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Brain ethanol metabolism by astrocytic ALDH2 drives the behavioural effects of ethanol intoxication

Nature Metabolism volume 3, pages 337–351 (2021)Cite this article

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Abstract

Alcohol is among the most widely used psychoactive substances worldwide. Ethanol metabolites such as acetate, thought to be primarily the result of ethanol breakdown by hepatic aldehyde dehydrogenase 2 (ALDH2), contribute to alcohol’s behavioural effects and alcoholism. Here, we show that ALDH2 is expressed in astrocytes in the mouse cerebellum and that ethanol metabolism by astrocytic ALDH2 mediates behavioural effects associated with ethanol intoxication. We show that ALDH2 is expressed in astrocytes in specific brain regions and that astrocytic, but not hepatocytic, ALDH2 is required to produce ethanol-derived acetate in the mouse cerebellum. Cerebellar astrocytic ALDH2 mediates low-dose ethanol-induced elevation of GABA levels, enhancement of tonic inhibition and impairment of balance and coordination skills. Thus, astrocytic ALDH2 controls the production, cellular and behavioural effects of alcohol metabolites in a brain-region-specific manner. Our data indicate that astrocytic ALDH2 is an important, but previously under-recognized, target in the brain to alter alcohol pharmacokinetics and potentially treat alcohol use disorder.

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Fig. 1: Astrocytic ALDH2 expression in the cerebellum.
Fig. 2: Astrocytic ALDH2 selectively regulates ethanol metabolite acetate in cerebellar tissues.
Fig. 3: Astrocytic ALDH2 mediates ethanol-induced elevation in cerebellar acetate and GABA levels.
Fig. 4: Astrocytic ALDH2 mediates ethanol enhancement of GABA tonic inhibition.
Fig. 5: Acetate enhancement of GABA synthesis and GABA tonic inhibition in cerebellum.
Fig. 6: Single-astrocyte analysis of the effects of ethanol and acetate on GABA synthesis.
Fig. 7: Astrocytic ALDH2 mediates ethanol-induced discoordination.

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Data availability

All data from these studies are contained within this manuscript or are available from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank F. Langevin and K. J. Patel (University of Cambridge) for Aldh2 flox mice. We especially thank M. Chen, X. Sun and X. Li (Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland) for their technical support for in vivo MRS experiments and data analysis. We thank A. Salinas, G. Luo, T. Ren, A. Guillot and H.-J. White (NIAAA, NIH) for technical assistance and comments on the manuscript. We thank the Human Brain Collection Core at the National Institute of Mental Health for providing human cerebellar tissues. This work was supported by grants 1UL1TR003098 (to Q. Cao) from the University of Maryland, Baltimore, Institute for Clinical & Translational Research (ICTR) and the National Center for Advancing Translational Sciences (NCATS) Clinical Translational Science Award (CTSA), K08AA024895-01A1 to (Q. Cao) from the National Institue on Alcohol Abuse and Alcoholism (NIAAA), National Institutes of Health (NIH), chairman seed grant award (to Q. Cao) of the Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, Baltimore and 81801938 (to S.J.) from the National Natural Science Foundation of China.

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Author notes

  1. These authors contributed equally: Shiyun Jin, Qi Cao, Fanghan Yang.

Authors and Affiliations

  1. Laboratory for Integrative Neuroscience, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Shiyun Jin, Fanghan Yang, David M. Lovinger & Li Zhang

  2. Department of Anesthesiology, Second Affiliated Hospital, Anhui Medical University, Hefei, PR China

    Shiyun Jin & Ye Zhang

  3. Department of Diagnostic Radiology and Nuclear Medicine, University of Maryland, School of Medicine, Baltimore, MD, USA

    Qi Cao & Su Xu

  4. Department of Neuroscience, University of Science and Technology of China, Hefei, PR China

    Hongying Zhu, Qi Chen, Ziyi Wang & Wei Xiong

  5. Laboratory for Liver Diseases, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Yuhong Lin & Bin Gao

  6. Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Resat Cinar

  7. Laboratory for Metabolic Control, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Robert J. Pawlosky

  8. National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA

    George F. Koob

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  1. Shiyun Jin
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Contributions

L.Z. conceived, designed and supervised the study. S.J., L.Z., F.Y., Y.L., R.C. and R.J.P. contributed to initial data collection and analysis. Q. Cao and S.X. conducted the MRS experiment and analysed the data. Q. Chen, Z.W., H.Z. and W.X. conducted high-resolution single-cell GC–MS analysis and analysed the data. S.J. and L.Z. performed the final data analysis. S.J. made the figures and tables. L.Z. wrote the manuscript. Y.Z., D.M.L., B.G., Q. Cao and G.F.K. helped with data interpretation and revision of the manuscript.

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Correspondence to Li Zhang.

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The authors declare no competing interests.

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Peer review information Primary Handling Editors: Pooja Jha; Isabella Samuelson. Nature Metabolism thanks Juan Bolaños and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Astrocyte-specific distribution of ALDH2 in the cerebellum.

a, Bar graphs of ALDH2 activity in cerebellar anterior, vermis and posterior regions. (n=6 mice/group). Data are presented as means±s.e.m. Analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey’s test. b, Epifluorescence images of ALDH2 (green), GFAP (violet) and L7/PC (red) gene in a cerebellar sagittal slice. Red scale bar, 200 μm; White scale bars, 50 μm. c, Epifluorescence images of ALDH2 (green), MAP2 (violet) and L7/PC (red) gene. No colocalization across these genes. Red scale bar, 200 μm; White scale bars, 50 μm. d, Images showing a colocalization of ALDH2 (green) with GFAP (red) but not MAP2 (violet) gene in cerebellum. Red scale bar, 200 μm; White scale bars, 50 μm. For b, c and d, Experiments were repeated four times for each biologically independent mouse, with similar results obtained. e, Quantification of cluster colocalization between ALDH2 and ALDH1L1 in cerebellum. The experiment was repeated four times for each biologically independent mouse, with similar results obtained (n=3 mice). Quantitative data was provided in Supplementary Table 2.YES: colocalization; NO: no colocalization; White scale bars, 50 μm.

Source data

Extended Data Fig. 2 ALDH2 expression in different brain regions of Aldh2Gfap-/- mice.

a, the schematic diagram shows the generation of astrocytic specific Aldh2 deficient mice. b, A representative image of PCR genotyping result. The image of agar gel migration shows flox (homozygous: one band, 365 bp; heterozygous: two bands, 365 and 300 bp) and Cre (negative: one band, 350 bp; positive: two bands, 350 and 200 bp) gene. c, RNAscope images of ALDH2 signals in cerebellum and liver slices from conditional and global Aldh2-/- mice. Noted that ALDH2 was selectively depleted from microdomains surrounding PC signal in cerebellar slice from Aldh2Gfap-/- mice. Liver ALDH2 mRNA was intact in Aldh2Gfap-/- mice, whereas liver ALDH2 signal was abolished in Aldh2-/- mice. Each experiment was repeated four times with similar results obtained. White scale bars, 100 μm. d, e, Representative peaks from electrophoresis densitometry of cerebellar (n=7, 7 and 3 mice, respectively) and liver (n=5, 5 and 3 mice, respectively) ALDH2 proteins from Aldh2Gfap+/+ (black line or rectangle), Aldh2Gfap-/- and Aldh2-/- mice. f, Summary statistics of cerebellar ALDH2 enzymatic activity detected in Aldh2L7-/- and Aldh2Camk2a-/- mice (n=6, 6, 7 and 7 mice, respectively). g, Representative electropherogram traces of ALDH2 protein samples collected from different brain areas of Aldh2Gfap+/+ and Aldh2Gfap-/- mice. h, Summary statistics of brain ALDH2 protein detected in difference brain areas from Aldh2Gfap+/+ (n=5, 4, 6, 4, 4 and 7 mice, respectively) and Aldh2Gfap-/- (n=5, 4, 6, 4, 4 and 7 mice, respectively) mice. i, Representative electropherogram traces of protein samples collected from different brain areas from Aldh2Camk2a+/+ and Aldh2Camk2a-/- mice. j, Summary statistics of brain ALDH2 protein detected in difference brain areas from Aldh2Camk2a+/+ and Aldh2Camk2a-/- mice (n=4 mice/group). k, l, Representative electropherogram traces and summary statistics of brain ALDH2 protein detected in different brain areas from astrocytic ALDH2 deficient mice generated by crossing Aldh2 flox with GFAPCre (Jax, 024098), Aldh2Gfap-/- mice and their wildtype littermates, Aldh2Gfap+/+ (n=3 mice/group). Data are presented as means ±s.e.m. Analysis was performed using unpaired two tailed student’s t test (h, j, f, l) or one-way analysis of variance (ANOVA) followed by Tukey’s test (d, e). PFC, Prefrontal cortex; CE, Cerebellum; HIPP, Hippocampus; CeA, Central nucleus of the amygdala; VTA, Ventral tegmental area; BLA, Basolateral amygdala; NAc, Nucleus accumbent.

Source data

Extended Data Fig. 3 Astrocytic Aldh2 deficiency does not alter serum and brain (cerebellar cortex) ethanol and acetaldehyde contents.

a, b, Serum and cerebellum ethanol concentrations measured 50 min after ethanol (2 g/kg, i.p.) in WT and Aldh2*2KI mice (n=4 mice/group). c, d, Graphs showing ethanol concentrations in the serum and cerebellum 50 min after ethanol in Aldh2Gfap+/+ and Aldh2Gfap-/- mice (0 g/kg ethanol: n=4 mice/group; 1 g/kg ethanol: n=12 and 13 from Aldh2Gfap+/+ and Aldh2Gfap-/- mice respectively (serum), n=7 and 8 from Aldh2Gfap+/+ and Aldh2Gfap-/- mice respectively (cerebellum); 2 g/kg ethanol: n=5 mice/group; 3.6 g/kg ethanol: n=5 mice/group). e, f, Serum and cerebellum acetaldehyde contents measured after ethanol (1 g/kg, i.p.) in Aldh2Gfap+/+ and Aldh2Gfap-/- mice (0 min: n=4 mice/group; 10 min: n=9 mice/group (serum), n=4 mice/group (cerebellum); 50 min: n=11 mice/group (serum), n=7 and 8 from Aldh2Gfap+/+ and Aldh2Gfap-/- mice respectively (cerebellum)). g, h, Serum and cerebellum ethanol contents measured after ethanol (1 g/kg, i.p.) in Aldh2Gfap+/+ and Aldh2Gfap-/- mice (0 min: n=4 mice/group; 10 min: n=9 mice/group (serum), n=4 mice/group (cerebellum); 50 min: n=12 and 13 from Aldh2Gfap+/+ and Aldh2Gfap-/- mice respectively (serum), n=7 and 8 from Aldh2Gfap+/+ and Aldh2Gfap-/- mice respectively (cerebellum)). Data are presented as mean±s.e.m. Analysis was performed using unpaired two tailed student’s t test (a, b) or two-way analysis of variance (ANOVA) (c-h).

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Extended Data Fig. 4 In vivo MRS quantitative measurement of brain ethanol metabolites and neurochemicals.

a, b, Representative peaks and Summary statistics of brain ALDH2 protein in cerebellum and liver from Aldh2Hep+/+ and Aldh2Hep-/-mice (n=4 mice/group).c, d, In vivo MRS measurement of cerebellar acetate and GABA contents after systemic ethanol (1-2 g/kg, i.p.) in Aldh2Gfap+/+ mice (n=5, 3 and 5 mice, respectively). e, Ethanol enhancement of cerebellar GABA, Glutamine, Glutamate and NAA in Aldh2 flox mice (n=9 mice/group). f, g, Statistic summaries of cerebellar ethanol by MRS after systemic administration of ethanol (1-2 g/kg, i.p.) in astrocytic ALDH2 deficient mice (f: n=3 mice/group; g: n=5 mice/group), hepatocytic ALDH2 deficient mice (Aldh2Hep+/+: n=4 mice and Aldh2Hep-/-: n=6 mice) and their wild type littermates mice. g, Bar graphs showing cerebellar ethanol concentrations after ethanol (2 g/kg, i.p.) by in vivo MRS measurement in Aldh2Hep+/+ (n=4) and Aldh2Hep-/- (n=4) mice. h, Bar graph showing the basal levels of GABA, Glutamine, Glutamate and NAA in cerebellum in Aldh2Gfap+/+ and Aldh2Gfap-/- mice (n=5 mice/group). i, Bar graph showing the basal levels of GABA, Glutamine, Glutamate and NAA in cerebellum in Aldh2Hep+/+ (n=4) and Aldh2Hep-/- (n=6) mice. Data are presented as mean±s.e.m. Groups were compared by unpaired two tailed student’s t test (b, e-i), or one-way analysis of variance (ANOVA) followed by Tukey’s test (c, d).

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Extended Data Fig. 5 Ethanol does not significantly alter GABAergic mIPSCs and GABA-activated current in GCs.

a, b, Trace records and summary statistics of tonic current recorded using whole cell recording in cerebellar GCs from Aldh2Gfap+/+ and Aldh2Gfap-/- mice (ACSF: n=8 cells, 6 mice). c, Summary statistics of the cell membrane capacitance with and without ethanol and acetate (n=8, 8, 7, 7, 7, 8, 9, 9, 9 and 10 cells, respectively). d, e, Left, Trace records of GABA (20 µM) activated currents in cerebellar GCs from Aldh2Gfap+/+ and Aldh2Gfap-/- mice. Right, Summary of the average amplitudes of GABA-activated currents (n=4 cells/group, 4 mice). f, Statistic summary of the average levels of cerebellar GABA contents measured by LC-MS/MS in Aldh2Gfap+/+ and Aldh2Gfap-/- mice (n=8 mice/group). g-j, Trace records and summary statistics of mIPSCs in GCs from Aldh2Gfap+/+ and Aldh2Gfap-/- mice (n=8 cells/group). k-n, The effect of ethanol on GABAergic IPSCs (n=9 cells/group, 6 mice). Data are presented as mean±s.e.m. Groups were compared by one-way analysis of variance (ANOVA) (c), or unpaired two tailed student’s t test (b, c, e, f, h, i, l, m).

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Extended Data Fig. 6 Characterization of unilateral conditional deletion of cerebellar astrocytic ALDH2.

a, Bar graph showing the rotarod performance of Aldh2Camk2a+/+ and Aldh2Camk2a-/- mice (n=10 mice/group) after 10 min 1 g/kg ethanol i.p. injection. b, Time course of the basal levels of the rotarod performance in consecutive trails in Aldh2Gfap+/+ (n=9) and Aldh2Gfap-/- (n=10) mice. c, Time courses of core-body temperature after ethanol (2 g/kg, i.p.) in Aldh2Gfap+/+ (n=9) and Aldh2Gfap-/- (n=13) mice. d,e, Florescent microscopy images of ALDH2 signal (green) in cerebellar slices from Aldh2 flox mice previously injected with AAV-GFAPCre and AAV-Synt1Cre virus (4 weeks after injection). Experiments were repeated four times for each biologically independent mouse, with similar results obtained (n=3 mice). Red scale bar, 500 μm; White scale bars, 50 μm. f, Bar graph showing the baseline levels of rotarod performance in cerebellar virus injected mice (n=15 mice for AAV-Synt1Cre; n=16 mice for AAV-GFAPCre). g, Time courses of the levels of rotarod performance after i.c.v. injection of CGP (200 ng, n=7 mice) or Bicuculline (200 ng, n=5 mice). Data are presented as mean±s.e.m. Analysis was performed using unpaired two tailed student’s t test (f) or two-way analysis of variance (ANOVA) followed by Turkey’s test (a), or two-way repeated-measures analysis of variance (ANOVA) (b,c,g).

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Extended Data Fig. 7 A hypothetical mechanism for astrocytic ALDH2 control of alcohol metabolism and action in the brain.

Simplified diagram of ALDH2-dependent pathways that mediate ethanol-induced elevation in cerebellar GABA levels and discoordination.

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Jin, S., Cao, Q., Yang, F. et al. Brain ethanol metabolism by astrocytic ALDH2 drives the behavioural effects of ethanol intoxication. Nat Metab 3, 337–351 (2021). https://doi.org/10.1038/s42255-021-00357-z

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