoleculares/genética , Camundongos Transgênicos , Dependovirus/genética , Modelos Animais de Doenças , Masculino , Mutação/genética , Feminino , Dobramento de Proteína
2.
J Neuroinflammation ; 21(1): 17, 2024 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-38212835

RESUMO

Amyotrophic lateral sclerosis (ALS) is an adult-onset progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons in the brain and spinal cord. Accumulating evidence suggests that ALS is not solely a neuronal cell- or brain tissue-autonomous disease and that neuroinflammation plays a key role in disease progression. Furthermore, whereas both CD4 and CD8 T cells were observed in spinal cords of ALS patients and in mouse models of the disease, their role in the neuroinflammatory process, especially considering their functional changes with age, is not fully explored. In this study, we revealed the structure of the CD4 T-cell compartment during disease progression of early-onset SOD1G93A and late-onset SOD1G37R mouse models of ALS. We show age-related changes in the CD4 T-cell subset organization between these mutant SOD1 mouse models towards increased frequency of effector T cells in spleens of SOD1G37R mice and robust infiltration of CD4 T cells expressing activation markers and the checkpoint molecule PD1 into the spinal cord. The frequency of infiltrating CD4 T cells correlated with the frequency of infiltrating CD8 T cells which displayed a more exhausted phenotype. Moreover, RNA-Seq and immunohistochemistry analyses of spinal cords from SOD1G37R mice with early clinical symptoms demonstrated immunological trajectories reminiscent of a neurotoxic inflammatory response which involved proinflammatory T cells and antigen presentation related pathways. Overall, our findings suggest that age-related changes of the CD4 T cell landscape is indicative of a chronic inflammatory response, which aggravates the disease process and can be therapeutically targeted.


Assuntos
Esclerose Lateral Amiotrófica , Humanos , Camundongos , Animais , Esclerose Lateral Amiotrófica/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Camundongos Transgênicos , Doenças Neuroinflamatórias , Senescência de Células T , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Medula Espinal/metabolismo , Progressão da Doença , Modelos Animais de Doenças
3.
STAR Protoc ; 2(3): 100719, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34401785

RESUMO

Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019.


Assuntos
Imunoterapia Adotiva/métodos , Transdução Genética/métodos , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo/métodos , Vetores Genéticos/genética , Camundongos , Retroviridae/genética
4.
Sci Rep ; 8(1): 2430, 2018 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-29403068

RESUMO

The pancrustacean theory groups crustaceans and hexapods (once thought to comprise separate clades within the Arthropoda) into a single clade. A key feature common to all pancrustaceans is their chitinous exoskeleton, with a major contribution by cuticular proteins. Among these, are the CPAP3's, a family of cuticular proteins, first identified in the hexapod Drosophila melanogaster and characterized by an N-terminal signaling peptide and three chitin-binding domains. In this study, CPAP3 proteins were mined from a transcriptomic library of a decapod crustacean, the crayfish Cherax quadricarinatus. Phylogenetic analysis of other CPAP3 proteins from hexapods and other crustaceans showed a high degree of conservation. Characterization of the crayfish proteins, designated CqCPAP3's, suggested a major role for CPAP3'sin cuticle formation. Loss-of-function experiments using RNAi supported such a notion by demonstrating crucial roles for several CqCPAP3 proteins during molting. A putative mode of action for the CqCPAP3 proteins -theoretically binding three chitin strands- was suggested by the structural data obtained from a representative recombinant CqCPAP3. The similarities between the CqCPAP3 proteins and their hexapod homologues further demonstrated common genetic and proteinaceous features of cuticle formation in pancrustaceans, thereby reinforcing the linkage between these two highly important phylogenetic groups.


Assuntos
Proteínas de Artrópodes/química , Astacoidea/genética , Quitina/química , Insetos/genética , Filogenia , Transcriptoma , Exoesqueleto/química , Exoesqueleto/metabolismo , Animais , Proteínas de Artrópodes/antagonistas & inibidores , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/metabolismo , Astacoidea/classificação , Astacoidea/metabolismo , Biomineralização/genética , Quitina/biossíntese , Quitina/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Insetos/classificação , Insetos/metabolismo , Muda , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
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