Genome-wide association analysis identifies APOE as a mitophagy modifier in Lewy body disease

Abstract

Introduction: Phosphorylated ubiquitin (p-S65-Ub) is generated during PINK1-PRKN mitophagy as a specific marker of mitochondrial damage. Despite the widespread deposition of p-S65-Ub in aged and diseased human brain, the genetic contribution to its accumulation remains unclear.

Methods: To identify novel mitophagy regulators, we performed a genome-wide association study using p-S65-Ub level as a quantitative trait in 1012 autopsy-confirmed Lewy body disease (LBD) samples.

Results: We identified a significant genome-wide association with p-S65-Ub for rs429358 (apolipoprotein E ε4 [APOE4]) and a suggestive association for rs6480922 (ZMIZ1). APOE4 was associated with higher p-S65-Ub levels and greater neuropathological burden. Functional validation in mouse and human induced pluripotent stem cell (iPSC) models confirmed APOE4-mediated mitophagy alterations. Intriguingly, ZMIZ1 rs6480922 was associated with lower p-S65-Ub levels, reduced neuropathological load, and increased brain weight, indicating a potential protective role.

Discussion: Our findings underscore the importance of mitochondrial quality control in LBD pathogenesis and nominate regulators that may contribute to disease risk or resilience.

Highlights: p-S65-Ub levels were used as a quantitative marker of mitochondrial damage. A GWAS identified two genetic variants that modify mitophagy in LBD autopsy brain. APOE4 was associated with increased p-S65-Ub accumulation and neuropathology. APOE4 altered mitophagy via pathology-dependent and pathology-independent mechanisms. ZMIZ1 was linked to reduced p-S65-Ub and neuropathology indicative of protection.

Keywords: GWAS; PINK1; PRKN; Parkin; Parkinson's disease; ZMIZ1; autophagy; mitochondria; phosphorylated ubiquitin; ubiquitin.

FIGURE 1

Workflow of GWAS analysis using hippocampal p‐S65‐Ub levels as quantitative trait. A total of 1021 autophagy brains with pathological confirmed LBD were obtained from the brain bank. Hippocampal region was dissected from the fixed hemibrain during autopsy and immunostained for p‐S65‐Ub by autostainer. Stained hippocampal sections were then scanned and p‐S65‐Ub‐positive cell density were quantified using unbiased digital pathology. Representative images of p‐S65‐Ub‐positive cells and levels of hippocampal p‐S65‐Ub cell density in control and LBD cases were shown. DNA was extracted from frozen cerebellar tissue from the same cohort. In the discovery phase, genome‐wide SNP array was performed in 754 LBD cases. p‐S65‐Ub levels were used a quantitative trait for genome‐wide association analysis to identify regulators of PINK1‐PRKN‐directed mitophagy. In the replication phase, significant SNPs were genotyped in the remaining 258 LBD cases. Finally, a combined analysis was performed in the entire cohort. This figure was created with BioRender.com. GWAS, genome‐wide association study; LBD, Lewy body disease; p‐S65‐Ub, phosphorylated ubiquitin.

FIGURE 2

GWAS analysis identified two distinct mitophagy modifiers in LBD autopsy brain. (A) Manhattan plot showing genome‐wide p values of association with p‐S65‐Ub levels. Y‐axis: −log10 p values of 8,696,291 SNPs; x‐axis: their chromosomal positions; red solid line: p value threshold used to define genome‐wide significant association (p < 5 × 10⁻⁸); blue solid line: p value threshold used to define “suggestive” association (p < 1 × 10⁻⁶). (B and C) Locus Zoom plot showing association of variants at APOE (B) and ZMIZ1 (C) loci with p‐S65‐Ub levels. The most significant variants (rs429358 and rs6480922) are indicated in purple. The association p value is shown on the y‐axis and linear position on the chromosome on the x‐axis. Each point on plot represents one variant; colors of points indicate linkage disequilibrium (r ²) value with index variant (rs429358 or rs6480922). Points that are missing linkage disequilibrium information are shown in gray. Our analysis code is available at https://github.com/ORossLab/PhosphoUb‐GWAS. GWAS, genome‐wide association study; LBD, Lewy body disease; p‐S65‐Ub, phosphorylated ubiquitin.

FIGURE 3

Significant association of APOE and ZMIZ1 variants with p‐S65‐Ub levels, brain weight, and neuropathological measures in LBD autopsy brain. (A) Violin plot of pS65‐Ub level (natural logarithm scale) according to number of minor alleles of APOE rs429358 and ZMIZ1 rs6480922 in combined (discovery and replication) series, together with the corresponding representative images of p‐S65‐Ub staining in hippocampus in LBD cases. Scale bar: 50 µm. (B) Violin plot of brain weight, αSyn burden (by NACP staining) (natural logarithm scale) and densities of SPs and NFTs (count per microscope field) (cube root scale) according to number of minor alleles of APOE rs429358 and ZMIZ1 rs6480922 in the combined (discovery and replication) series. The associations were examined using linear regression models with adjustment for age at death and sex. αSyn, alpha‐synuclein; β, regression coefficient beta; LBD, Lewy body disease; NFT, neurofibrillary tangle; p‐S65‐Ub, phosphorylated ubiquitin; SP, senile plaque.

FIGURE 4

Functional validation of APOE4 effect on mitophagy changes in mouse and iPSC models. (A) Brain lysates from 3‐ and 22‐month‐old APOE‐TR mice are used to measure p‐S65‐Ub levels. (B) MSD shows significantly increased p‐S65‐Ub levels in brain lysates from 3‐months‐old APOE4 mice compared to age‐matched APOE3 mice. An age‐dependent increase of p‐S65‐Ub levels is also observed in APOE3 mice. n = 7 to 8 mice/genotype/age group. (C) Dermal fibroblasts collected from individuals carrying homozygous APOE3 or APOE4 are converted into iPSCs, which are then differentiated into astrocytes. (D) Representative Western blot images of human iPSC‐derived astrocytes. (E) Western blot results showed that human iPSC‐derived homozygous APOE4 astrocytes have significantly increased p‐S65‐Ub levels but similar PINK1 levels compared to homozygous APOE3 astrocytes upon mitochondrial depolarization (8 h CCCP treatment). n = 3 independent differentiation experiments. Unpaired t‐tests were used for comparison between APOE3 and APOE4 groups, and p < 0.05 was considered statistically significant. Panels (A) and (C) were created with BioRender.com. APOE‐TR, APOE‐targeted replacement; CCCP, carbonyl cyanide m‐chlorophenyl hydrazone; iPSC, induced pluripotent stem cell; p‐S65‐Ub, phosphorylated ubiquitin.