RESUMEN
The R47H variant of the microglial membrane receptor TREM2 is linked to increased risk of late onset Alzheimer's disease. Human induced pluripotent stem cell derived microglia (iPS-Mg) from patient iPSC lines expressing the AD-linked R47Hhet TREM2 variant, common variant (Cv) or an R47Hhom CRISPR edited line and its isogeneic control, demonstrated that R47H-expressing iPS-Mg expressed a deficit in signal transduction in response to the TREM2 endogenous ligand phosphatidylserine with reduced pSYK-pERK1/2 signalling and a reduced NLRP3 inflammasome response, (including ASC speck formation, Caspase-1 activation and IL-1beta secretion). Apoptotic cell phagocytosis and soluble TREM2 shedding were unaltered, suggesting a disjoint between these pathways and the signalling cascades downstream of TREM2 in R47H-expressing iPS-Mg, whilst metabolic deficits in glycolytic capacity and maximum respiration were reversed when R47H expressing iPS-Mg were exposed to PS+ expressing cells. These findings suggest that R47H-expressing microglia are unable to respond fully to cell damage signals such as phosphatidylserine, which may contribute to the progression of neurodegeneration in late-onset AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Inflamasomas/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/metabolismo , Receptores Inmunológicos/metabolismo , Transducción de Señal/fisiología , Encéfalo/metabolismo , Células Cultivadas , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Fagocitosis/fisiologíaRESUMEN
Mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration, including Alzheimer's disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signaling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterized. Here, we present phage display generated antibody single-chain variable fragments (scFvs) to human TREM2 immunoglobulin-like domain. Co-crystal structures revealed the binding of two scFvs to an epitope on the TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species, which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterization of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.
Asunto(s)
Epítopos/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores Inmunológicos/metabolismo , Células HEK293 , Humanos , Fagocitosis/fisiología , Anticuerpos de Cadena ÚnicaRESUMEN
c-Jun N-terminal kinases (JNKs) are implicated in cell death in neurodegenerative disorders. Therefore, JNK inhibitors could act as neuroprotective agents. To evaluate potential candidates, reproducible and quantitative CNS in vivo models are required. To that end, a pentylenetetrazole-induced seizure model was explored. c-Jun phosphorylation was detected in hippocampal extracts by blotting c-Jun immunoprecipitates with phosphorylation-specific antibodies. Pentylenetetrazole administration induced rapid and reproducible increases in c-Jun phosphorylation. However, special attention had to be paid to the composition of the extraction buffer to ensure stabilization of protein phosphorylation, as demonstrated using internal standards of phosphorylated recombinant c-Jun. As JNK and its upstream activator MKK4 are activated by phosphorylation, these events were also evaluated. In principle, kinase inhibitors could act at the level of JNK or upstream kinases to inhibit c-Jun phosphorylation. MKK4 phosphorylation was dramatically increased in response to pentylenetetrazole but, again, only when appropriate phosphatase inhibitors were in the extraction buffer. In contrast, JNK was found to be constitutively phosphorylated and unaltered upon pentylenetetrazole treatment. The JNK inhibitor SP600125 was shown to inhibit c-Jun phosphorylation without affecting MKK4 phosphorylation. Our procedures enable analysis of JNK pathway signalling in a CNS model and, also, should be applicable to that of other protein phosphorylation events in vivo.
Asunto(s)
Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Hipocampo/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Pentilenotetrazol/toxicidad , Convulsiones/inducido químicamente , Animales , Electroforesis en Gel de Poliacrilamida , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Immunoblotting , Inmunohistoquímica , MAP Quinasa Quinasa 4/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-jun/efectos de los fármacos , Proteínas Proto-Oncogénicas c-jun/metabolismoRESUMEN
Developing nerve growth factor (NGF)-dependent sympathetic neurons are one of the best-studied in vitro models of neuronal apoptosis and have been used to identify key components of the neuronal cell death pathway. This chapter describes how to prepare purified cultures of primary sympathetic neurons and how to induce apoptosis by NGF deprivation. In addition, a simple method for measuring neuronal viability based on the live/dead assay is also described. This can be used for assessing the effect of small molecule inhibitors of protein kinases, caspases and other enzymes, on NGF withdrawal-induced death.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Supervivencia Celular , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citología , Neuronas/metabolismo , Sistema Nervioso Simpático/citología , Animales , Apoptosis/fisiología , Bioensayo/métodos , Tamaño de la Célula , Células Cultivadas , Medios de Cultivo/química , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismoRESUMEN
Developing sympathetic neurons, which depend on nerve growth factor for survival, are one of the best studied in vitro models of neuronal apoptosis and have been extensively used for cellular and molecular studies of the neuronal death pathway. Important apoptotic events after nerve growth factor withdrawal include the release of proapoptotic proteins, such as cytochrome c, from the mitochondria and the activation of caspases, followed by nuclear DNA fragmentation and chromatin condensation. In this chapter, we describe immunocytochemical techniques for studying apoptotic DNA fragmentation, changes in nuclear morphology, and mitochondrial cytochrome c release at the single cell level using sympathetic neurons cultured on glass coverslips.
Asunto(s)
Apoptosis/fisiología , Inmunohistoquímica/métodos , Neuronas/fisiología , Sistema Nervioso Simpático/citología , Animales , Bisbenzimidazol/metabolismo , Células Cultivadas , Citocromos c/metabolismo , Fragmentación del ADN , Colorantes Fluorescentes/metabolismo , Etiquetado Corte-Fin in Situ , Factor de Crecimiento Nervioso/metabolismo , Neuronas/citologíaRESUMEN
Imidazole-based structures of p38 inhibitors served as a starting point for the design of JNK3 inhibitors. Construction of a 6,7-dihydro-5H-pyrrolo[1,2-a]imidazole scaffold led to the synthesis of the (S)-enantiomers, which exhibited p38/JNK3 IC50 ratio of up to 10 and were up to 20 times more potent inhibitors of JNK3 than the relevant (R)-enantiomers. The JNK3 inhibitory potency correlated well with inhibition of c-Jun phosphorylation and neuroprotective properties of the compounds in low K+-induced cell death of rat cerebellar granule neurones.