Simultaneous isolation of intact brain cells and cell-specific extracellular vesicles from cryopreserved Alzheimer's disease cortex.

Astrocytes Dissociation Exosomes Extracellular vesicles Flow cytometry Microglia Neurons Oligodendrocytes

Journal

Journal of neuroscience methods
ISSN: 1872-678X
Titre abrégé: J Neurosci Methods
Pays: Netherlands
ID NLM: 7905558

Informations de publication

Date de publication:
14 Apr 2024
Historique:
received: 15 12 2023
revised: 25 03 2024
accepted: 12 04 2024
pubmed: 17 4 2024
medline: 17 4 2024
entrez: 16 4 2024
Statut: aheadofprint

Résumé

The neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD). Postmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation. Neuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers. We optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Sections du résumé

BACKGROUND BACKGROUND
The neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD).
NEW METHOD METHODS
Postmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation.
RESULTS RESULTS
Neuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers.
COMPARISON WITH EXISTING METHODS AND CONCLUSIONS CONCLUSIONS
We optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Identifiants

DOI: 10.1016/j.jneumeth.2024.110137 PMID: 38626853
pubmed: 38626853
pii: S0165-0270(24)00082-7
doi: 10.1016/j.jneumeth.2024.110137
pii:
doi:

Types de publication

Journal Article

Langues

eng

Sous-ensembles de citation

IM

Pagination

110137

Informations de copyright

Copyright © 2024. Published by Elsevier B.V.

Déclaration de conflit d'intérêts

Declaration of Competing Interest None.

Auteurs

Mikhail Melnik (M)

UCLA School of Nursing, Los Angeles, CA 90095, USA; Neuroscience Interdepartmental Program, UCLA School of Medicine, Los Angeles, CA 90095, USA.

Emily Miyoshi (E)

UCLA School of Nursing, Los Angeles, CA 90095, USA.

Ricky Ma (R)

UCLA School of Nursing, Los Angeles, CA 90095, USA.

Maria Corrada (M)

Departments of Neurology, Irvine, CA 92697, USA; Institute for Memory Impairments and Neurological Disorders, UC Irvine, Irvine, CA 92697, USA.

Claudia Kawas (C)

Departments of Neurology, Irvine, CA 92697, USA; Neurobiology & Behavior, Irvine, CA 92697, USA; Institute for Memory Impairments and Neurological Disorders, UC Irvine, Irvine, CA 92697, USA.

Ryan Bohannan (R)

Institute for Memory Impairments and Neurological Disorders, UC Irvine, Irvine, CA 92697, USA.

Chad Caraway (C)

Institute for Memory Impairments and Neurological Disorders, UC Irvine, Irvine, CA 92697, USA.

Carol A Miller (CA)

Keck USC School of Medicine, Los Angeles, CA 90033, USA.

Jason D Hinman (JD)

Mary S. Easton Center for Alzheimer's Research at UCLA, Los Angeles, CA 90073, USA; Departments of Neurology, UCLA School of Medicine, Los Angeles, CA 90095, USA.

Varghese John (V)

Mary S. Easton Center for Alzheimer's Research at UCLA, Los Angeles, CA 90073, USA; Departments of Neurology, UCLA School of Medicine, Los Angeles, CA 90095, USA.

Tina Bilousova (T)

UCLA School of Nursing, Los Angeles, CA 90095, USA; Mary S. Easton Center for Alzheimer's Research at UCLA, Los Angeles, CA 90073, USA; Departments of Neurology, UCLA School of Medicine, Los Angeles, CA 90095, USA. Electronic address: tina.bilousova@ucsf.edu.

Karen H Gylys (KH)

UCLA School of Nursing, Los Angeles, CA 90095, USA; Mary S. Easton Center for Alzheimer's Research at UCLA, Los Angeles, CA 90073, USA; Neuroscience Interdepartmental Program, UCLA School of Medicine, Los Angeles, CA 90095, USA.

Classifications MeSH