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1.
Cell Death Dis ; 15(6): 405, 2024 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-38858390

RESUMO

Genetic mutations causing primary mitochondrial disease (i.e those compromising oxidative phosphorylation [OxPhos]) resulting in reduced bioenergetic output display great variability in their clinical features, but the reason for this is unknown. We hypothesized that disruption of the communication between endoplasmic reticulum (ER) and mitochondria at mitochondria-associated ER membranes (MAM) might play a role in this variability. To test this, we assayed MAM function and ER-mitochondrial communication in OxPhos-deficient cells, including cybrids from patients with selected pathogenic mtDNA mutations. Our results show that each of the various mutations studied indeed altered MAM functions, but notably, each disorder presented with a different MAM "signature". We also found that mitochondrial membrane potential is a key driver of ER-mitochondrial connectivity. Moreover, our findings demonstrate that disruption in ER-mitochondrial communication has consequences for cell survivability that go well beyond that of reduced ATP output. The findings of a "MAM-OxPhos" axis, the role of mitochondrial membrane potential in controlling this process, and the contribution of MAM dysfunction to cell death, reveal a new relationship between mitochondria and the rest of the cell, as well as providing new insights into the diagnosis and treatment of these devastating disorders.


Assuntos
Retículo Endoplasmático , Potencial da Membrana Mitocondrial , Mitocôndrias , Doenças Mitocondriais , Fosforilação Oxidativa , Humanos , Retículo Endoplasmático/metabolismo , Mitocôndrias/metabolismo , Doenças Mitocondriais/metabolismo , Doenças Mitocondriais/genética , Doenças Mitocondriais/patologia , Mutação/genética , DNA Mitocondrial/metabolismo , DNA Mitocondrial/genética
2.
bioRxiv ; 2024 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-38645234

RESUMO

The immune system has a dynamic role in neurodegenerative diseases, and purinergic receptors allow immune cells to recognize neuronal signaling, cell injury, or stress. Purinergic Receptor 7 (P2RX7) can modulate inflammatory cascades and its expression is upregulated in Alzheimer's disease (AD) brain tissue. P2RX7 expression is enriched in microglia, and elevated levels are found in microglia surrounding amyloid-beta plaques in the brain. While P2RX7 is thought to play a role in neurodegenerative diseases, how it modulates pathology and disease progression is not well understood. Here, we utilize a human monocyte-derived microglia-like cell (MDMi) model to interrogate P2RX7 activation and downstream consequences on microglia function. By using MDMi derived from human donors, we can examine how human donor variation impacts microglia function. We assessed P2RX7-driven IL1ß and IL18 production and amyloid-beta peptide 1-42 (Aß1-42) uptake levels. Our results show that ATP-stimulation of MDMi triggers upregulation of IL1ß and IL18 expression. This upregulation of cytokine gene expression is blocked with the A740003 P2RX7 antagonist. We find that high extracellular ATP conditions also reduced MDMi capacity for Aß1-42 uptake, and this loss of function is prevented through A740003 inhibition of P2RX7. In addition, pretreatment of MDMi with IL-1RA limited ATP-driven IL1ß and IL18 gene expression upregulation, indicating that ATP immunomodulation of P2RX7 is IL-1R dependent. Aß1-42 uptake was higher with IL-1RA pretreatment compared to ATP treatment alone, suggesting P2RX7 regulates phagocytic engulfment through IL-1 signaling. Overall, our results demonstrate that P2RX7 is a key response protein for high extracellular ATP in human microglia-like cells, and its function can be modulated by IL-1 signaling. This work opens the door to future studies examining anti-IL-1 biologics to increase the clearance of amyloid-beta.

3.
Neuroscience ; 450: 48-56, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-32615233

RESUMO

Reliable and consistent pluripotent stem cell reporter systems for efficient purification and visualization of motor neurons are essential reagents for the study of normal motor neuron biology and for effective disease modeling. To overcome the inherent noisiness of transgene-based reporters, we developed a new series of human induced pluripotent stem cell lines by knocking in tdTomato, Cre, or CreERT2 recombinase into the HB9 (MNX1) or VACHT (SLC18A3) genomic loci. The new lines were validated by directed differentiation into spinal motor neurons and immunostaining for motor neuron markers HB9 and ISL1. To facilitate efficient purification of spinal motor neurons, we further engineered the VACHT-Cre cell line with a validated, conditional CD14-GFP construct that allows for both fluorescence-based identification of motor neurons, as well as magnetic-activated cell sorting (MACS) to isolate differentiated motor neurons at scale. The targeting strategies developed here offer a standardized platform for reproducible comparison of motor neurons across independently derived pluripotent cell lines.


Assuntos
Células-Tronco Pluripotentes Induzidas , Células-Tronco Pluripotentes , Diferenciação Celular , Colinérgicos , Proteínas de Homeodomínio , Humanos , Neurônios Motores , Fatores de Transcrição
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