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1.
Biomolecules ; 14(3)2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38540709

RESUMO

Today, neurodegenerative disorders like Alzheimer's disease (AD), Parkinson's disease (PD), frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) affect millions of people worldwide, and as the average human lifespan increases, similarly grows the number of patients. For many decades, cognitive and motoric decline has been explained by the very apparent deterioration of neurons in various regions of the brain and spinal cord. However, more recent studies show that disease progression is greatly influenced by the vast population of glial cells. Astrocytes are traditionally considered star-shaped cells on which neurons rely heavily for their optimal homeostasis and survival. Increasing amounts of evidence depict how astrocytes lose their supportive functions while simultaneously gaining toxic properties during neurodegeneration. Many of these changes are similar across various neurodegenerative diseases, and in this review, we highlight these commonalities. We discuss how astrocyte dysfunction drives neuronal demise across a wide range of neurodegenerative diseases, but rather than categorizing based on disease, we aim to provide an overview based on currently known mechanisms. As such, this review delivers a different perspective on the disease causes of neurodegeneration in the hope to encourage further cross-disease studies into shared disease mechanisms, which might ultimately disclose potentially common therapeutic entry points across a wide panel of neurodegenerative diseases.


Assuntos
Doença de Alzheimer , Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Astrócitos/fisiologia , Esclerose Lateral Amiotrófica/terapia
2.
Acta Neuropathol ; 147(1): 41, 2024 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-38363426

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease which currently lacks effective treatments. Mutations in the RNA-binding protein FUS are a common cause of familial ALS, accounting for around 4% of the cases. Understanding the mechanisms by which mutant FUS becomes toxic to neurons can provide insight into the pathogenesis of both familial and sporadic ALS. We have previously observed that overexpression of wild-type or ALS-mutant FUS in Drosophila motor neurons is toxic, which allowed us to screen for novel genetic modifiers of the disease. Using a genome-wide screening approach, we identified Protein Phosphatase 2A (PP2A) and Glycogen Synthase Kinase 3 (GSK3) as novel modifiers of FUS-ALS. Loss of function or pharmacological inhibition of either protein rescued FUS-associated lethality in Drosophila. Consistent with a conserved role in disease pathogenesis, pharmacological inhibition of both proteins rescued disease-relevant phenotypes, including mitochondrial trafficking defects and neuromuscular junction failure, in patient iPSC-derived spinal motor neurons (iPSC-sMNs). In FUS-ALS flies, mice, and human iPSC-sMNs, we observed reduced GSK3 inhibitory phosphorylation, suggesting that FUS dysfunction results in GSK3 hyperactivity. Furthermore, we found that PP2A acts upstream of GSK3, affecting its inhibitory phosphorylation. GSK3 has previously been linked to kinesin-1 hyperphosphorylation. We observed this in both flies and iPSC-sMNs, and we rescued this hyperphosphorylation by inhibiting GSK3 or PP2A. Moreover, increasing the level of kinesin-1 expression in our Drosophila model strongly rescued toxicity, confirming the relevance of kinesin-1 hyperphosphorylation. Our data provide in vivo evidence that PP2A and GSK3 are disease modifiers, and reveal an unexplored mechanistic link between PP2A, GSK3, and kinesin-1, that may be central to the pathogenesis of FUS-ALS and sporadic forms of the disease.


Assuntos
Esclerose Lateral Amiotrófica , Doenças Neurodegenerativas , Animais , Humanos , Camundongos , Esclerose Lateral Amiotrófica/patologia , Quinase 3 da Glicogênio Sintase/genética , Quinase 3 da Glicogênio Sintase/metabolismo , Proteína Fosfatase 2/genética , Proteína Fosfatase 2/metabolismo , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Doenças Neurodegenerativas/patologia , Cinesinas/genética , Cinesinas/metabolismo , Neurônios Motores/metabolismo , Drosophila/genética , Drosophila/metabolismo , Mutação/genética
3.
Mol Neurodegener ; 18(1): 5, 2023 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-36653804

RESUMO

BACKGROUND: Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. METHODS: In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. RESULTS: We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/ß-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. CONCLUSIONS: Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms.


Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Humanos , Esclerose Lateral Amiotrófica/metabolismo , Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios Motores/metabolismo , Junção Neuromuscular , Proteína FUS de Ligação a RNA/fisiologia
4.
J Vis Exp ; (175)2021 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-34570099

RESUMO

Neuromuscular junctions (NMJs) are specialized synapses between the axon of the lower motor neuron and the muscle facilitating the engagement of muscle contraction. In motor neuron disorders, such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), NMJs degenerate, resulting in muscle atrophy and progressive paralysis. The underlying mechanism of NMJ degeneration is unknown, largely due to the lack of translatable research models. This study aimed to create a versatile and reproducible in vitro model of a human motor unit with functional NMJs. Therefore, human induced pluripotent stem cell (hiPSC)-derived motor neurons and human primary mesoangioblast (MAB)-derived myotubes were co-cultured in commercially available microfluidic devices. The use of fluidically isolated micro-compartments allows for the maintenance of cell-specific microenvironments while permitting cell-to-cell contact through microgrooves. By applying a chemotactic and volumetric gradient, the growth of motor neuron-neurites through the microgrooves promoting myotube interaction and the formation of NMJs were stimulated. These NMJs were identified immunocytochemically through co-localization of motor neuron presynaptic marker synaptophysin (SYP) and postsynaptic acetylcholine receptor (AChR) marker α-bungarotoxin (Btx) on myotubes and characterized morphologically using scanning electron microscopy (SEM). The functionality of the NMJs was confirmed by measuring calcium responses in myotubes upon depolarization of the motor neurons. The motor unit generated using standard microfluidic devices and stem cell technology can aid future research focusing on NMJs in health and disease.


Assuntos
Células-Tronco Pluripotentes Induzidas , Dispositivos Lab-On-A-Chip , Humanos , Neurônios Motores , Músculo Esquelético , Junção Neuromuscular
5.
Stem Cell Reports ; 16(9): 2213-2227, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-33891869

RESUMO

Neuromuscular junctions (NMJs) ensure communication between motor neurons (MNs) and muscle; however, in MN disorders, such as amyotrophic lateral sclerosis (ALS), NMJs degenerate resulting in muscle atrophy. The aim of this study was to establish a versatile and reproducible in vitro model of a human motor unit to investigate the effects of ALS-causing mutations. Therefore, we generated a co-culture of human induced pluripotent stem cell (iPSC)-derived MNs and human primary mesoangioblast-derived myotubes in microfluidic devices. A chemotactic and volumetric gradient facilitated the growth of MN neurites through microgrooves resulting in the interaction with myotubes and the formation of NMJs. We observed that ALS-causing FUS mutations resulted in reduced neurite outgrowth as well as an impaired neurite regrowth upon axotomy. NMJ numbers were likewise reduced in the FUS-ALS model. Interestingly, the selective HDAC6 inhibitor, Tubastatin A, improved the neurite outgrowth, regrowth, and NMJ morphology, prompting HDAC6 inhibition as a potential therapeutic strategy for ALS.


Assuntos
Desacetilase 6 de Histona/antagonistas & inibidores , Inibidores de Histona Desacetilases/farmacologia , Dispositivos Lab-On-A-Chip , Mutação , Junção Neuromuscular/genética , Junção Neuromuscular/fisiopatologia , Proteína FUS de Ligação a RNA/genética , Agrina/metabolismo , Esclerose Lateral Amiotrófica/etiologia , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Biomarcadores , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Técnicas de Cocultura , Imunofluorescência , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Laminina/metabolismo , Técnicas Analíticas Microfluídicas , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Fibras Musculares Esqueléticas/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos
6.
Semin Cell Dev Biol ; 99: 133-150, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31542222

RESUMO

Because of the extremely polarized morphology, the proper functioning of neurons largely relies on the efficient cargo transport along the axon. Axonal transport defects have been reported in multiple neurodegenerative diseases as an early pathological feature. The discovery of mutations in human genes involved in the transport machinery provide a direct causative relationship between axonal transport defects and neurodegeneration. Here, we summarize the current genetic findings related to axonal transport in neurodegenerative diseases, and we discuss the relationship between axonal transport defects and other pathological changes observed in neurodegeneration. In addition, we summarize the therapeutic approaches targeting the axonal transport machinery in studies of neurodegenerative diseases. Finally, we review the technical advances in tracking axonal transport both in vivo and in vitro.


Assuntos
Transporte Axonal/efeitos dos fármacos , Transporte Axonal/genética , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Animais , Humanos , Mutação , Doenças Neurodegenerativas/metabolismo
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