Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease DOI Creative Commons

Jillian E J Cox,

Kevin D Pham,

Alex W Keck

и другие.

BIO-PROTOCOL, Год журнала: 2024, Номер 14(1347)

Опубликована: Янв. 1, 2024

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental neurodegenerative processes. Recent advances single-cell transcriptomic profiling have allowed for increased granularity better separation of distinct microglial states. While techniques such as immunofluorescence RNA sequencing (scRNA-seq) are available to differentiate functions, methods present notable limitations, including challenging quantification methods, high cost, advanced analytical techniques. This protocol addresses limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation a flow cytometry panel distinguish four from murine brain tissue. Following preparation, fluorescent antibodies were applied label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), 4) lipid-droplet accumulating (LDAM) microglia, based gene markers identified previous scRNA-Seq studies. Stained cells analyzed assess distribution function age sex. A key advantage this is its adaptability, allowing provided be enhanced using additional with appropriate analyzer (i.e., Cytek Aurora 5 laser spectral cytometer) interrogating different regions or disease models. Additionally, does not require sorting, resulting relatively quick straightforward experiment. Ultimately, compare experimental groups, state age, lower cost higher throughput than scRNA-seq. Key features • Analysis without need imaging, (LDAM), (IRM) microglia any region and/or model interest. modified incorporate interest dyes when capable multiple color detections.

Язык: Английский

Microglial senescence in neurodegeneration: Insights, implications, and therapeutic opportunities DOI Creative Commons
Tobiloba Samuel Olajide, Toheeb O. Oyerinde, Omolabake I. Omotosho

и другие.

Neuroprotection/Neuroprotection (Chichester, England. Print), Год журнала: 2024, Номер 2(3), С. 182 - 195

Опубликована: Сен. 1, 2024

The existing literature on neurodegenerative diseases (NDDs) reveals a common pathological feature: the accumulation of misfolded proteins. However, heterogeneity in disease onset mechanisms and specific brain regions affected complicates understanding diverse clinical manifestations individual NDDs. Dementia, hallmark symptom across various NDDs, serves as multifaceted denominator, contributing to these disorders. There is compelling hypothesis that therapeutic strategies capable mitigating protein disrupting ongoing pathogenic processes may slow or even halt progression. Recent research has linked disease-associated microglia their transition into senescent state-characterized by irreversible cell cycle arrest-in aging populations Although are consistently observed few studies have utilized animal models explore role pathology. Emerging evidence from experimental rat suggests exhibit characteristics senescence, indicating deeper exploration microglial senescence could enhance our NDD pathogenesis reveal novel targets. This review underscores importance investigating its potential contributions pathophysiology including Alzheimer's disease, Parkinson's Huntington's amyotrophic lateral sclerosis. Additionally, it highlights targeting through iron chelation senolytic therapies innovative approaches for treating age-related

Язык: Английский

Процитировано

1
Specificity and efficiency of tamoxifen-mediated Cre induction is equivalent regardless of age DOI Creative Commons

Collyn M. Kellogg,

Kevin D. Pham,

Sung-Hwan Ko

и другие.

iScience, Год журнала: 2023, Номер 26(12), С. 108413 - 108413

Опубликована: Ноя. 9, 2023

Temporally controlling Cre recombination through tamoxifen (Tam) induction has many advantages for biomedical research. Most studies report early post-natal/juvenile (<2 m.o.) Tam induction, but age-related neurodegeneration and aging can require in older mice (>12 m.o.). While anecdotally reported as problematic, there are no published comparisons of Tam-mediated at late ages. Here, microglial-specific Cx3cr1creERT2 were crossed to a floxed NuTRAP reporter compare (3-6 (20 Specificity efficiency microglial labeling 21-22 m.o. identical induced with Age-related translatomic changes also similar regardless age. Each flox mouse line should be independently validated, however, these findings demonstrate that performed even into ages generalizable other inducible models.

Язык: Английский

Процитировано

2
Selective agonism of GPR34 stimulates microglial uptake and clearance of amyloid β fibrils DOI Open Access

Hayato Etani,

Sho Takatori, Wenbo Wang

и другие.

bioRxiv (Cold Spring Harbor Laboratory), Год журнала: 2024, Номер unknown

Опубликована: Май 9, 2024

Abstract Microglia, the primary immune cells of central nervous system, play a crucial role in maintaining brain homeostasis through phagocytosis various substrates, including amyloid-β (Aβ) fibrils, hallmark Alzheimer disease (AD) pathology. However, molecular mechanisms regulating microglial Aβ uptake remain poorly understood. Here, we identified GPR34, Gi/o-coupled receptor highly expressed microglia, as novel regulator fibrillar phagocytosis. Treatment with selective GPR34 agonist, M1, specifically enhanced but not its monomer or oligomer, both mouse and human microglia. Mechanistically, M1 reduced intracellular cAMP levels, which inversely correlated activity. Importantly, single intrahippocampal injection an AD model significantly increased vivo. Furthermore, single-nucleus RNA-sequencing analysis Japanese patient samples revealed significant reduction expression microglia from patients compared to controls. We also observed age-dependent decline datasets, suggesting potential contribution downregulation age-related accumulation risk. Collectively, our findings identify promising target for modulating clearance highlight therapeutic agonists AD. Significance statement is characterized by brain. brain’s cells, metabolism Aβ. discovered that activating agonist enhances key pathogenic form decreases aging progression, potentially contributing impaired clearance. Our AD, boosting activity could promote slow progression. This study provides valuable insights into function offers strategy developing disease-modifying therapies.

Язык: Английский

Процитировано

0
Phosphodiesterase 8 (PDE8): Distribution and Cellular Expression and Association with Alzheimer’s Disease DOI
Nianzhuang Qiu,

Hui-mei Hou,

Tianyang Guo

и другие.

Neurochemical Research, Год журнала: 2024, Номер 49(8), С. 1993 - 2004

Опубликована: Май 23, 2024

Язык: Английский

Процитировано

0
Flow Cytometry Analysis of Microglial Phenotypes in the Murine Brain During Aging and Disease DOI Creative Commons

Jillian E J Cox,

Kevin D Pham,

Alex W Keck

и другие.

BIO-PROTOCOL, Год журнала: 2024, Номер 14(1347)

Опубликована: Янв. 1, 2024

Microglia, the brain's primary resident immune cell, exists in various phenotypic states depending on intrinsic and extrinsic signaling. Distinguishing between these phenotypes can offer valuable biological insights into neurodevelopmental neurodegenerative processes. Recent advances single-cell transcriptomic profiling have allowed for increased granularity better separation of distinct microglial states. While techniques such as immunofluorescence RNA sequencing (scRNA-seq) are available to differentiate functions, methods present notable limitations, including challenging quantification methods, high cost, advanced analytical techniques. This protocol addresses limitations by presenting an optimized cell preparation procedure that prevents ex vivo activation a flow cytometry panel distinguish four from murine brain tissue. Following preparation, fluorescent antibodies were applied label 1) homeostatic, 2) disease-associated (DAM), 3) interferon response (IRM), 4) lipid-droplet accumulating (LDAM) microglia, based gene markers identified previous scRNA-Seq studies. Stained cells analyzed assess distribution function age sex. A key advantage this is its adaptability, allowing provided be enhanced using additional with appropriate analyzer (i.e., Cytek Aurora 5 laser spectral cytometer) interrogating different regions or disease models. Additionally, does not require sorting, resulting relatively quick straightforward experiment. Ultimately, compare experimental groups, state age, lower cost higher throughput than scRNA-seq. Key features • Analysis without need imaging, (LDAM), (IRM) microglia any region and/or model interest. modified incorporate interest dyes when capable multiple color detections.

Язык: Английский

Процитировано

0