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1.
J Med Chem ; 65(18): 12445-12459, 2022 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-36098485

RESUMO

Huntington's disease (HD) is a lethal autosomal dominant neurodegenerative disorder resulting from a CAG repeat expansion in the huntingtin (HTT) gene. The product of translation of this gene is a highly aggregation-prone protein containing a polyglutamine tract >35 repeats (mHTT) that has been shown to colocalize with histone deacetylase 4 (HDAC4) in cytoplasmic inclusions in HD mouse models. Genetic reduction of HDAC4 in an HD mouse model resulted in delayed aggregation of mHTT, along with amelioration of neurological phenotypes and extended lifespan. To further investigate the role of HDAC4 in cellular models of HD, we have developed bifunctional degraders of the protein and report the first potent and selective degraders of HDAC4 that show an effect in multiple cell lines, including HD mouse model-derived cortical neurons. These degraders act via the ubiquitin-proteasomal pathway and selectively degrade HDAC4 over other class IIa HDAC isoforms (HDAC5, HDAC7, and HDAC9).


Assuntos
Histona Desacetilases , Doença de Huntington , Animais , Modelos Animais de Doenças , Desenvolvimento de Medicamentos , Histona Desacetilases/metabolismo , Proteína Huntingtina/genética , Proteína Huntingtina/metabolismo , Doença de Huntington/genética , Camundongos , Neurônios/metabolismo , Proteólise , Ubiquitinas
2.
J Med Chem ; 62(6): 2988-3008, 2019 03 28.
Artigo em Inglês | MEDLINE | ID: mdl-30840447

RESUMO

Genetic and pharmacological evidence indicates that the reduction of ataxia telangiectasia-mutated (ATM) kinase activity can ameliorate mutant huntingtin (mHTT) toxicity in cellular and animal models of Huntington's disease (HD), suggesting that selective inhibition of ATM could provide a novel clinical intervention to treat HD. Here, we describe the development and characterization of ATM inhibitor molecules to enable in vivo proof-of-concept studies in HD animal models. Starting from previously reported ATM inhibitors, we aimed with few modifications to increase brain exposure by decreasing P-glycoprotein liability while maintaining potency and selectivity. Here, we report brain-penetrant ATM inhibitors that have robust pharmacodynamic (PD) effects consistent with ATM kinase inhibition in the mouse brain and an understandable pharmacokinetic/PD (PK/PD) relationship. Compound 17 engages ATM kinase and shows robust dose-dependent inhibition of X-ray irradiation-induced KAP1 phosphorylation in the mouse brain. Furthermore, compound 17 protects against mHTT (Q73)-induced cytotoxicity in a cortical-striatal cell model of HD.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/antagonistas & inibidores , Doença de Huntington/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Modelos Animais de Doenças , Cães , Humanos , Células Madin Darby de Rim Canino , Camundongos , Fármacos Neuroprotetores/metabolismo , Fármacos Neuroprotetores/farmacocinética , Estudo de Prova de Conceito
3.
J Cell Sci ; 130(11): 1877-1889, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28404788

RESUMO

Neuronal dense-core vesicles (DCVs) transport and secrete neuropeptides necessary for development, plasticity and survival, but little is known about their fusion mechanism. We show that Snap-25-null mutant (SNAP-25 KO) neurons, previously shown to degenerate after 4 days in vitro (DIV), contain fewer DCVs and have reduced DCV fusion probability in surviving neurons at DIV14. At DIV3, before degeneration, SNAP-25 KO neurons show normal DCV fusion, but one day later fusion is significantly reduced. To test if other SNAP homologs support DCV fusion, we expressed SNAP-23, SNAP-29 or SNAP-47 in SNAP-25 KO neurons. SNAP-23 and SNAP-29 rescued viability and supported DCV fusion in SNAP-25 KO neurons, but SNAP-23 did so more efficiently. SNAP-23 also rescued synaptic vesicle (SV) fusion while SNAP-29 did not. SNAP-47 failed to rescue viability and did not support DCV or SV fusion. These data demonstrate a developmental switch, in hippocampal neurons between DIV3 and DIV4, where DCV fusion becomes SNAP-25 dependent. Furthermore, SNAP-25 homologs support DCV and SV fusion and neuronal viability to variable extents - SNAP-23 most effectively, SNAP-29 less so and SNAP-47 ineffectively.


Assuntos
Hipocampo/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Secretórias/metabolismo , Proteína 25 Associada a Sinaptossoma/genética , Animais , Transporte Biológico , Morte Celular/genética , Embrião não Mamífero , Exocitose , Regulação da Expressão Gênica , Teste de Complementação Genética , Hipocampo/patologia , Fusão de Membrana , Camundongos , Camundongos Knockout , Neurônios/patologia , Terminações Pré-Sinápticas/patologia , Cultura Primária de Células , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Qb-SNARE/genética , Proteínas Qb-SNARE/metabolismo , Proteínas Qc-SNARE/genética , Proteínas Qc-SNARE/metabolismo , Vesículas Secretórias/patologia , Transmissão Sináptica , Proteína 25 Associada a Sinaptossoma/deficiência
5.
Elife ; 42015 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-25719439

RESUMO

Neuropeptides released from dense-core vesicles (DCVs) modulate neuronal activity, but the molecules driving DCV secretion in mammalian neurons are largely unknown. We studied the role of calcium-activator protein for secretion (CAPS) proteins in neuronal DCV secretion at single vesicle resolution. Endogenous CAPS-1 co-localized with synaptic markers but was not enriched at every synapse. Deletion of CAPS-1 and CAPS-2 did not affect DCV biogenesis, loading, transport or docking, but DCV secretion was reduced by 70% in CAPS-1/CAPS-2 double null mutant (DKO) neurons and remaining fusion events required prolonged stimulation. CAPS deletion specifically reduced secretion of stationary DCVs. CAPS-1-EYFP expression in DKO neurons restored DCV secretion, but CAPS-1-EYFP and DCVs rarely traveled together. Synaptic localization of CAPS-1-EYFP in DKO neurons was calcium dependent and DCV fusion probability correlated with synaptic CAPS-1-EYFP expression. These data indicate that CAPS-1 promotes fusion competence of immobile (tethered) DCVs in presynaptic terminals and that CAPS-1 localization to DCVs is probably not essential for this role.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Terminações Pré-Sinápticas/fisiologia , Animais , Hipocampo/citologia , Camundongos , Neurônios/citologia , Transmissão Sináptica
6.
PLoS One ; 9(3): e91697, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24614299

RESUMO

Protein modification by Small Ubiquitin-like MOdifier (SUMO) entities is involved in a number of neuronal functions, including synaptogenesis and synaptic plasticity. Tomosyn-1 (syntaxin-binding protein 5; STXPB5) binds to t-SNARE (Soluble NSF Attachment Protein Receptor) proteins to regulate neurotransmission and is one of the few neuronal SUMO substrate proteins identified. Here we used yeast two-hybrid screening to show that tomosyn-1 interacts with the SUMO E3 ligase PIASγ (Protein Inhibitor of Activated STAT; PIAS4 or ZMIZ6). This novel interaction involved the C-terminus of tomosyn-1 and the N-terminus of PIASγ. It was confirmed by two-way immunoprecipitation experiments using the full-length proteins expressed in HEK293T cells. Tomosyn-1 was preferentially modified by the SUMO-2/3 isoform. PIASγ-dependent modification of tomosyn-1 with SUMO-2/3 presents a novel mechanism to adapt secretory strength to the dynamic synaptic environment.


Assuntos
Proteínas do Tecido Nervoso/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas R-SNARE/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Etilmaleimida/farmacologia , Células HEK293 , Humanos , Masculino , Camundongos Endogâmicos BALB C , Proteínas de Ligação a Poli-ADP-Ribose , Ligação Proteica/efeitos dos fármacos , Técnicas do Sistema de Duplo-Híbrido
7.
PLoS One ; 9(2): e87923, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24503862

RESUMO

Huntington's disease (HD) is a devastating, genetic neurodegenerative disease caused by a tri-nucleotide expansion in exon 1 of the huntingtin gene. HD is clinically characterized by chorea, emotional and psychiatric disturbances and cognitive deficits with later symptoms including rigidity and dementia. Pathologically, the cortico-striatal pathway is severely dysfunctional as reflected by striatal and cortical atrophy in late-stage disease. Brain-derived neurotrophic factor (BDNF) is a neuroprotective, secreted protein that binds with high affinity to the extracellular domain of the tropomyosin-receptor kinase B (TrkB) receptor promoting neuronal cell survival by activating the receptor and down-stream signaling proteins. Reduced cortical BDNF production and transport to the striatum have been implicated in HD pathogenesis; the ability to enhance TrkB signaling using a BDNF mimetic might be beneficial in disease progression, so we explored this as a therapeutic strategy for HD. Using recombinant and native assay formats, we report here the evaluation of TrkB antibodies and a panel of reported small molecule TrkB agonists, and identify the best candidate, from those tested, for in vivo proof of concept studies in transgenic HD models.


Assuntos
Anticorpos Monoclonais/farmacologia , Doença de Huntington/metabolismo , Receptor trkB/agonistas , Receptor trkB/metabolismo , Animais , Anticorpos Monoclonais/química , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/efeitos dos fármacos , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Humanos , Doença de Huntington/tratamento farmacológico , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacos
8.
J Cell Biol ; 199(6): 883-91, 2012 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-23229896

RESUMO

Neuronal dense-core vesicles (DCVs) contain diverse cargo crucial for brain development and function, but the mechanisms that control their release are largely unknown. We quantified activity-dependent DCV release in hippocampal neurons at single vesicle resolution. DCVs fused preferentially at synaptic terminals. DCVs also fused at extrasynaptic sites but only after prolonged stimulation. In munc13-1/2-null mutant neurons, synaptic DCV release was reduced but not abolished, and synaptic preference was lost. The remaining fusion required prolonged stimulation, similar to extrasynaptic fusion in wild-type neurons. Conversely, Munc13-1 overexpression (M13OE) promoted extrasynaptic DCV release, also without prolonged stimulation. Thus, Munc13-1/2 facilitate DCV fusion but, unlike for synaptic vesicles, are not essential for DCV release, and M13OE is sufficient to produce efficient DCV release extrasynaptically.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios/fisiologia , Vesículas Secretórias/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Terminações Pré-Sinápticas/fisiologia
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