The Hepatic Mitochondrial Alterations Exacerbate Meta-Inflammation in Autism Spectrum Disorders

Abstract

The role of the liver in autism spectrum disorders (ASD), developmental disabilities characterized by impairments in social interactions and repetitive behavioral patterns, has been poorly investigated. In ASD, it has been shown a dysregulation of gut-brain crosstalk, a communication system able to influence metabolic homeostasis, as well as brain development, mood and cognitive functions. The liver, with its key role in inflammatory and metabolic states, represents the crucial metabolic organ in this crosstalk. Indeed, through the portal vein, the liver receives not only nutrients but also numerous factors derived from the gut and visceral adipose tissue, which modulate metabolism and hepatic mitochondrial functions. Here, we investigated, in an animal model of ASD (BTBR mice), the involvement of hepatic mitochondria in the regulation of inflammatory state and liver damage. We observed increased inflammation and oxidative stress linked to hepatic mitochondrial dysfunction, steatotic hepatocytes, and marked mitochondrial fission in BTBR mice. Our preliminary study provides a better understanding of the pathophysiology of ASD and could open the way to identifying hepatic mitochondria as targets for innovative therapeutic strategies for the disease.

Keywords: autism spectrum disorders; inflammation; liver; mitochondrial dysfunction.

Figure 1

Metabolic and inflammatory profile in control and BTBR mice. Body weight and daily food intake at the end of treatment (A). Body energy content and gross efficiency (B), body lipid, protein, and water percentages (C). Serum levels of triglycerides, cholesterol, alanine aminotransferase (ALT), aspartate aminotransferase (AST), monocyte chemoattractant protein-1 (MCP-1) (D), leptin, adiponectin, lipopolysaccharide (LPS), tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6) (E). Hepatic levels of TNF-α, IL-1β, IL-6 (F). Data are presented as means ± SEM from n = 7 animals/group. * p < 0.05, ** p < 0.01, *** p < 0.001 BTBR vs. control (CTR).

Figure 2

Hepatocytes morphological features of control and BTBR mice. Control group (CTR) (A,C); BTBR group (B,D). Toluidine blue staining (A,B). PAS staining on resin sections (C,D). CTR shows hepatocytes (H) arranged around the centrilobular vein (Cv) (A). BTBR group shows an increase in lipid drops (arrow), irregularly shaped nuclei (N), nucleolar hypertrophy (n) (B). PAS staining of CTR revealed widespread positivity (asterisk) (C). BTBR hepatocytes show different grade of positivity (asterisk). K: Kupffer cell, I: Ito cell (D).(A–D): 100× magnification. Hepatic lipid content (E), and average Kupffer cells (F). ** p < 0.01, *** p < 0.001 BTBR vs. Control (CTR).

Figure 3

Morphological features of control and BTBR mice liver. PAS staining of BTBR mice liver (A–D). Some hepatocytes show nuclear inclusions (arrow), with PAS-positive cytoplasm and lipid drops, which at some point maintains contact with the nuclear envelope (double arrow). Transmission Electron Microscopy micrographs of BTBR mice liver (E,F) highlighting nuclear inclusions (double asterisks) with glycogen (g) or lipid droplets (L). N: nucleus; n: nucleolus; asterisk: PAS positivity. (A–D): 100× magnification; (E,F): 7000× magnification.

Figure 4

Transmission Electron Microscopy micrographs of control and BTBR mice liver. Control (CTR) (A,B) and BTBR mice liver (C–F). CTR liver shows regular nucleus (N), few lipids drop (L), well-preserved mitochondria (m) associated with rough (RER) or smooth reticulum (SER) (A). Greater magnification of mitochondria, surrounded by RER (B). BTBR liver shows irregular nuclear envelope, hypertrophic nucleoli and RER with congested cisterns, increased number and size of lipid drops (C). Mitochondria, mostly small in size (C–E) and mitochondrial fission phenomena ((F) and insert) are recognizable. Asterisk: bile canaliculus; (g) glycogen; (arrow) nuclear pore. Magnification: (A) 2550×, (B) 9900× (C) 4000×, (D) 7800×, (E) 7000×, (F) 9900×.

Figure 5

Hepatic markers of the endoplasmic reticulum stress and mitochondrial dynamics in control and BTBR mice. Percentage and length of mitochondria detected by histological analyses (A). Hepatic expression levels of endoplasmic reticulum stress markers: Binding Immunoglobulin Protein (BIP) and phosphorylated eukaryotic translation initiation factor 2A (pEIF2α), as ratio pEIF2α/EIF2α (B). Expression levels of markers of mitochondrial dynamics: fusion-related proteins mitofusin 1 (MFN1), mitofusin 2 (MFN2) and optic atrophy protein (OPA1) (C), and fission-related dynamin-related protein (DRP1) and fission 1 protein (FIS1) (D). (n = 4 animal/group). * p < 0.05, *** p < 0.001, BTBR vs. Control (CTR).

Figure 6

Hepatic mitochondrial function and efficiency, AMPK/ACC and CPT1 expression in control and BTBR mice. Hepatic mitochondrial respiration rates were evaluated with succinate + rotenone (A) or palmitoyl-carnitine + malate (B) as substrates. CPT activity was measured in isolated mitochondria (C). Mitochondrial oxygen consumption in the presence of oligomycin, uncoupled by carbonyl-cyanide-4(trifluoromethoxy)phenylhydrazone (FCCP) and the degree of coupling (D) were shown. Hepatic expression levels of phosphorylated adenosine-monophosphate-activated protein kinase-α (pAMPK), as ratio pAMPK/AMPK (E), phosphorylated acetyl-CoA-carboxylase (pACC) as ratio pACC/ACC, and carnitine-palmitoyl-transferase 1 (CPT-1) (F). n = 4. Data are presented as means ± SEM from n = 7 animals/group. * p < 0.05, ** p < 0.01, *** p < 0.001, BTBR vs. Control (CTR).

Figure 7

Hepatic oxidative stress profile and antioxidant/detoxifying defenses in control and BTBR mice. Hydrogen peroxide (H₂O₂) release (A), basal/total aconitase (B) and superoxide dismutase (SOD) activity (C) were determined in hepatic isolated mitochondria. Reduced glutathione (GSH), glutathione oxidized (GSSG) content ((D), left Y axis) and ratio GSH/GSSG ((D), right Y axis), glutathione transferase (GST) and NAD(P)H Quinone Dehydrogenase (NQO1) levels (E) were evaluated in hepatic tissue. Hepatic levels of malondialdehyde (MDA) and protein carbonyls (PC) (F). Data are presented as means ± SEM from n = 7 animals/group. * p < 0.05, *** p < 0.001, BTBR vs. Control (CTR).