Alleviation of Autophagic Deficits and Neuroinflammation by Histamine H3 Receptor Antagonist E159 Ameliorates Autism-Related Behaviors in BTBR Mice

Abstract

Background/objectives: Autism spectrum disorder (ASD) is a neurodevelopmental condition marked by social interaction difficulties, repetitive behaviors, and immune dysregulation with elevated pro-inflammatory markers. Autophagic deficiency also contributes to social behavior deficits in ASD. Histamine H3 receptor (H3R) antagonism is a potential treatment strategy for brain disorders with features overlapping ASD, such as schizophrenia and Alzheimer's disease.

Methods: This study investigated the effects of sub-chronic systemic treatment with the H3R antagonist E159 on social deficits, repetitive behaviors, neuroinflammation, and autophagic disruption in male BTBR mice.

Results: E159 (2.5, 5, and 10 mg/kg, i.p.) improved stereotypic repetitive behavior by reducing self-grooming time and enhancing spontaneous alternation in addition to attenuating social deficits. It also decreased pro-inflammatory cytokines in the cerebellum and hippocampus of treated BTBR mice. In BTBR mice, reduced expression of autophagy-related proteins LC3A/B and Beclin 1 was observed, which was elevated following treatment with E159, attenuating the disruption in autophagy. The co-administration with the H3R agonist MHA (10 mg/kg, i.p.) reversed these effects, highlighting the role of histaminergic neurotransmission in observed behavioral improvements.

Conclusions: These preliminary findings suggest the therapeutic potential of H3R antagonists in targeting neuroinflammation and autophagic disruption to improve ASD-like behaviors.

Keywords: BTBR mice; H3 receptor antagonists; autism spectrum disorder; autophagy; mTOR; neuroinflammation; repetitive behavior; social features.

Figure 1

Chemical structure and pharmacological in vitro binding affinity profile of E159 on selected human histamine receptor subtypes.

Figure 2

Left panel: Predicted binding mode of E159 (left) within histamine H3R receptor binding site. Hydrogen bonds are shown with yellow dashed lines, salt bridges with magenta lines, cation-π interactions with green lines, and π−π interactions with blue lines. Roman numerals indicate the respective TMs; Right panel: Summary of ligand-protein contacts from MD simulation (top; hydrogen bond is shown with a purple dashed line, π−π interactions as green and cation-π as red lines), and contacts histogram (bottom; green for hydrogen bonds, violet for hydrophobic contacts, blue for water bridges; X-axis represents interaction fraction (1.0 = 100% simulation time), Y-axis represents particular interacting amino acids.

Figure 3

(Left panel): Orientation of E159 during the 250 ns MD simulation. Different colors represent distinct frames: 0 ns is shown in blue, transitioning through the violet spectrum (dark to light: 25–100 ns) to grey (125 ns) and vice versa through the orange spectrum (light to dark: 150–225 ns) to red (250 ns). (Right panel): Time evolution of RMSD for ligand (magenta) and protein (grey blue and dark red) for specific frames relative to the reference frame at 0 ns.

Figure 4

E159 mitigated compulsive grooming in BTBR mice. Both B6 and BTBR mice, were administered an intraperitoneal injection of a vehicle, E159 or ARP before the assessment of self-grooming. BTBR mice exhibited a significantly higher grooming duration in comparison to B6 mice. E159 as well as ARP, significantly decreased self-grooming in the autistic model. Additionally, the impact of co-injection of (R)-α-methylhistamine (MHA) on E159 (2.5 mg/kg)-induced reduction in grooming duration in BTBR mice was evaluated. * p < 0.001 relative to B6 mice treated with vehicle, ^## p < 0.001 relative to BTBR mice treated with vehicle, ^$$ p < 0.01 relative to E159 (2.5 mg)-treated autistic mice, (n = 6). (mean ± SEM, n = 6/group).

Figure 5

E159 treatment enhanced the alternation behavior in autistic mice. A significantly lower alternation behavior was seen in BTBR mice compared to B6 mice. However, E159 or ARP considerably improved the alternation in BTBR mice. The impact of MHA (10 mg/kg) co-administration on the enhancement of alternation behavior induced by E159 (2.5 mg) in autistic mice was evaluated. * p < 0.001 relative to B6 mice treated with vehicle. ** p < 0.01, ^## p < 0.05 relative to BTBR mice treated with vehicle. ^$ p < 0.01 relative to E159 (2.5 mg/kg, i.p.)-treated BTBR mice. (mean ± SEM, n = 6/group).

Figure 6

E159 improved impaired sociability in the autistic mice. Mice explored all three chambers for two consecutive 10 min sessions. Results measured included (A) the Sociability Index (SI) and (B) the Social Novelty Index (SNI). BTBR mice were administered E159 or ARP. The impact of co-administering MHA (10 mg/kg) on the enhancement of SI and SNI induced by 2.5 mg of E159 in the autistic model was evaluated. (A) SI: * p < 0.001 relative to B6 mice treated with vehicle. ** p < 0.001, ^# p < 0.01 relative to BTBR mice treated with vehicle, ^$ p < 0.05 relative to E159 (2.5 mg)-treated BTBR mice. (B) SNI: * p < 0.01 relative to B6 mice treated with vehicle, ** p < 0.01, ^## p < 0.05 relative to BTBR mice treated with vehicle, ^$ p < 0.01 relative to E159 (2.5 mg)-treated BTBR mice, (n = 6). (mean ± SEM, n = 6/group).

Figure 6

E159 improved impaired sociability in the autistic mice. Mice explored all three chambers for two consecutive 10 min sessions. Results measured included (A) the Sociability Index (SI) and (B) the Social Novelty Index (SNI). BTBR mice were administered E159 or ARP. The impact of co-administering MHA (10 mg/kg) on the enhancement of SI and SNI induced by 2.5 mg of E159 in the autistic model was evaluated. (A) SI: * p < 0.001 relative to B6 mice treated with vehicle. ** p < 0.001, ^# p < 0.01 relative to BTBR mice treated with vehicle, ^$ p < 0.05 relative to E159 (2.5 mg)-treated BTBR mice. (B) SNI: * p < 0.01 relative to B6 mice treated with vehicle, ** p < 0.01, ^## p < 0.05 relative to BTBR mice treated with vehicle, ^$ p < 0.01 relative to E159 (2.5 mg)-treated BTBR mice, (n = 6). (mean ± SEM, n = 6/group).

Figure 7

Treatment with E159 had no discernible impact on locomotor ability in autistic mice. (A) BTBR mice showed significantly greater distances travelled relative to B6 mice. (B) Pretreatment with E159 or ARP did not significantly affect the duration spent in periphery in autistic mice. (C) Additionally, BTBR mice spent less duration in the center relative to B6 mice. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01 relative to B6 mice treated with vehicle. ^# p < 0.05, ^## p < 0.01 relative to BTBR mice treated with vehicle. ^$ p < 0.01 versus BTBR mice treated with E159 (2.5 mg) (n = 6).

Figure 8

The proteins p-mTOR, mTOR, LC3A/B and Beclin-1 were determined by Western blotting (A). BTBR mice showed decreased expression level of Beclin-1 and LC3A/B in the cerebellum with increase in the levels of p-mTOR/ mTOR which further suggests autophagic deficiency. E159 ameliorates autophagic deficits with reduced levels of p-mTOR/ mTOR (B), and increased Beclin-1 (C) and LC3A/B (D). ^# p < 0.05 versus B6 mice treated with vehicle. * p < 0.05 versus BTBR mice treated with vehicle. ** p < 0.05,*** p < 0.01 versus E159 (2.5 mg)-treated BTBR mice (n = 3).

Figure 8

The proteins p-mTOR, mTOR, LC3A/B and Beclin-1 were determined by Western blotting (A). BTBR mice showed decreased expression level of Beclin-1 and LC3A/B in the cerebellum with increase in the levels of p-mTOR/ mTOR which further suggests autophagic deficiency. E159 ameliorates autophagic deficits with reduced levels of p-mTOR/ mTOR (B), and increased Beclin-1 (C) and LC3A/B (D). ^# p < 0.05 versus B6 mice treated with vehicle. * p < 0.05 versus BTBR mice treated with vehicle. ** p < 0.05,*** p < 0.01 versus E159 (2.5 mg)-treated BTBR mice (n = 3).