Ethanol-activated microglial exosomes induce MCP1 signaling mediated death of stress-regulatory proopiomelanocortin neurons in the developing hypothalamus.
Animals
Ethanol
/ toxicity
Rats
Exosomes
/ metabolism
Neurons
/ drug effects
Pro-Opiomelanocortin
/ metabolism
Chemokine CCL2
/ metabolism
Hypothalamus
/ metabolism
Microglia
/ drug effects
Signal Transduction
/ drug effects
Rats, Sprague-Dawley
Animals, Newborn
Cell Death
/ drug effects
Cells, Cultured
Female
Central Nervous System Depressants
/ pharmacology
beta-Endorphin
/ metabolism
CCR2
Developmental alcohol exposure
Exosomes
Fetal alcohol spectrum disorder
MCP1
Microglia
Neuronal apoptosis
Proopiomelanocortin neuron
Journal
Journal of neuroinflammation
ISSN: 1742-2094
Titre abrégé: J Neuroinflammation
Pays: England
ID NLM: 101222974
Informations de publication
Date de publication:
30 Oct 2024
30 Oct 2024
Historique:
received:
07
08
2024
accepted:
23
10
2024
medline:
31
10
2024
pubmed:
31
10
2024
entrez:
31
10
2024
Statut:
epublish
Résumé
Microglia, a type of resident immune cells within the central nervous system, have been implicated in ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined if microglial extracellular vesicles (exosomes) are involved in the ethanol-induced neuronal death of the β-endorphin neuron via secreting elevated levels of the chemokine monocyte chemoattractant protein 1 (MCP1), a key regulator of neuroinflammation. We employed an in vitro model, consisting of primary culture of hypothalamic microglia prepared from postnatal day 2 (PND2) rat hypothalami and treated with or without 50 mM ethanol for 24 h, and an in vivo animal model in which microglia were obtained from hypothalami of PND6 rats fed daily with 2.5 mg/kg ethanol or control milk formula for five days prior to use. Exosomes were extracted and characterized with nanosight tracking analysis (NTA), transmission electron microscopy and western blot. Chemokine multiplex immunoassay and ELISA were used for quantitative estimation of MCP1 level. Neurotoxic ability of exosome was tested using primary cultures of β-endorphin neurons and employing nucleosome assay and immunocytochemistry. Elevated plus maze, open field and restraint tests were used to assess anxiety-related behaviors. Ethanol elevated MCP1 levels in microglial exosomes both in vitro and in vivo models. Ethanol-activated microglial exosomes when introduced into primary cultures of β-endorphin neurons, increased cellular levels of MCP1 and the chemokine receptor CCR2 related signaling molecules including inflammatory cytokines and apoptotic genes as well as apoptotic death of β-endorphin neurons. These effects of microglial exosomes on β-endorphin neurons were suppressed by a CCR2 antagonist RS504393. Furthermore, RS504393 when injected in postnatal rats prior to feeding with ethanol it reduced alcohol-induced β-endorphin neuronal death in the hypothalamus. RS504393 also suppressed corticosterone response to stress and anxiety-like behaviors in postnatally alcohol-fed rats during adult period. These data suggest that alcohol exposures during the developmental period elevates MCP1 levels in microglial exosomes that promote MCP1/CCR2 signaling to increase the apoptosis of β-endorphin neurons and resulting in hormonal and behavioral stress responses.
Sections du résumé
BACKGROUND
BACKGROUND
Microglia, a type of resident immune cells within the central nervous system, have been implicated in ethanol-activated neuronal death of the stress regulatory proopiomelanocortin (POMC) neuron-producing β-endorphin peptides in the hypothalamus in a postnatal rat model of fetal alcohol spectrum disorders. We determined if microglial extracellular vesicles (exosomes) are involved in the ethanol-induced neuronal death of the β-endorphin neuron via secreting elevated levels of the chemokine monocyte chemoattractant protein 1 (MCP1), a key regulator of neuroinflammation.
METHODS
METHODS
We employed an in vitro model, consisting of primary culture of hypothalamic microglia prepared from postnatal day 2 (PND2) rat hypothalami and treated with or without 50 mM ethanol for 24 h, and an in vivo animal model in which microglia were obtained from hypothalami of PND6 rats fed daily with 2.5 mg/kg ethanol or control milk formula for five days prior to use. Exosomes were extracted and characterized with nanosight tracking analysis (NTA), transmission electron microscopy and western blot. Chemokine multiplex immunoassay and ELISA were used for quantitative estimation of MCP1 level. Neurotoxic ability of exosome was tested using primary cultures of β-endorphin neurons and employing nucleosome assay and immunocytochemistry. Elevated plus maze, open field and restraint tests were used to assess anxiety-related behaviors.
RESULTS
RESULTS
Ethanol elevated MCP1 levels in microglial exosomes both in vitro and in vivo models. Ethanol-activated microglial exosomes when introduced into primary cultures of β-endorphin neurons, increased cellular levels of MCP1 and the chemokine receptor CCR2 related signaling molecules including inflammatory cytokines and apoptotic genes as well as apoptotic death of β-endorphin neurons. These effects of microglial exosomes on β-endorphin neurons were suppressed by a CCR2 antagonist RS504393. Furthermore, RS504393 when injected in postnatal rats prior to feeding with ethanol it reduced alcohol-induced β-endorphin neuronal death in the hypothalamus. RS504393 also suppressed corticosterone response to stress and anxiety-like behaviors in postnatally alcohol-fed rats during adult period.
CONCLUSION
CONCLUSIONS
These data suggest that alcohol exposures during the developmental period elevates MCP1 levels in microglial exosomes that promote MCP1/CCR2 signaling to increase the apoptosis of β-endorphin neurons and resulting in hormonal and behavioral stress responses.
Identifiants
pubmed: 39478585
doi: 10.1186/s12974-024-03274-6
pii: 10.1186/s12974-024-03274-6
doi:
Substances chimiques
Ethanol
3K9958V90M
Pro-Opiomelanocortin
66796-54-1
Chemokine CCL2
0
Ccl2 protein, rat
0
Central Nervous System Depressants
0
beta-Endorphin
60617-12-1
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
279Subventions
Organisme : NIAAA NIH HHS
ID : R01AA028767
Pays : United States
Informations de copyright
© 2024. The Author(s).
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