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1.
Proc Natl Acad Sci U S A ; 117(52): 33597-33607, 2020 12 29.
Artigo em Inglês | MEDLINE | ID: mdl-33318207

RESUMO

Axon injury is a hallmark of many neurodegenerative diseases, often resulting in neuronal cell death and functional impairment. Dual leucine zipper kinase (DLK) has emerged as a key mediator of this process. However, while DLK inhibition is robustly protective in a wide range of neurodegenerative disease models, it also inhibits axonal regeneration. Indeed, there are no genetic perturbations that are known to both improve long-term survival and promote regeneration. To identify such a neuroprotective target, we conducted a set of complementary high-throughput screens using a protein kinase inhibitor library in human stem cell-derived retinal ganglion cells (hRGCs). Overlapping compounds that promoted both neuroprotection and neurite outgrowth were bioinformatically deconvoluted to identify specific kinases that regulated neuronal death and axon regeneration. This work identified the role of germinal cell kinase four (GCK-IV) kinases in cell death and additionally revealed their unexpected activity in suppressing axon regeneration. Using an adeno-associated virus (AAV) approach, coupled with genome editing, we validated that GCK-IV kinase knockout improves neuronal survival, comparable to that of DLK knockout, while simultaneously promoting axon regeneration. Finally, we also found that GCK-IV kinase inhibition also prevented the attrition of RGCs in developing retinal organoid cultures without compromising axon outgrowth, addressing a major issue in the field of stem cell-derived retinas. Together, these results demonstrate a role for the GCK-IV kinases in dissociating the cell death and axonal outgrowth in neurons and their druggability provides for therapeutic options for neurodegenerative diseases.


Assuntos
Axônios/enzimologia , Axônios/patologia , Sistema Nervoso Central/patologia , Quinases do Centro Germinativo/metabolismo , Regeneração Nervosa , Animais , Sequência de Bases , Sistemas CRISPR-Cas/genética , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dependovirus/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Traumatismos do Nervo Óptico/metabolismo , Traumatismos do Nervo Óptico/patologia , Organoides/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/metabolismo , Transdução de Sinais/efeitos dos fármacos
2.
Elife ; 92020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32369015

RESUMO

The intermediate filament protein keratin 14 (K14) provides vital structural support in basal keratinocytes of epidermis. Recent studies evidenced a role for K14-dependent disulfide bonding in the organization and dynamics of keratin IFs in skin keratinocytes. Here we report that knock-in mice harboring a cysteine-to-alanine substitution at Krt14's codon 373 (C373A) exhibit alterations in disulfide-bonded K14 species and a barrier defect secondary to enhanced proliferation, faster transit time and altered differentiation in epidermis. A proteomics screen identified 14-3-3 as K14 interacting proteins. Follow-up studies showed that YAP1, a transcriptional effector of Hippo signaling regulated by 14-3-3sigma in skin keratinocytes, shows aberrant subcellular partitioning and function in differentiating Krt14 C373A keratinocytes. Residue C373 in K14, which is conserved in a subset of keratins, is revealed as a novel regulator of keratin organization and YAP function in early differentiating keratinocytes, with an impact on cell mechanics, homeostasis and barrier function in epidermis.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Epiderme/metabolismo , Queratina-14/metabolismo , Proteínas 14-3-3/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Proteínas de Ciclo Celular/fisiologia , Epiderme/fisiologia , Epiderme/ultraestrutura , Feminino , Técnicas de Introdução de Genes , Homeostase , Queratina-14/fisiologia , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas de Sinalização YAP
3.
Nat Genet ; 51(1): 42-50, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30455415

RESUMO

Bicuspid aortic valve (BAV) is a common congenital heart defect (population incidence, 1-2%)1-3 that frequently presents with ascending aortic aneurysm (AscAA)4. BAV/AscAA shows autosomal dominant inheritance with incomplete penetrance and male predominance. Causative gene mutations (for example, NOTCH1, SMAD6) are known for ≤1% of nonsyndromic BAV cases with and without AscAA5-8, impeding mechanistic insight and development of therapeutic strategies. Here, we report the identification of variants in ROBO4 (which encodes a factor known to contribute to endothelial performance) that segregate with disease in two families. Targeted sequencing of ROBO4 showed enrichment for rare variants in BAV/AscAA probands compared with controls. Targeted silencing of ROBO4 or mutant ROBO4 expression in endothelial cell lines results in impaired barrier function and a synthetic repertoire suggestive of endothelial-to-mesenchymal transition. This is consistent with BAV/AscAA-associated findings in patients and in animal models deficient for ROBO4. These data identify a novel endothelial etiology for this common human disease phenotype.


Assuntos
Aneurisma da Aorta Torácica/genética , Valva Aórtica/anormalidades , Doenças das Valvas Cardíacas/genética , Mutação/genética , Receptores de Superfície Celular/genética , Animais , Doença da Válvula Aórtica Bicúspide , Células Cultivadas , Modelos Animais de Doenças , Células Endoteliais/fisiologia , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Peixe-Zebra
4.
Neuron ; 94(6): 1142-1154.e6, 2017 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-28641113

RESUMO

Dual leucine zipper kinase (DLK) has been implicated in cell death signaling secondary to axonal damage in retinal ganglion cells (RGCs) and other neurons. To better understand the pathway through which DLK acts, we developed enhanced functional genomic screens in primary RGCs, including use of arrayed, whole-genome, small interfering RNA libraries. Explaining why DLK inhibition is only partially protective, we identify leucine zipper kinase (LZK) as cooperating with DLK to activate downstream signaling and cell death in RGCs, including in a mouse model of optic nerve injury, and show that the same pathway is active in human stem cell-derived RGCs. Moreover, we identify four transcription factors, JUN, activating transcription factor 2 (ATF2), myocyte-specific enhancer factor 2A (MEF2A), and SRY-Box 11 (SOX11), as being the major downstream mediators through which DLK/LZK activation leads to RGC cell death. Increased understanding of the DLK pathway has implications for understanding and treating neurodegenerative diseases.


Assuntos
Sobrevivência Celular/genética , MAP Quinase Quinase Quinases/genética , MAP Quinase Quinase Quinases/metabolismo , Traumatismos do Nervo Óptico/genética , Células Ganglionares da Retina/metabolismo , Animais , Morte Celular , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Citometria de Fluxo , Células-Tronco Embrionárias Humanas/citologia , Humanos , Imunoprecipitação , Camundongos , Camundongos Knockout , Neuritos , Neurônios , Traumatismos do Nervo Óptico/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/patologia
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