ACSL3 is a promising therapeutic target for alleviating anxiety and depression in Alzheimer's disease

Abstract

Alzheimer's disease (AD), the leading cause of dementia, affects over 55 million people worldwide and is often accompanied by depression and anxiety. Both significantly impact patients' quality of life and impose substantial societal and economic burdens on healthcare systems. Identifying the complex regulatory mechanisms that contribute to the psychological and emotional deficits in AD will provide promising therapeutic targets. Biosynthesis of omega-3 (ω3) and omega-6 fatty acids (ω6-FA) through long-chain acyl-CoA synthetases (ACSL) is crucial for cell function and survival. This is due to ω3/6-FA's imperative role in modulating the plasma membrane, energy production, and inflammation. While ACSL dysfunction is known to cause heart, liver, and kidney diseases, the role of ACSL in pathological conditions in the central nervous system (e.g., depression and anxiety) remains largely unexplored. The impact of ACSLs on AD-related depression and anxiety was investigated in a mouse model of Alzheimer's disease (3xTg-AD). ACSL3 levels were significantly reduced in the hippocampus of aged 3xTg-AD mice (via capillary-based immunoassay). This reduction in ACAL3 was closely associated with increased depression and anxiety-like behavior (via forced swim, tail suspension, elevated plus maze, and sucrose preference test). Upregulation of ACSL3 via adenovirus in aged 3xTg-AD mice led to increased protein levels of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor C (VEGF-C) (via brain histology, capillary-based immunoassay), resulting in alleviation of depression and anxiety symptoms. The present study highlights a novel neuroprotective role of ACSL3 in the brain. Targeting ACSL3 will offer an innovative approach for treating AD-related depression and anxiety.

Keywords: Alzheimer’s disease; Anxiety; Brain-derived neurotrophic factor; Depression; Long-chain acyl-CoA synthetases; Vascular endothelial growth factor C.

Fig. 1

Expression of ACSL3 was significantly decreased in the brain of aged 3xTg-AD female mice, while treatment with AAV-ACSL3 or GW3965 increased ACSL3 protein levels (A) Relative protein levels of ACSL3 in the hippocampus of 3xTg-AD mice were measured by capillary-based immunoassay. ACSL3 bands at 66 kDa. Results were normalized with total protein and summarized in panel B. (C) Colocalization of ACSL3-immunoreactivity with NeuN in the mouse hippocampus and cortex. Neurons were stained for ACSL3 (green) and the pan-neuronal marker NeuN (red). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI, blue). Scale bar = 100 μm. The vast majority of the neurons are positive for ACSL3. (D) GFP fluorescent imaging of a coronal brain section 30 days after a single retro-orbital injection of AAV-ACSL3. Scale bar = 500 µm. Green fluorescence indicates the presence of GFP-tagged AAV-ACSL3 particles. *p ≤ 0.05 indicates significantly different versus aged 3xTg-AD female mice, evaluated by one-way ANOVA with Tukey’s post-hoc

Fig. 2

Overexpression of ACSL3 via AAV-ACSL3 alleviated depression-like behavior in 3xTg-AD mice. Aged female 3xTg-AD mice (9–12 months) received single retro-orbital injection of AAV-ACSL3 (1 × 10¹¹ Viral particles). Forced swimming test (A) and tail suspension test (B) were implemented in mice 30 days after AAV-ACSL3 treatment. Results from forced swimming and tail suspension tests were summarized in panels B and D, respectively. *p ≤ 0.05 versus aged 3xTg-AD female mice, evaluated by one-way ANOVA with Tukey’s post-hoc

Fig. 3

Anxiety-like behavior of aged 3xTg-AD mice was alleviated with AAV-ACSL3 treatment. The elevated plus maze (A) and sucrose preference test (C) were used to study the impact of ACSL3 on anxiety-like behavior in young (1–3 months old) and aged 3xTg-AD (9–12 months old) mice with and without AAV-ACSL3 treatment. Results from elevated plus maze and sucrose preference test were summarized in panels B and D. *p ≤ 0.05 versus aged 3xTg-AD female mice via one-way ANOVA with Tukey’s post-hoc

Fig. 4

BDNF and VEGFc protein levels were reduced in aged 3xTg-AD mice but increased following AAV-ACSL3 treatment. (A) Representative immunofluorescence images of BDNF (green) and VEGF-C (red) in the CA1 region of the hippocampus and cortex. Quantification of the results were summarized in panels B and C. Scale bar = 50 µm. *p ≤ 0.05 versus aged 3xTg-AD female mice via one-way ANOVA with Tukey’s post-hoc

Fig. 5

Overexpression of ACSL3 via AAV increased protein levels of proBDNF and VEGF-C in aged 3xTg-AD female mice. (A) Protein levels of pro and mature BDNF in the hippocampus of AD mice were measured via ELISA according to the manufacturer’s instructions. (B) Relative protein levels of VEGF-C in the hippocampus of 3xTg-AD mice were measured by capillary-based immunoassay. VEGF-C bands at 47 kDa. VEGF-C protein levels were normalized with total protein and summarized in panel C. *p ≤ 0.05 versus aged 3xTg-AD female mice via one-way ANOVA with Tukey’s post-hoc

Fig. 6

Gene expression profiling revealed that BDNF signaling pathways were involved in ACSL3-mediated anti-depression and anti-anxiety effects. RNA-sequencing of the mouse hippocampus was utilized to identify potential pathways involved ACSL3-mediated neuroprotection against AD. (A) Heatmap of 65 anxiety-related genes was generated from seven distinct mouse hippocampal tissues (three 3xTg-AD and four 3xTg-AD + AAV-ACSL3). (B) Differential gene expression was analyzed by Ingenuity Pathway Analysis (IPA). Genes shaded in green represent downregulated genes, while those in red are upregulated. Dashed lines indicate indirect interactions between genes. Blue and orange lines predict inhibition and activation, respectively. Grey lines indicate that the effects between two genes are not predicted based on the Ingenuity Knowledge Base