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1.
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
2.
Cell Tissue Res ; 378(3): 499-511, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31352550

RESUMO

Autophagy (cellular self-consumption) is an adaptive stress response and an important aspect of adaption to mechanical loading. If mechanical forces are associated with autophagy regulation in periodontal ligament (PDL) fibroblasts is still unknown. The aim of this study was to analyze the influence of force magnitude on autophagy regulation and subsequently on cell death in human PDL fibroblasts. Autophagy-associated genes were analyzed with a specific PrimePCR assay after 24 h of stimulation with high (STSH) and low magnitudes (STSL) of static tensile strain applied to PDL fibroblasts. Based on the results, targets were selected for further real-time PCR analysis. The autophagic flux was assessed by immunoblotting for autophagy marker microtubule-associated protein 1, light chain 3, and by autophagosome staining. Cell death was determined by TUNEL assay and Cell Death Detection ELISAPLUS. Autophagy was induced pharmacologically by rapamycin and inhibited by chloroquine. For statistical analysis, the Kruskal Wallis test followed by the post-hoc Dunnett's test was used. Static tensile strain had regulatory effects on mRNA expression of multiple autophagy-associated targets. Stimulation with STSH induced mRNA expression changes in more autophagy-associated targets than STSL. The autophagic flux was induced by STSH while STSL had no significant effect on autophagosome formation. Furthermore, autophagy inhibition led to increased cell death. Low magnitudes of tensile strain seem to have cell-protective properties. Taken together, our findings provide novel insights about autophagy regulation by biomechanical loading in human PDL fibroblasts. Our results suggest a gradual response of autophagy to static tensile strain in human PDL fibroblasts.


Assuntos
Biomarcadores/metabolismo , Fibroblastos/metabolismo , Ligamento Periodontal/metabolismo , Adolescente , Adulto , Autofagia , Fibroblastos/citologia , Voluntários Saudáveis , Humanos , Ligamento Periodontal/citologia , Estresse Mecânico , Resistência à Tração , Adulto Jovem
3.
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
4.
NPJ Parkinsons Dis ; 8(1): 118, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36114228

RESUMO

α-Synuclein (αSyn) aggregation in Lewy bodies and neurites defines both familial and 'sporadic' Parkinson's disease. We previously identified α-helically folded αSyn tetramers, in addition to the long-known unfolded monomers, in normal cells. PD-causing αSyn mutations decrease the tetramer:monomer (T:M) ratio, associated with αSyn hyperphosphorylation and cytotoxicity in neurons and a motor syndrome of tremor and gait deficits in transgenic mice that responds in part to L-DOPA. Here, we asked whether LRRK2 mutations, the most common genetic cause of cases previously considered sporadic PD, also alter tetramer homeostasis. Patient neurons carrying G2019S, the most prevalent LRRK2 mutation, or R1441C each had decreased T:M ratios and pSer129 hyperphosphorylation of their endogenous αSyn along with increased phosphorylation of Rab10, a widely reported substrate of LRRK2 kinase activity. Two LRRK2 kinase inhibitors normalized the T:M ratio and the hyperphosphorylation in the G2019S and R1441C patient neurons. An inhibitor of stearoyl-CoA desaturase, the rate-limiting enzyme for monounsaturated fatty acid synthesis, also restored the αSyn T:M ratio and reversed pSer129 hyperphosphorylation in both mutants. Coupled with the recent discovery that PD-causing mutations of glucocerebrosidase in Gaucher's neurons also decrease T:M ratios, our findings indicate that three dominant genetic forms of PD involve life-long destabilization of αSyn physiological tetramers as a common pathogenic mechanism that can occur upstream of progressive neuronal synucleinopathy. Based on αSyn's finely-tuned interaction with certain vesicles, we hypothesize that the fatty acid composition and fluidity of membranes regulate αSyn's correct binding to highly curved membranes and subsequent assembly into metastable tetramers.

5.
Cells ; 11(9)2022 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-35563695

RESUMO

Understanding early nervous system stress response mechanisms is crucial for studying developmental neurotoxicity and devising neuroprotective treatments. We used hiPSC-derived long-term self-renewing neuroepithelial stem (lt-NES) cells differentiated for up to 12 weeks as an in vitro model of human neural development. Following a transcriptome analysis to identify pathway alterations, we induced acute oxidative stress (OS) using tert-butyl hydroperoxide (TBHP) and assessed cell viability at different stages of neural differentiation. We studied NRF2 activation, autophagy, and proteasomal function to explore the contribution and interplay of these pathways in the acute stress response. With increasing differentiation, lt-NES cells showed changes in the expression of metabolic pathway-associated genes with engagement of the pentose phosphate pathway after 6 weeks, this was accompanied by a decreased susceptibility to TBHP-induced stress. Microarray analysis revealed upregulation of target genes of the antioxidant response KEAP1-NRF2-ARE pathway after 6 weeks of differentiation. Pharmacological inhibition of NRF2 confirmed its vital role in the increased resistance to stress. While autophagy was upregulated alongside differentiation, it was not further increased upon oxidative stress and had no effect on stress-induced cell loss and the activation of NRF2 downstream genes. In contrast, proteasome inhibition led to the aggravation of the stress response resulting in decreased cell viability, derangement of NRF2 and KEAP1 protein levels, and lacking NRF2-pathway activation. Our data provide detailed insight into the dynamic regulation and interaction of pathways involved in modulating stress responses across defined time points of neural differentiation.


Assuntos
Fator 2 Relacionado a NF-E2 , Sistema Nervoso , Proliferação de Células , Humanos , Células-Tronco Pluripotentes Induzidas , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Redes e Vias Metabólicas , Fator 2 Relacionado a NF-E2/metabolismo , Sistema Nervoso/metabolismo
6.
Stem Cell Reports ; 17(10): 2349-2364, 2022 10 11.
Artigo em Inglês | MEDLINE | ID: mdl-36179692

RESUMO

Combining multiple Parkinson's disease (PD) relevant cellular phenotypes might increase the accuracy of midbrain dopaminergic neuron (mDAN) in vitro models. We differentiated patient-derived induced pluripotent stem cells (iPSCs) with a LRRK2 G2019S mutation, isogenic control, and genetically unrelated iPSCs into mDANs. Using automated fluorescence microscopy in 384-well-plate format, we identified elevated levels of α-synuclein (αSyn) and serine 129 phosphorylation, reduced dendritic complexity, and mitochondrial dysfunction. Next, we measured additional image-based phenotypes and used machine learning (ML) to accurately classify mDANs according to their genotype. Additionally, we show that chemical compound treatments, targeting LRRK2 kinase activity or αSyn levels, are detectable when using ML classification based on multiple image-based phenotypes. We validated our approach using a second isogenic patient-derived SNCA gene triplication mDAN model which overexpresses αSyn. This phenotyping and classification strategy improves the practical exploitability of mDANs for disease modeling and the identification of novel LRRK2-associated drug targets.


Assuntos
Células-Tronco Pluripotentes Induzidas , Doença de Parkinson , Neurônios Dopaminérgicos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Aprendizado de Máquina , Mesencéfalo/metabolismo , Mutação , Doença de Parkinson/genética , Doença de Parkinson/terapia , Serina , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo
7.
Elife ; 82019 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-30689541

RESUMO

Midbrain dopaminergic (mDA) neurons migrate to form the laterally-located substantia nigra pars compacta (SN) and medially-located ventral tegmental area (VTA), but little is known about the underlying cellular and molecular processes. Here we visualize the dynamic cell morphologies of tangentially migrating SN-mDA neurons in 3D and identify two distinct migration modes. Slow migration is the default mode in SN-mDA neurons, while fast, laterally-directed migration occurs infrequently and is strongly associated with bipolar cell morphology. Tangential migration of SN-mDA neurons is altered in absence of Reelin signaling, but it is unclear whether Reelin acts directly on migrating SN-mDA neurons and how it affects their cell morphology and migratory behavior. By specifically inactivating Reelin signaling in mDA neurons we demonstrate its direct role in SN-mDA tangential migration. Reelin promotes laterally-biased movements in mDA neurons during their slow migration mode, stabilizes leading process morphology and increases the probability of fast, laterally-directed migration.


Assuntos
Moléculas de Adesão Celular Neuronais/metabolismo , Movimento Celular , Neurônios Dopaminérgicos/citologia , Proteínas da Matriz Extracelular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Serina Endopeptidases/metabolismo , Substância Negra/citologia , Animais , Forma Celular , Neurônios Dopaminérgicos/metabolismo , Mesencéfalo/citologia , Camundongos , Fosforilação , Proteína Reelina , Transdução de Sinais , Área Tegmentar Ventral/citologia
8.
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
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