The metabesity factor HMG20A potentiates astrocyte survival and reactive astrogliosis preserving neuronal integrity

Abstract

Rationale: We recently demonstrated that the 'Metabesity' factor HMG20A regulates islet beta-cell functional maturity and adaptation to physiological stress such as pregnancy and pre-diabetes. HMG20A also dictates central nervous system (CNS) development via inhibition of the LSD1-CoREST complex but its expression pattern and function in adult brain remains unknown. Herein we sought to determine whether HMG20A is expressed in the adult CNS, specifically in hypothalamic astrocytes that are key in glucose homeostasis and whether similar to islets, HMG20A potentiates astrocyte function in response to environmental cues. Methods: HMG20A expression profile was assessed by quantitative PCR (QT-PCR), Western blotting and/or immunofluorescence in: 1) the hypothalamus of mice exposed or not to either a high-fat diet or a high-fat high-sucrose regimen, 2) human blood leukocytes and adipose tissue obtained from healthy or diabetic individuals and 3) primary mouse hypothalamic astrocytes exposed to either high glucose or palmitate. RNA-seq and cell metabolic parameters were performed on astrocytes treated or not with a siHMG20A. Astrocyte-mediated neuronal survival was evaluated using conditioned media from siHMG20A-treated astrocytes. The impact of ORY1001, an inhibitor of the LSD1-CoREST complex, on HMG20A expression, reactive astrogliosis and glucose metabolism was evaluated in vitro and in vivo in high-fat high-sucrose fed mice. Results: We show that Hmg20a is predominantly expressed in hypothalamic astrocytes, the main nutrient-sensing cell type of the brain. HMG20A expression was upregulated in diet-induced obesity and glucose intolerant mice, correlating with increased transcript levels of Gfap and Il1b indicative of inflammation and reactive astrogliosis. Hmg20a transcript levels were also increased in adipose tissue of obese non-diabetic individuals as compared to obese diabetic patients. HMG20A silencing in astrocytes resulted in repression of inflammatory, cholesterol biogenesis and epithelial-to-mesenchymal transition pathways which are hallmarks of reactive astrogliosis. Accordingly, HMG20A depleted astrocytes exhibited reduced mitochondrial bioenergetics and increased susceptibility to apoptosis. Neuron viability was also hindered in HMG20A-depleted astrocyte-derived conditioned media. ORY1001 treatment rescued expression of reactive astrogliosis-linked genes in HMG20A ablated astrocytes while enhancing cell surface area, GFAP intensity and STAT3 expression in healthy astrocytes, mimicking the effect of HMG20A. Furthermore, ORY1001 treatment protected against obesity-associated glucose intolerance in mice correlating with a regression of hypothalamic HMG20A expression, indicative of reactive astrogliosis attenuation with improved health status. Conclusion: HMG20A coordinates the astrocyte polarization state. Under physiological pressure such as obesity and insulin resistance that induces low grade inflammation, HMG20A expression is increased to induce reactive astrogliosis in an attempt to preserve the neuronal network and re-establish glucose homeostasis. Nonetheless, a chronic metabesity state or functional mutations will result in lower levels of HMG20A, failure to promote reactive astrogliosis and increase susceptibility of neurons to stress-induced apoptosis. Such effects could be reversed by ORY1001 treatment both in vitro and in vivo, paving the way for a new therapeutic approach for Type 2 Diabetes Mellitus.

Keywords: HMG20A; ORY1001.; astrocytes; inflammation; metabesity; metabolism.

Figure 1

HMG20A is expressed in hypothalamic astrocytes. (A) Representative confocal microscopy images of free-floating mouse brain sections immunostained for HMG20A (red) and GFAP, an astrocyte marker (green, upper panel left) or (B) NeuN a neuronal marker (green, upper panel right). Nuclei were stained using DAPI (blue). The bottom panel in (A) provides higher magnification images for the detection of HMG20A (red) and GFAP (green) co-expression in hypothalamic astrocytes. Arrows point to astrocytes expressing HMG20A. White bar corresponds to 50 μm. (C) Relative expression levels of Hmg20a in mouse islets, astrocytes, brain and white adipose tissue (WAT). Hmg20a transcript levels were normalized to those of the housekeeping gene Gapdh and/or Cyclophilin. n = 5-6 biological replicates analyzed in duplicate.

Figure 2

HMG20A levels are increased by physiological/metabolic stresses in both mouse and human. (A) Relative expression levels of hmg20a, Gfap, Vimentin and (B) Il1b in hypothalamus extracted from mice fed either a normal diet (CT) or a high fat diet (HFD). Transcript levels were normalized to those of the housekeeping gene b-Actin. Values are referred to the average expression levels detected in CT mice. n = 5-6 biological replicates analyzed in duplicate. *p < 0.05, unpaired t-test CT versus HFD. (C) HMG20A protein levels (left panel) and quantification (right panel) in hypothalamus extracted from mice fed either a normal diet (CT) or a high fat diet (HFD). Each lane represents a protein extract of one hypothalamus isolated from either CT or HFD mouse. GAPDH was used as the housekeeping protein for normalization. **p < 0.01, unpaired t-test CT versus HFD. Relative expression levels of Hmg20a in isolated mouse astrocytes cultured in either (D) increasing concentration of glucose or (E) 0.5 mM palmitate for 1, 24 and 48 h. Transcript levels were normalized to those of the housekeeping gene Gapdh and/or Cyclophilin. Values are referred to the average expression levels detected in astrocytes cultured either in 17.5 mM glucose (D) or untreated (vehicle, E). n = 4-5 biological replicates analyzed in duplicate. *p < 0.05, unpaired t-test 1 versus 24 h. (F) HMG20A expression levels were measured in subcutaneous white adipose tissue biopsies that were obtained from non-obese/non-diabetic (NO ND), obese/non-diabetic (O ND), obese/diabetic (O DM) and non-obese/diabetic (NO DM). The expression of HMG20A was corrected to those of the housekeeping gene HISTONE. Values are referred to the average expression detected NO ND individuals. n = 3-8 individuals per group, analyzed in duplicate. ** p<0.01, unpaired t-test NO ND versus O ND or O DM. (G) Leukocytes were isolated from blood samples extracted from non-obese/non-diabetic (NO ND), obese/non-diabetic (O ND), obese/diabetic (O DM) and non-obese/diabetic (NO DM) and relative expression levels of HMG20A were assessed by quantitative PCR. The expression of HMG20A was corrected to those of the housekeeping genes HISTONE and B-ACTIN. n = 10-58 individuals per group analyzed in duplicate.

Figure 3

HMG20A silencing represses signaling pathways linked to reactive astrogliosis. (A) Relative expression levels of Hmg20a in astrocytes exposed to either a control luciferase siRNA (siCT) or a siRNA targeted to HMG20A (siHMG20A). Transcript levels were normalized to those of the housekeeping gene Gapdh and/or Cyclophilin. Values are referred to the average expression levels detected in siCT. n = 3 biological replicates of primary astrocyte preparations pooled from 3-6 mice and analyzed in duplicate. *p < 0.05, unpaired t-test siCT versus siHMG20A. (B) Volcano plot representation of differentially regulated genes after HMG20A silencing, analyzed by RNA-seq. Genes significantly (p-value < 0.05) downregulated are marked in blue (Log2FC ≥ - 0.5) and the upregulated ones in purple (Log2FC ≥ 0.5). Hmg20A is highlighted as a red dot. (C) Biological process (BP)-GO categories enriched after HMG20A silencing, analyzed using the David software. (D), (E) and (F) Representative enriched pathways (upper panels) related to reactive astrogliosis. The analysis was performed using the GSEA software. Normalized Enrichment Score (NES) and False Discovery Rate (FDR) are indicated within the plots. Lower panels depict heatmaps of the relative expression of those genes significantly downregulated after HMG20A silencing in each enriched pathways in two independent RNAseq. (G) Confirmatory QT-PRC of selected target genes from the various pathways altered by HMG20A repression. Transcript levels were normalized to those of the housekeeping gene Gapdh and/or Cyclophilin.

Figure 4

HMG20A silencing alters mitochondrial bioenergetics sensitizing astrocytes to apoptosis. (A) Bcl2l11 transcript levels in siCT or siHMG20A treated astrocytes. Expression levels of Bcl2l11 were normalized to the housekeeping gene Gapdh. n = 5 independent experiments, ** p < 0.01 unpaired Student t-test siCT versus siHMG20A. (B) Knockdown of HMG20A in astrocytes significantly decreased mitochondrial metabolism as assessed by MTT assay. Seahorse analysis showing (C) OCR profile plot, (D) Basal and maximal respiration and (E) ECAR in astrocytes silenced or not for HMG20A. Oligo; Oligomycin, FCCP; Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, Rot; Rotenone, Ant, Antimycin A. n = 5 (independent primary cultures obtained from 4 male pups of 2-4 days old), * p < 0.05 unpaired t-test siCT versus siHMG20A. (F) Lactate secretion and (G) Apoptosis was assessed subsequent to siRNA-mediated HMG20A silencing in astrocytes. (B), (F) and (G) Data are referred to the average value detected in non-silenced astrocytes (siCT). For each condition, 3-4 biological replicates (independent primary cultures obtained from 4 male pups of 2-4 days old) were analyzed in duplicate. **p < 0.01 unpaired Student t-test siCT versus siHMG20A.

Figure 5

HMG20A coordinates expression of astrocyte derived extracellular factors involved in neuronal survival. (A) Representative immunofluorescent images of mouse spinal cord motoneurons cultured for 24 h in increasing percentage of conditioned media derived from primary astrocytes silenced for HMG20A (siHMG20A) or not (siCT). Live cells are stained with Calcein (green) while dying cells label for Eth-D1 (red). (B) Quantification of motoneuron cell death after growth in the indicated % of astrocyte conditioned media; control siCT astrocytes (black circles) or HMG20A silenced siHMG20A astrocytes (Black triangles). n = 6 biological replicates (independent spinal cord motoneurons primary cultures). * p < 0.05 and ** p < 0.01, unpaired t-test siCT versus siHMG20A. (C) GO Cellular compartment of genes differentially regulated by siHMG20A in astrocytes. % of genes of the indicated categories is represented. p-value of the enrichment is also provided. (D) Vulcano plot showing expression change (logs(FC) and p-value (-log10(p-value)) (siHMG20a versus siCT) of all expressed growth factors encoding genes.

Figure 6

The LSD1 inhibitor ORY1001 mimics HMG20A-mediated reactive astrogliosis and improves hypothalamic neurons survival. (A) Representative immunofluorescent images of primary astrocytes treated or not for 24, 48 and 72 h with ORY1001. Cells were immunostained for GFAP (red) and nuclei with DAPI (blue). Two images are provided for each time point. Scale bar: 500 um. (B) Quantification of reactive astrocytes (i.e. reactive astrogliosis) in (A) as assessed by GFAP expression and average cell surface area. n = 46-56 dot representing an image from which the area of at least 40 GFAP⁺ cells were measured from 3 independent slides/experiments. (C) Quantification of reactive astrocytes in (A) as assessed by GFAP intensity. n=337-396 cells from 3 independent slides/experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01 and *p < 0.05 unpaired two-tailed t-test CT versus 48 and 72 h. (D) Protein levels (left panel) and quantification (right panels) of STAT3 and phosphorylated STAT3 (pSTAT3) were assessed in whole cell extracts of astrocytes cultured or not with ORY1001 for 72 h. Relative expression levels were normalized to GADPH. Each lane represents an independent experiment. Transcript levels of (E) Hmg20a (F) Slc1a2 and (G) Igfbp3 were determined in astrocytes silenced or not for HMG20A and treated or not with ORY1001. n = 6 independent experiments. **** p < 0.0001, *** p < 0.001 and ** p < 0.01 unpaired two-tailed t-test siCT versus siHMG20A and siHMG20A/ORY1001. (H) Quantification of primary hypothalamic neuron cell death after growth in media containing 75 % of astrocyte conditioned media obtained from siCT-treated astrocytes, siHMG20A-treated astrocytes and ORY1001 supplemented siHMG20A-treated astrocytes. n = 5 biological replicates (independent hypothalamic neuron primary cultures). ** p < 0.01, ANOVA, Turkey's multiple comparison test.

Figure 7

ORY1001 improves glucose sensitivity in HF-HS fed mice correlating with decreased hypothalamic HMG20A levels. An oral glucose tolerance test was performed on either normal chow (NC) or high-fat high-sucrose (HF-HS) fed mice for 6 weeks that were either (A) untreated or (B) treated with ORY1001 (0.0125 mg/Kg body weight). Inset depict area under the curve (AUC). ****p < 0.0001, **p < 0.01 and *p < 0.05 unpaired t-test CT versus HF-HS. (C) Representative Western blot depicting HMG20A protein levels in hypothalamus extracted from 3 mice for each condition depicted in (A) and (B). Right panels depict the quantification of blots for n = 3-10. GAPDH was used as the housekeeping protein for normalization. **** p< 0.0001 and *p < 0.05, unpaired t-test CT versus HFD. (D) Relative expression levels of Hmg20a in isolated primary astrocytes treated with ORY1001 for up to 72 h. Transcript levels were normalized to those of the housekeeping gene Gapdh. n = 3 independent experiments. ***p < 0.001, ****p < 0.0001, unpaired t-test CT versus experimental time of OR1001 treatment. (E) Representative Western blot depicting pSTAT3 protein levels in hypothalamus extracted from 3 mice for each condition depicted in (A) and (B). Right panels depict the quantification of blots for n = 5-8. GAPDH was used as the housekeeping protein for normalization. **p < 0.01, unpaired t-test CT versus HF-HS.

Figure 8

Proposed model for HMG20A/ORY1001 mechanism of action. Under stress conditions, HMG20A expression is enhanced in hypothalamic astrocytes resulting in the inhibition of the LSD1-CoREST complex and downstream activation of the genetic program for reactive astrogliosis, enhanced mitochondrial function and increased cell survival. This phenotypic switch will promote neuronal protection and survival with the goal to resolve the stress response, re-establish a physiological steady state and resorption of reactive astrogliosis. If unresolved, sustained reactive astrogliosis will precipitate both astrocyte and neuron dismay and apoptosis. Treatment of astrocytes with the LSD1 inhibitor ORY1001 mimics HMG20A action, opening the possibility of its pharmacologic use for neuronal protection in metabesity conditions.