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1.
Cell Stem Cell ; 31(8): 1162-1174.e8, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38917806

RESUMO

Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APPswe/swe mutation. Aged APPswe/swe but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSENM146V/M146V cortical and LRRK2G2019S/G2019S midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.


Assuntos
Doença de Alzheimer , Humanos , Doença de Alzheimer/patologia , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Senescência Celular/genética , Neurônios/metabolismo , Neurônios/patologia , Proteína NEDD8/metabolismo , Proteína NEDD8/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas tau/metabolismo , Proteínas tau/genética , Doença de Parkinson/genética , Doença de Parkinson/patologia , Doença de Parkinson/metabolismo , Envelhecimento/genética , Envelhecimento/patologia , Envelhecimento/metabolismo , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Sistemas CRISPR-Cas/genética
2.
Cell Rep ; 43(4): 114031, 2024 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-38583153

RESUMO

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.


Assuntos
Diferenciação Celular , Ventrículos Laterais , Fator Inibidor de Leucemia , Organoides , Células-Tronco Pluripotentes , Humanos , Organoides/metabolismo , Organoides/citologia , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/citologia , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Fator de Transcrição STAT3/metabolismo , Neuroglia/metabolismo , Neuroglia/citologia , Transdução de Sinais
3.
bioRxiv ; 2023 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-36824730

RESUMO

Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.

5.
Cell Stem Cell ; 28(9): 1566-1581.e8, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-33951478

RESUMO

The biological function and disease association of human endogenous retroviruses (HERVs) are largely elusive. HERV-K(HML-2) has been associated with neurotoxicity, but there is no clear understanding of its role or mechanistic basis. We addressed the physiological functions of HERV-K(HML-2) in neuronal differentiation using CRISPR engineering to activate or repress its expression levels in a human-pluripotent-stem-cell-based system. We found that elevated HERV-K(HML-2) transcription is detrimental for the development and function of cortical neurons. These effects are cell-type-specific, as dopaminergic neurons are unaffected. Moreover, high HERV-K(HML-2) transcription alters cortical layer formation in forebrain organoids. HERV-K(HML-2) transcriptional activation leads to hyperactivation of NTRK3 expression and other neurodegeneration-related genes. Direct activation of NTRK3 phenotypically resembles HERV-K(HML-2) induction, and reducing NTRK3 levels in context of HERV-K(HML-2) induction restores cortical neuron differentiation. Hence, these findings unravel a cell-type-specific role for HERV-K(HML-2) in cortical neuron development.


Assuntos
Retrovirus Endógenos , Diferenciação Celular , Humanos , Ativação Transcricional
6.
Sci Rep ; 9(1): 9615, 2019 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-31270336

RESUMO

Axonal degeneration is a key pathology of neurodegenerative diseases, including hereditary spastic paraplegia (HSP), a disorder characterized by spasticity in the lower limbs. Treatments for HSP and other neurodegenerative diseases are mainly symptomatic. While iPSC-derived neurons are valuable for drug discovery and target identification, these applications require robust differentiation paradigms and rapid phenotypic read-outs ranging between hours and a few days. Using spastic paraplegia type 4 (SPG4, the most frequent HSP subtype) as an exemplar, we here present three rapid phenotypic assays for uncovering neuronal process pathologies in iPSC-derived glutamatergic cortical neurons. Specifically, these assays detected a 51% reduction in neurite outgrowth and a 60% increase in growth cone area already 24 hours after plating; axonal swellings, a hallmark of HSP pathology, was discernible after only 5 days. Remarkably, the identified phenotypes were neuron subtype-specific and not detectable in SPG4-derived GABAergic forebrain neurons. We transferred all three phenotypic assays to a 96-well setup, applied small molecules and found that a liver X receptor (LXR) agonist rescued all three phenotypes in HSP neurons, providing a potential drug target for HSP treatment. We expect this multiparametric and rapid phenotyping approach to accelerate development of therapeutic compounds for HSP and other neurodegenerative diseases.


Assuntos
Biomarcadores , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Diferenciação Celular , Células Cultivadas , Haploinsuficiência , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Crescimento Neuronal , Fenótipo , Paraplegia Espástica Hereditária/tratamento farmacológico , Paraplegia Espástica Hereditária/etiologia , Paraplegia Espástica Hereditária/metabolismo , Espastina/genética
7.
Mol Neurobiol ; 56(12): 8168-8202, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31201651

RESUMO

Spinocerebellar ataxia type 3 (SCA3/MJD) is a polyQ neurodegenerative disease where the presymptomatic phase of pathogenesis is unknown. Therefore, we investigated the molecular network of transcriptomic and proteomic triggers in young presymptomatic SCA3/MJD brain from Ki91 knock-in mouse. We found that transcriptional dysregulations resulting from mutant ataxin-3 are not occurring in young Ki91 mice, while old Ki91 mice and also postmitotic patient SCA3 neurons demonstrate the late transcriptomic changes. Unlike the lack of early mRNA changes, we have identified numerous early changes of total proteins and phosphoproteins in 2-month-old Ki91 mouse cortex and cerebellum. We discovered the network of processes in presymptomatic SCA3 with three main groups of disturbed processes comprising altered proteins: (I) modulation of protein levels and DNA damage (Pabpc1, Ddb1, Nedd8), (II) formation of neuronal cellular structures (Tubb3, Nefh, p-Tau), and (III) neuronal function affected by processes following perturbed cytoskeletal formation (Mt-Co3, Stx1b, p-Syn1). Phosphoproteins downregulate in the young Ki91 mouse brain and their phosphosites are associated with kinases that interact with ATXN3 such as casein kinase, Camk2, and kinases controlled by another Atxn3 interactor p21 such as Gsk3, Pka, and Cdk kinases. We conclude that the onset of SCA3 pathology occurs without altered transcript level and is characterized by changed levels of proteins responsible for termination of translation, DNA damage, spliceosome, and protein phosphorylation. This disturbs global cellular processes such as cytoskeleton and transport of vesicles and mitochondria along axons causing energy deficit and neurodegeneration also manifesting in an altered level of transcripts at later ages.


Assuntos
Ataxina-3/metabolismo , Encéfalo/metabolismo , Doença de Machado-Joseph/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/metabolismo , Transcrição Gênica/fisiologia , Fatores Etários , Animais , Ataxina-3/genética , Encéfalo/patologia , Células Cultivadas , Humanos , Doença de Machado-Joseph/genética , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Fosfoproteínas/genética
8.
Eur J Neurosci ; 49(4): 561-589, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30656775

RESUMO

Recent advances in cell reprogramming have enabled assessment of disease-related cellular traits in patient-derived somatic cells, thus providing a versatile platform for disease modeling and drug development. Given the limited access to vital human brain cells, this technology is especially relevant for neurodegenerative disorders such as Parkinson's disease (PD) as a tool to decipher underlying pathomechanisms. Importantly, recent progress in genome-editing technologies has provided an ability to analyze isogenic induced pluripotent stem cell (iPSC) pairs that differ only in a single genetic change, thus allowing a thorough assessment of the molecular and cellular phenotypes that result from monogenetic risk factors. In this review, we summarize the current state of iPSC-based modeling of PD with a focus on leucine-rich repeat kinase 2 (LRRK2), one of the most prominent monogenetic risk factors for PD linked to both familial and idiopathic forms. The LRRK2 protein is a primarily cytosolic multi-domain protein contributing to regulation of several pathways including autophagy, mitochondrial function, vesicle transport, nuclear architecture and cell morphology. We summarize iPSC-based studies that contributed to improving our understanding of the function of LRRK2 and its variants in the context of PD etiopathology. These data, along with results obtained in our own studies, underscore the multifaceted role of LRRK2 in regulating cellular homeostasis on several levels, including proteostasis, mitochondrial dynamics and regulation of the cytoskeleton. Finally, we expound advantages and limitations of reprogramming technologies for disease modeling and drug development and provide an outlook on future challenges and expectations offered by this exciting technology.


Assuntos
Células-Tronco Pluripotentes Induzidas , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Mitofagia , Modelos Neurológicos , Doença de Parkinson , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Doença de Parkinson/genética , Doença de Parkinson/terapia
9.
Nat Commun ; 9(1): 4047, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279449

RESUMO

Recent reports suggest that induced neurons (iNs), but not induced pluripotent stem cell (iPSC)-derived neurons, largely preserve age-associated traits. Here, we report on the extent of preserved epigenetic and transcriptional aging signatures in directly converted induced neural stem cells (iNSCs). Employing restricted and integration-free expression of SOX2 and c-MYC, we generated a fully functional, bona fide NSC population from adult blood cells that remains highly responsive to regional patterning cues. Upon conversion, low passage iNSCs display a profound loss of age-related DNA methylation signatures, which further erode across extended passaging, thereby approximating the DNA methylation age of isogenic iPSC-derived neural precursors. This epigenetic rejuvenation is accompanied by a lack of age-associated transcriptional signatures and absence of cellular aging hallmarks. We find iNSCs to be competent for modeling pathological protein aggregation and for neurotransplantation, depicting blood-to-NSC conversion as a rapid alternative route for both disease modeling and neuroregeneration.


Assuntos
Envelhecimento/genética , Células-Tronco Pluripotentes Induzidas , Células-Tronco Neurais , Envelhecimento/metabolismo , Metilação de DNA , Epigênese Genética , Humanos , Doença de Machado-Joseph/sangue , Células-Tronco de Sangue Periférico
10.
PLoS One ; 13(8): e0201794, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30086154

RESUMO

Molecular chaperones are important regulators of protein folding and proteasomal removal of misfolded proteins. In spinocerebellar ataxia type 3 (SCA3), the co-chaperone DnaJ homology subfamily B member 1 (DNAJB1 or heat shock protein 40) is recruited to protein aggregates formed by the disease-causing mutant polyglutamine (polyQ) protein ataxin-3 (ATXN3). Over-expression of DNAJB1 reduces polyQ protein toxicity. Here, we identified two miRNAs, miR-370 and miR-543, that function in posttranscriptional regulation of DNAJB1 expression. MiRNAs are small endogenously produced RNAs controlling mRNA stability and play a role in polyQ disease pathogenesis. In human neuronal cultures derived from SCA3 patient-specific induced pluripotent stem cell (iPSC) lines, miR-370 and miR-543 levels are upregulated, while DNAJB1 expression is concurrently reduced. These findings suggest that downregulation of DNAJB1 by these two miRNAs is an early event that could contribute to SCA3 pathogenesis. Inhibition of these two miRNAs in turn could stabilize DNAJB1 and thereby be beneficial in SCA3 disease.


Assuntos
Proteínas de Choque Térmico HSP40/metabolismo , Doença de Machado-Joseph/metabolismo , MicroRNAs/metabolismo , Adulto , Idoso , Animais , Sítios de Ligação , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Camundongos Transgênicos , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Rombencéfalo/metabolismo , Adulto Jovem
11.
J Mol Med (Berl) ; 95(7): 705-718, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28593578

RESUMO

The advent of cell reprogramming has enabled the generation of induced pluripotent stem cells (iPSCs) from patient skin fibroblasts or blood cells and their subsequent differentiation into tissue-specific cells, including neurons and glia. This approach can be used to recapitulate disease-specific phenotypes in classical cell culture paradigms and thus represents an invaluable asset for disease modeling and drug validation in the framework of personalized medicine. The autophagy pathway is a ubiquitous eukaryotic degradation and recycling system, which relies on lysosomal degradation of unwanted and potentially cytotoxic components. The relevance of autophagy in the pathogenesis of neurodegenerative diseases is underlined by the observation that disease-linked genetic variants of susceptibility factors frequently result in dysregulation of autophagic-lysosomal pathways. In particular, disrupted autophagy is implied in the accumulation of potentially neurotoxic products such as protein aggregates and their precursors and defective turnover of dysfunctional mitochondria. Here, we review the current state of iPSC-based assessment of autophagic dysfunction in the context of neurodegenerative disease modeling. The collected data show that iPSC technology is capable to reveal even subtle alterations in subcellular homeostatic processes, which form the molecular basis for disease manifestation.


Assuntos
Autofagia , Células-Tronco Pluripotentes Induzidas/patologia , Doenças Neurodegenerativas/patologia , Animais , Reprogramação Celular , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo
12.
Stem Cell Reports ; 7(2): 207-19, 2016 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-27426040

RESUMO

Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9(∗) has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9(∗) decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9(∗) depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9(∗) via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9(∗)_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9(∗) and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells.


Assuntos
Diferenciação Celular/genética , Autorrenovação Celular/genética , MicroRNAs/genética , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transcrição Gênica , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/metabolismo , Regulação da Expressão Gênica , Loci Gênicos , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , MicroRNAs/metabolismo , Complexos Multiproteicos/metabolismo , Ligação Proteica , Transdução de Sinais/genética
13.
Cell Mol Life Sci ; 73(10): 2089-104, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26573968

RESUMO

Spinal muscular atrophy (SMA) is a devastating motoneuron (MN) disorder caused by homozygous loss of SMN1. Rarely, SMN1-deleted individuals are fully asymptomatic despite carrying identical SMN2 copies as their SMA III-affected siblings suggesting protection by genetic modifiers other than SMN2. High plastin 3 (PLS3) expression has previously been found in lymphoblastoid cells but not in fibroblasts of asymptomatic compared to symptomatic siblings. To find out whether PLS3 is also upregulated in MNs of asymptomatic individuals and thus a convincing SMA protective modifier, we generated induced pluripotent stem cells (iPSCs) from fibroblasts of three asymptomatic and three SMA III-affected siblings from two families and compared these to iPSCs from a SMA I patient and control individuals. MNs were differentiated from iPSC-derived small molecule neural precursor cells (smNPCs). All four genotype classes showed similar capacity to differentiate into MNs at day 8. However, SMA I-derived MN survival was significantly decreased while SMA III- and asymptomatic-derived MN survival was moderately reduced compared to controls at day 27. SMN expression levels and concomitant gem numbers broadly matched SMN2 copy number distribution; SMA I presented the lowest levels, whereas SMA III and asymptomatic showed similar levels. In contrast, PLS3 was significantly upregulated in mixed MN cultures from asymptomatic individuals pinpointing a tissue-specific regulation. Evidence for strong PLS3 accumulation in shaft and rim of growth cones in MN cultures from asymptomatic individuals implies an important role in neuromuscular synapse formation and maintenance. These findings provide strong evidence that PLS3 is a genuine SMA protective modifier.


Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/patologia , Atrofia Muscular Espinal/genética , Células-Tronco Neurais/citologia , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Regulação para Cima , Biópsia , Diferenciação Celular , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Inativação Gênica , Vetores Genéticos , Genótipo , Humanos , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Cariotipagem , Linfócitos/citologia , Masculino , Microscopia Confocal , Mutação , Linhagem , Fenótipo , RNA Interferente Pequeno/metabolismo , Pele/patologia
14.
Brain ; 135(Pt 5): 1586-605, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22447120

RESUMO

During central nervous system autoimmunity, interactions between infiltrating immune cells and brain-resident cells are critical for disease progression and ultimately organ damage. Here, we demonstrate that local cross-talk between invading autoreactive T cells and auto-antigen-presenting myeloid cells within the central nervous system results in myeloid cell activation, which is crucial for disease progression during experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis. This T cell-mediated licensing of central nervous system myeloid cells triggered astrocytic CCL2-release and promoted recruitment of inflammatory CCR2(+)-monocytes, which are the main effectors of disease progression. By employing a cell-specific knockout model, we identify the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in myeloid cells as key regulator of their disease-determining interactions with autoreactive T cells and brain-resident cells, respectively. LysM-PPARγ(KO) mice exhibited disease exacerbation during the effector phase of experimental autoimmune encephalomyelitis characterized by enhanced activation of central nervous system myeloid cells accompanied by pronounced local CCL2 production and inflammatory monocyte invasion, which finally resulted in increased demyelination and neuronal damage. Pharmacological PPARγ activation decreased antigen-specific T cell-mediated licensing of central nervous system myeloid cells, reduced myeloid cell-mediated neurotoxicity and hence dampened central nervous system autoimmunity. Importantly, human monocytes derived from patients with multiple sclerosis clearly responded to PPARγ-mediated control of proinflammatory activation and production of neurotoxic mediators. Furthermore, PPARγ in human monocytes restricted their capacity to activate human astrocytes leading to dampened astrocytic CCL2 production. Together, interference with the disease-promoting cross-talk between central nervous system myeloid cells, autoreactive T cells and brain-resident cells represents a novel therapeutic approach that limits disease progression and lesion development during ongoing central nervous system autoimmunity.


Assuntos
Autoimunidade/fisiologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Encefalomielite Autoimune Experimental/patologia , Células Mieloides/fisiologia , PPAR gama/metabolismo , Análise de Variância , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Autoimunidade/imunologia , Linfócitos T CD4-Positivos/imunologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Cerebelo/citologia , Técnicas de Cocultura , Citocinas/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Encefalomielite Autoimune Experimental/imunologia , Citometria de Fluxo , Adjuvante de Freund/efeitos adversos , Técnicas de Silenciamento de Genes , Glicoproteínas/administração & dosagem , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Humanos , Hipoglicemiantes/administração & dosagem , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/fisiologia , Glicoproteína Mielina-Oligodendrócito , Células Mieloides/imunologia , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/metabolismo , PPAR gama/deficiência , Fragmentos de Peptídeos/administração & dosagem , Pioglitazona , RNA Interferente Pequeno/metabolismo , Receptores CCR2/metabolismo , Linfócitos T , Tiazolidinedionas/administração & dosagem
15.
Nature ; 480(7378): 543-6, 2011 Nov 23.
Artigo em Inglês | MEDLINE | ID: mdl-22113611

RESUMO

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.


Assuntos
Doença de Machado-Joseph/patologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Repressoras/metabolismo , Ataxina-3 , Cálcio/metabolismo , Calpaína/metabolismo , Células Cultivadas , Aminoácidos Excitatórios/farmacologia , Ácido Glutâmico/farmacologia , Humanos , Neurônios/efeitos dos fármacos
16.
Stem Cells ; 28(5): 894-902, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20333748

RESUMO

Combined application of DNA recombinases Cre and FLP enables tightly controlled independent and/or sequential gene regulations. However, in practice, such dual recombinase strategies are hampered by the comparably low efficiency of the FLP recombinase. Here, we present the engineering of a recombinant cell-permeant FLP protein (TAT-FLP) that induces recombination in >75% of fibroblasts and mouse as well as human embryonic stem (ES) cells. We show that TAT-FLP ideally complements the strength of cell-permeant Cre recombinase for genetic engineering as exemplified by FLP-ON-Cre-OFF, an inducible transgene expression cassette that enables tightly controlled expression in a reversible manner. We exemplify this concept by conditional overexpression of LacZ and the caudal-related homeobox transcription factor CDX2. We expect our FLP transduction system to become widely useful for numerous genetic interventions addressing complex biological questions and the generation of transgene-free therapeutically applicable ES cell-derived cells.


Assuntos
Permeabilidade da Membrana Celular , DNA Nucleotidiltransferases/genética , Células-Tronco Embrionárias/metabolismo , Produtos do Gene tat/genética , Engenharia Genética/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Animais , Desdiferenciação Celular/genética , Linhagem Celular , Linhagem da Célula/genética , Permeabilidade da Membrana Celular/genética , Células-Tronco Embrionárias/citologia , Regulação Enzimológica da Expressão Gênica/genética , Humanos , Integrases/genética , Camundongos , Camundongos Endogâmicos C57BL , Células NIH 3T3 , Transdução Genética/métodos
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