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2.
Mov Disord ; 29(9): 1201-4, 2014 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-24676999

RESUMEN

BACKGROUND: Depression, parkinsonism, and hypoventilation (Perry syndrome) or familial motor neuron disease have been linked to mutations in dynactin P150(Glued) (DCTN1). METHODS: We employed genealogic, clinical, neurologic, and MRI investigations, as well as analysis of genes implicated in parkinsonism. Cellular transfection, immunocytochemistry, and immunoprecipitation analysis of wild-type (WT) and mutant DCTN1 were also performed. RESULTS: A novel heterozygous mutation, DCTN1 c.156T>G, encoding p.Phe52Leu, segregates with parkinsonism in a Japanese family. The substitution was not observed in affected probands with familial parkinsonism or control subjects and is evolutionarily conserved. In contrast to Perry syndrome, affected carriers have late-onset disease and slower progression, with frontotemporal atrophy revealed by MRI. In vitro studies suggest the mutant protein has impaired microtubule binding, compared to WT dynactin p150(Glued) . CONCLUSIONS: DCTN1 mutations may contribute to disparate neurodegenerative diagnoses, including familial motor neuron disease, parkinsonism, and frontotemporal atrophy, and further studies of dynactin-mediated cargo transport may prove insightful.


Asunto(s)
Demencia Frontotemporal/genética , Proteínas Asociadas a Microtúbulos/genética , Trastornos Parkinsonianos/genética , Polimorfismo de Nucleótido Simple/genética , Edad de Inicio , Anciano , Atrofia , Citocinas/metabolismo , Complejo Dinactina , Femenino , Pruebas Genéticas , Células HEK293 , Humanos , Masculino , Microtúbulos/patología , Persona de Mediana Edad , Examen Neurológico , Transfección
3.
Alzheimers Dement (N Y) ; 4: 521-534, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30386817

RESUMEN

INTRODUCTION: The abnormal hyperphosphorylation of the microtubule-associated protein tau plays a crucial role in neurodegeneration in Alzheimer's disease (AD) and other tauopathies. METHODS: Highly specific and selective anti-pS396-tau antibodies have been generated using peptide immunization with screening against pathologic hyperphosphorylated tau from rTg4510 mouse and AD brains and selection in in vitro and in vivo tau seeding assays. RESULTS: The antibody C10.2 bound specifically to pS396-tau with an IC50 of 104 pM and detected preferentially hyperphosphorylated tau aggregates from AD brain with an IC50 of 1.2 nM. C10.2 significantly reduced tau seeding of P301L human tau in HEK293 cells, murine cortical neurons, and mice. AD brain extracts depleted with C10.2 were not able to seed tau in vitro and in vivo, demonstrating that C10.2 specifically recognized pathologic seeding-competent tau. DISCUSSION: Targeting pS396-tau with an antibody like C10.2 may provide therapeutic benefit in AD and other tauopathies.

4.
J Med Chem ; 60(21): 8945-8962, 2017 11 09.
Artículo en Inglés | MEDLINE | ID: mdl-29023112

RESUMEN

Mutations in leucine-rich repeat kinase 2 (LRRK2), such as G2019S, are associated with an increased risk of developing Parkinson's disease. Surrogates for the LRRK2 kinase domain based on checkpoint kinase 1 (CHK1) mutants were designed, expressed in insect cells infected with baculovirus, purified, and crystallized. X-ray structures of the surrogates complexed with known LRRK2 inhibitors rationalized compound potency and selectivity. The CHK1 10-point mutant was preferred, following assessment of surrogate binding affinity with LRRK2 inhibitors. Fragment hit-derived arylpyrrolo[2,3-b]pyridine LRRK2 inhibitors underwent structure-guided optimization using this crystallographic surrogate. LRRK2-pSer935 HEK293 IC50 data for 22 were consistent with binding to Ala2016 in LRRK2 (equivalent to Ala147 in CHK1 10-point mutant structure). Compound 22 was shown to be potent, moderately selective, orally available, and brain-penetrant in wild-type mice, and confirmation of target engagement was demonstrated, with LRRK2-pSer935 IC50 values for 22 in mouse brain and kidney being 1.3 and 5 nM, respectively.


Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Encéfalo/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Cristalografía/métodos , Células HEK293 , Humanos , Riñón/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Ratones , Mutación , Enfermedad de Parkinson/genética , Unión Proteica , Dominios Proteicos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacocinética
5.
Stem Cell Res ; 17(2): 306-317, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-27596958

RESUMEN

The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3) is caused by a CAG-repeat expansion in the ATXN3 gene. In this study, induced pluripotent stem cell (iPSC) lines were established from two SCA3 patients. Dermal fibroblasts were reprogrammed using an integration-free method and the resulting SCA3 iPSCs were differentiated into neurons. These neuronal lines harbored the disease causing mutation, expressed comparable levels of several neuronal markers and responded to the neurotransmitters, glutamate/glycine, GABA and acetylcholine. Additionally, all neuronal cultures formed networks displaying synchronized spontaneous calcium oscillations within 28days of maturation, and expressed the mature neuronal markers NeuN and Synapsin 1 implying a relatively advanced state of maturity, although not comparable to that of the adult human brain. Interestingly, we were not able to recapitulate the glutamate-induced ataxin-3 aggregation shown in a previously published iPSC-derived SCA3 model. In conclusion, we have generated a panel of SCA3 patient iPSCs and a robust protocol to derive neurons of relatively advanced maturity, which could potentially be valuable for the study of SCA3 disease mechanisms.


Asunto(s)
Reprogramación Celular , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Machado-Joseph/patología , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Ataxina-3/genética , Encéfalo/metabolismo , Calcio/metabolismo , Diferenciación Celular , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/trasplante , Ionomicina/farmacología , Cariotipo , Enfermedad de Machado-Joseph/genética , Enfermedad de Machado-Joseph/metabolismo , Microscopía Fluorescente , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Agregado de Proteínas/efectos de los fármacos , Proteínas Represoras/genética , Sinapsinas/genética , Sinapsinas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
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