Humanin variant P3S is associated with longevity in APOE4 carriers and resists APOE4-induced brain pathology

Abstract

The APOE4 allele is recognized as a significant genetic risk factor to Alzheimer's disease (AD) and influences longevity. Nonetheless, some APOE4 carriers exhibit resistance to AD even in advanced age. Humanin, a mitochondrial-derived peptide comprising 24 amino acids, has variants linked to cognitive resilience and longevity. Our research uncovered a unique humanin variant, P3S, specifically enriched in centenarians with the APOE4 allele. Through in silico analyses and subsequent experimental validation, we demonstrated a strong affinity between humanin P3S and APOE4. Utilizing an APOE4-centric mouse model of amyloidosis (APP/PS1/APOE4), we observed that humanin P3S significantly attenuated brain amyloid-beta accumulation compared to the wild-type humanin. Transcriptomic assessments of mice treated with humanin P3S highlighted its potential mechanism involving the enhancement of amyloid beta phagocytosis. Additionally, in vitro studies corroborated humanin P3S's efficacy in promoting amyloid-beta clearance. Notably, in the temporal cortex of APOE4 carriers, humanin expression is correlated with genes associated with phagocytosis. Our findings suggest a role of the rare humanin variant P3S, especially prevalent among individuals of Ashkenazi descent, in mitigating amyloid beta pathology and facilitating phagocytosis in APOE4-linked amyloidosis, underscoring its significance in longevity and cognitive health among APOE4 carriers.

Keywords: Alzheimer's disease; humanin; longevity; microprotein; mitochondrial DNA variation; small open reading frame.

FIGURE 1

The humanin variant P3S is enriched in APOE4 centenarians. (a) Mitochondrial genomic location of the humanin smORF and the P3S mutation. The frequency of P3S is below the location schematic. Below the frequency is the amino acid sequence of humanin and the 3rd residue change caused by P3S; n = 146. (b) APOE4 frequency as a function of age in the Albert Einstein Cohort (black line). The frequency of humanin P3S at age 100 in APOE4 was 30% (pink highlighted dot); n = 1139. (c) Pie graph illustrating enrichment of humanin P3S in APOE4 centenarians (p < 0.002; chi squared test); n = 146. (d) Box plot illustrating MMSE scores in individuals without and with APOE4, colored by those without (gray) and with (pink) P3S. APOE4 carriers with P3S had higher MMSE scores compared to APOE4 carriers without P3S (p < 0.05; nonparametric Mann–Whitney test).

FIGURE 2

Humanin binds APOE, but humanin P3S binds APOE4 with much higher affinity. (a, b) Surface plasmon resonance of APOE4:Humanin and APOE4:P3S, respectively, with kDs of 12.7 and 0.69. X axis is time in seconds and Y axis is RU. (c) Dot blow assay of human variants immobilized to a nitrocellulose membrane with APOE4 flow over. (d) MD simulation reveals that humanin P3S binds to APOE4 with both its C‐ (Asp35) and N‐terminus (Ser3). This is due, in part, to differences in the surface display of APOE Glu27 and Asp35.

FIGURE 3

Humanin P3S reduces brain Aβ burden in APP/PS1/APOE4 mice. Humanin wild type and P3S immuno‐based reduction of (a) hippocampal and (c) cortical amyloid beta (p < 0.05; Mann–Whitney test) with representative images above bar graphs. Humanin wild type and P3S thioflavin‐based reduction of (b) hippocampal and (d) cortical amyloid beta (p < 0.05; Mann–Whitney test) with representative images above the bar graphs, derived from the same field as HRP‐adjacent images in (a) and (c). Vehicle n = 12; Humanin wild type n = 16; Humanin P3S n = 16.

FIGURE 4

Humanin and humanin P3S differentiates the hippocampal transcriptome in APP/PS1/APOE4 mice. (a) Transcriptomic principal component analysis of vehicle (n = 5), humanin wild type (n = 5), and humanin P3S conditions (n = 3). (b) Venn diagram illustration showing the number of significant differentially expressed genes by humanin P3S and humanin wild type (false discovery rate <0.2). (c) WikiPathway enrichment of significant terms from humanin‐ and humanin‐P3S‐treated mice.

FIGURE 5

Humanin P3S increases Aβ phagocytosis in APOE4 glia. (a) Humanin P3S induces greater Aβ phagocytosis compared to humanin wild type (p < 0.05; nonparametric Mann–Whitney test). (b) Visual representation of panel.

FIGURE 6

Gene set enrichment analysis of humanin co‐expression with phagocytosis‐related genes. A total of 42 post‐mortem temporal cortex bulk transcriptomics (n Male = 14; n Female = 28; average age at death = 81.5 years) samples were analyzed.