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. 2021 Aug 31;6(1):325.
doi: 10.1038/s41392-021-00748-4.

A novel missense variant in ACAA1 contributes to early-onset Alzheimer's disease, impairs lysosomal function, and facilitates amyloid-β pathology and cognitive decline

Rongcan Luo #  1 , Yu Fan #  2 , Jing Yang  2   3 , Maosen Ye  2   3 , Deng-Feng Zhang  2 , Kun Guo  4 , Xiao Li  2   3 , Rui Bi  2 , Min Xu  2 , Lu-Xiu Yang  2 , Yu Li  2   3 , Xiaoqian Ran  2   3 , Hong-Yan Jiang  5 , Chen Zhang  6 , Liwen Tan  7 , Nengyin Sheng  4   8 , Yong-Gang Yao  9   10   11
Affiliations

A novel missense variant in ACAA1 contributes to early-onset Alzheimer's disease, impairs lysosomal function, and facilitates amyloid-β pathology and cognitive decline

Rongcan Luo et al. Signal Transduct Target Ther. .

Abstract

Alzheimer's disease (AD) is characterized by progressive synaptic dysfunction, neuronal death, and brain atrophy, with amyloid-β (Aβ) plaque deposits and hyperphosphorylated tau neurofibrillary tangle accumulation in the brain tissue, which all lead to loss of cognitive function. Pathogenic mutations in the well-known AD causal genes including APP, PSEN1, and PSEN2 impair a variety of pathways, including protein processing, axonal transport, and metabolic homeostasis. Here we identified a missense variant rs117916664 (c.896T>C, p.Asn299Ser [p.N299S]) of the acetyl-CoA acyltransferase 1 (ACAA1) gene in a Han Chinese AD family by whole-genome sequencing and validated its association with early-onset familial AD in an independent cohort. Further in vitro and in vivo evidence showed that ACAA1 p.N299S contributes to AD by disturbing its enzymatic activity, impairing lysosomal function, and aggravating the Aβ pathology and neuronal loss, which finally caused cognitive impairment in a murine model. Our findings reveal a fundamental role of peroxisome-mediated lysosomal dysfunction in AD pathogenesis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
The ACAA1 c.896T>C (p.N299S) variant identified in a Han Chinese pedigree with familiar AD disturbed acyltransferase activity. a Pedigree of a Han Chinese family with AD. Individuals who underwent whole-genome sequencing are indicated by asterisks (*). The subject with heterozygous or homozygous allele of rs117916664 was marked by T/C or C/C in the pedigree. b ACAA1 p.N299S protein (N299S) has reduced enzyme activity compared to the wild-type ACAA1 (WT). Purified ACAA1 p.N299S and ACAA1 WT were used for detection of acetyltransferase activity, with ACAA1 WT as the reference for normalization (n = 3 biological replicates for each group). Results are mean ± SD. **P < 0.01, Student’s t test
Fig. 2
Fig. 2
Overexpression of ACAA1 p.N299S disturbed global gene expression pattern and inhibited lysosomal and synaptic proteins in human cells. a KEGG pathway and GO biological processes analyses of differentially expressed genes in the HM and U251-APP cells overexpressing ACAA1 p.N299S and ACAA1 WT. b Enrichment of neuron development genes in cells with overexpression of ACAA1 p.N299S versus ACAA1 WT (upper, HM cells; below, U251-APP cells) based on gene set enrichment analyses (GSEA). c Overexpression of ACAA1 p.N299S versus ACAA1 WT affects co-expression network constructed using human AD brain tissues. d, e Overexpression of ACAA1 p.N299S in HM (d) and U251-APP cells (e) reduced the levels of lysosomal and postsynaptic proteins and increased LC3-II:LC3-I ratio and SQSTM1 protein level. The GAPDH was used as the loading control. Data are representative of three independent experiments with similar results. Bars represent mean ± SD of the three experiments. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test
Fig. 3
Fig. 3
ACAA1 p.N299S aggravated memory impairments in APP/PS1ΔE9 mice. a Impaired habituation in the exploratory open field for APP/PS1 mice (8-month-old) and wild-type littermates with delivery of AAV-ACAA1 N299S versus AAV-ACAA1 WT or empty vector. The changes of distance traveled on days 2 and 3 were normalized to the distance traveled on day 1 of training. Data are mean ± SEM. ns, not significant; *P < 0.05; **P < 0.01, two-way repeated-measures ANOVA. b ACAA1 p.N299S accelerated memory retrieval impairment of APP/PS1 and WT mice in fear conditioning tests. Shown data are the percentages of freezing time after 1 day (left) and 7 days (right) of electric shocks. Data are mean ± SEM. ns, not significant; *P < 0.05; **P < 0.01, one-way ANOVA with the Tukey’s post hoc test. c, d Morris water maze tests of APP/PS1ΔE9 mice or WT littermates with delivery of AAV empty vector or AAV-mediated expression of ACAA1 WT and ACAA1 N299S. The APP/PS1ΔE9 mice or WT littermates with AAV-Vector, AAV-ACAA1 WT, or AAV-ACAA1 N299S injection showed differences in escape latency, path length, and swim speed during learning session (c) and in probe trial performance at 4 h (short-term memory; left panel) and at 72 h (right panel) (d). TQ target quadrant (percentage of time and percentage of distance in the target quadrant), OQ opposite quadrant. Bars represent mean ± SEM. *P < 0.05; **P < 0.01; one-way ANOVA with the Tukey’s post hoc test
Fig. 4
Fig. 4
Overexpression of ACAA1 p.N299S via adeno-associated virus delivery exacerbated amyloid-β (Aβ) pathology. APP/PS1∆E9 mice and WT littermates were injected with AAV-Vector, AAV-ACAA1 WT, or AAV-ACAA1 N299S at 3 months of age and sacrificed at 9 months of age. a Fluorescent signals in the brain section of a 9-month-old mouse receiving AAV-Vector, AAV-ACAA1 WT, or AAV-ACAA1 N299S. b Quantification of soluble and insoluble Aβ40 and Aβ42 in the hippocampus and cortex tissues of mice in a by ELISA. c, d Representative microphotographs of hippocampal sections stained with an 4G8-specific Aβ antibody (c), and quantitative analysis of the number of Aβ plaques shown by 4G8 immunoreactivity in hippocampal and cortex tissues in APP/PS1ΔE9 mice (d). Bars represent mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test
Fig. 5
Fig. 5
Overexpression of ACAA1 p.N299S in APP/PS1ΔE9 mice and WT littermates induced neuronal losses. a, b Neuronal loss in the CA3 region of hippocampus in WT littermates (a) and APP/PS1ΔE9 mice (b) after delivery of the AAV-Vector, AAV-ACAA1 WT, and AAV-ACAA1 N299S. Higher neuronal density, marked by NeuN antibody, was noted in mice overexpressing ACAA1 p.N299S, suggesting a potential decrease of neurogenesis or an increase of neuron loss. Immunostaining of brain sections was performed using NeuN antibody. Shown results are representative hematoxylin and eosin (H&E) staining (top), Nissl staining (middle), and NeuN staining (bottom) of mouse hippocampus tissues in each group. c, d Decreased protein levels of structural plasticity markers SYP, PSD95, NeuN, GluR1, GluR1 (pS831), and GRIN2B in the hippocampus tissues of WT littermates (c) and APP/PS1ΔE9 (d) mice with AAV-mediated expression of ACAA1 and its mutant. Bars represent mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; Student’s t test
Fig. 6
Fig. 6
Effect of ACAA1 p.N299S on Aβ pathology was mediated by lysosomal dysfunction. a, b Western blot analysis of lysosomal proteins LAMP1 and LAMP2A and autophagy markers LC3-II:LC3-I ratio and SQSTM1 in the hippocampus tissues of WT littermates (a) and APP/PS1∆E9 (b) injected with AAV-ACAAA WT or AAV-ACAA1 N299S. Bars represent mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test. (c) ACAA1 knockout (KO) or overexpression of ACAA1 p.N299S in U251-APP cells decreased NAG activity. Cells were treated with BAFA1 (200 nM), NH4CL (10 mM), or without treatment (Control) or were transfected with empty vector (Vector) and expression vector of ACAA1 WT, or ACAA1 p.N299S. d Levels of extracellular Aβ42, Aβ40, and Aβ42:Aβ40 ratio in the culture supernatants of U251-APP cells and U251-APP ACAA1 KO cells. e–g Overexpression of ACAA1 WT, but not ACAA1 p.N299S, in U251-APP ACAA1 KO cells had a rescuing effect on the altered protein levels of autophagy markers (e), lysosomal markers (f), and the levels of extracellular Aβ42 and Aβ40 in the culture supernatant (g). The U251-APP cells without ACAA1 knockout was used as a control, and cells were transfected with empty vector (Vector) and expression vector of ACAA1 WT or ACAA1 p.N299S. Data in e–g were based on three independent experiments. Bars represent mean ± SD. ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test
Fig. 7
Fig. 7
ACAA1 p.N299S disturbs its physiological regulation of excitatory synaptic transmission. a–d Rat hippocampal slice cultures were biolistically transfected with expression vector of ACAA1 WT or ACAA1 p.N299S. Simultaneous dual whole-cell recordings were performed in a transfected CA1 pyramidal neuron (green trace) and a neighboring wild-type one (black trace). The evoked AMPA (a, b) and NMDA (c, d) EPSCs were measured, and open and filled circles represent amplitudes for single pairs and mean ± SEM, respectively. Sample current traces from control (black) and experimental (green) cells are shown as insets. Bar graphs show normalized EPSC amplitudes (mean ± SEM) of −70 mV (a, ***P < 0.001; b, P > 0.05) and +40 mV (c, **P < 0.005; d, P > 0.05) presented in scatter plots. The scale bars for representative EPSC traces are 100 pA/25 ms (a) and 50 pA/25 ms (b–d). e, f Difference of AMPA/NMDA ratios recorded from neurons overexpressing ACAA1 WT (*P < 0.05) or ACAA1 p.N299S (P > 0.05) compared to the respective wild-type (Control) ones. g, h No change in paired-pulse ratio of the second EPSC over the first EPSC from neurons overexpressing ACAA1 WT (P > 0.05) or ACAA1 p.N299S (P > 0.05) relative to the control neurons. All the statistical differences are estimated relative to the respective control neurons, with a two-tailed Wilcoxon signed-rank sum test
Fig. 8
Fig. 8
A proposed role of the ACAA1 p.N299S-mediated lysosomal dysfunction and impaired autophagy in the development of AD. ACAA1 p.N299S has an impaired enzymatic activity, affects autophagy and lysosomal function, subsequently contributes to the aggravation of the Aβ pathology and neuronal loss, and finally causes cognitive impairment and other AD-related symptoms
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