Galantamine prevents and reverses neuroimmune induction and loss of adult hippocampal neurogenesis following adolescent alcohol exposure

Abstract

Background Binge ethanol exposure during adolescence reduces hippocampal neurogenesis, a reduction which persists throughout adulthood despite abstinence. This loss of neurogenesis, indicated by reduced doublecortin+ immunoreactivity (DCX+IR), is paralleled by an increase in hippocampal proinflammatory signaling cascades. As galantamine, a cholinesterase inhibitor, has anti-inflammatory actions, we tested the hypothesis that galantamine would prevent (study 1) or restore (study 2) AIE induction of proinflammatory signals within the hippocampus as well as AIE-induced loss of hippocampal neurogenesis. Methods Galantamine (4 mg/kg) or vehicle (saline) was administered to Wistar rats during adolescent intermittent ethanol (AIE; 5.0 g/kg ethanol, 2 days on/2 days off, postnatal day [P] 25-54) (study 1, prevention) or after AIE during abstinent maturation to adulthood (study 2, restoration). Results Results indicate AIE reduced DCX+IR and induced cleaved caspase3 (Casp3) in DCX-expressing immature neurons. Excitingly, AIE induction of activated Casp3 in DCX-expressing neurons is both prevented and reversed by galantamine treatment, which also resulted in prevention and restoration of neurogenesis (DCX+IR). Similarly, galantamine prevented and/or reversed AIE induction of proinflammatory markers, including the chemokine (C-C motif) ligand 2 (CCL2), cyclooxygenase-2 (COX-2), and high mobility group box 1 (HMGB1) protein, suggesting that AIE induction of proinflammatory signaling mediates both cell death cascades and hippocampal neurogenesis. Interestingly, galantamine treatment increased Ki67+IR generally as well as increased pan-Trk expression specifically in AIE-treated rats but failed to reverse AIE induction of NADPH-oxidase (gp91phox). Conclusions Collectively, our studies suggest that (1) loss of neurogenesis after AIE is mediated by persistent induction of proinflammatory cascades which drive activation of cell death machinery in immature neurons, and (2) galantamine can prevent and restore AIE disruptions in the hippocampal environmental milieu to then prevent and restore AIE-mediated loss of neurogenesis.

Cholinergic and Neuroimmune Signaling Interact to Impact Adult Hippocampal Neurogenesis and Alcohol Pathology Across Development

“…Thus, induction of HMGB1→TLR4/ RAGE→pNFκB p65 signaling cascades may be a critical mediator in AIE-induced cell death of neuroprogenitors, as expression of hippocampal pNFκB p65 and cleaved caspase-3 are highly correlated (He and Crews, 2007). Furthermore, reversal of hippocampal proinflammatory cascades through the cholinesterase inhibitor galantamine prevents and reverses both AIE induction of HMGB1, COX-2, and CCL2 as well as reduces AIE induction of cleaved caspase-3 in DCX-labeled immature neurons in male rats (Macht et al, 2021), further highlighting that disruption of cholinergic signaling contributes to AIE induction of neuronal inflammatory proinflammatory cascades, cell death, and adult hippocampal neurogenesis.…”

“…Ki67 is expressed during mitosis across mammalian species and exhibits a very short half-life, making it a conservative but accurate estimate of actively dividing cells in the subgranular zone of the neurogenic niche. Some ( Nixon and Crews, 2002 ; Taffe et al, 2010 ; Vetreno and Crews, 2015 ; Liu and Crews, 2017 ) but not all ( Ehlers et al, 2013 ; Broadwater et al, 2014 ; Macht et al, 2021 ) studies have found that adolescent binge ethanol exposure decreases Ki67 immunoreactivity in adulthood. Conversely, adolescent binge ethanol exposure has consistently been found to induce activation of the apoptotic executioner caspase cleaved caspase-3 ( Ehlers et al, 2013 ; Broadwater et al, 2014 ; Vetreno and Crews, 2015 ; Liu and Crews, 2017 ; Macht et al, 2021 ), and/or to increase hippocampal necrotic cell death, as marked by Fluoro-Jade B ( Taffe et al, 2010 ; Ehlers et al, 2013 ), across species and across variations in route of administration.…”

“…Some ( Nixon and Crews, 2002 ; Taffe et al, 2010 ; Vetreno and Crews, 2015 ; Liu and Crews, 2017 ) but not all ( Ehlers et al, 2013 ; Broadwater et al, 2014 ; Macht et al, 2021 ) studies have found that adolescent binge ethanol exposure decreases Ki67 immunoreactivity in adulthood. Conversely, adolescent binge ethanol exposure has consistently been found to induce activation of the apoptotic executioner caspase cleaved caspase-3 ( Ehlers et al, 2013 ; Broadwater et al, 2014 ; Vetreno and Crews, 2015 ; Liu and Crews, 2017 ; Macht et al, 2021 ), and/or to increase hippocampal necrotic cell death, as marked by Fluoro-Jade B ( Taffe et al, 2010 ; Ehlers et al, 2013 ), across species and across variations in route of administration. Moreover, AIE increases expression of cleaved caspase-3 specifically within DCX-labeled neurons, suggesting that activation of cell death machinery directly contributes to loss of newborn hippocampal neurons ( Macht et al, 2021 ).…”

“…Conversely, adolescent binge ethanol exposure has consistently been found to induce activation of the apoptotic executioner caspase cleaved caspase-3 ( Ehlers et al, 2013 ; Broadwater et al, 2014 ; Vetreno and Crews, 2015 ; Liu and Crews, 2017 ; Macht et al, 2021 ), and/or to increase hippocampal necrotic cell death, as marked by Fluoro-Jade B ( Taffe et al, 2010 ; Ehlers et al, 2013 ), across species and across variations in route of administration. Moreover, AIE increases expression of cleaved caspase-3 specifically within DCX-labeled neurons, suggesting that activation of cell death machinery directly contributes to loss of newborn hippocampal neurons ( Macht et al, 2021 ). Collectively, these findings suggest that adolescent binge ethanol exposure disrupts the neurogenic niche in ways that interfere with the successful maturation of adult newborn neurons, potentially through mechanisms involving either 1 ) a failure to assimilate signals driving successful integration into existing hippocampal circuitry or 2 ) hypersensitivity to the stimulation of cell death executioner pathways.…”

“…One mechanism that activates cell death pathways in adult newborn neurons is induction of proinflammatory signaling cascades, resulting in cleavage of caspase-3 and consequential catalyzation and cleavage of critical cell proteins, chromatin condensation, DNA fragmentation, and ultimately cellular apoptosis ( Porter and Jänicke, 1999 ; Macht et al, 2021 ). Chronic induction of proinflammatory signaling cascades within the hippocampus are a persistent molecular consequence of adolescent ethanol exposure ( Crews et al, 2016 ), and AIE-induced upregulation of proinflammatory gene expression is evident across multiple signaling steps, including increasing the nuclear histone-binding protein high mobility group box protein 1 (HMGB1) ( Swartzwelder et al, 2019 ; Macht et al, 2021 ), CCL2 ( Macht et al, 2021 ), Toll-like receptor 4 (TLR4), the canonical neuroimmune gene transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells p65 (pNFκB p65) ( Li Q. et al, 2019 ), and cyclooxygenase-2 (COX-2) ( Pascual et al, 2007 ; Macht et al, 2021 ).…”