Aging Disrupts the Circadian Patterns of Protein Expression in the Murine Hippocampus

Abstract

Aging is associated with cognitive decline and dysregulation of the circadian system, which modulates hippocampal-dependent memory as well as biological processes underlying hippocampal function. While circadian dysfunction and memory impairment are common features of aging and several neurodegenerative brain disorders, how aging impacts the circadian expression patterns of proteins involved in processes that underlie hippocampal-dependent memory is not well understood. In this study, we profiled the hippocampal proteomes of young and middle-aged mice across two circadian cycles using quantitative mass spectrometry in order to explore aging-associated changes in the temporal orchestration of biological pathways. Of the ∼1,420 proteins that were accurately quantified, 15% (214 proteins) displayed circadian rhythms in abundance in the hippocampus of young mice, while only 1.6% (23 proteins) were rhythmic in middle-aged mice. Remarkably, aging disrupted the circadian regulation of proteins involved in cellular functions critical for hippocampal function and memory, including dozens of proteins participating in pathways of energy metabolism, neurotransmission, and synaptic plasticity. These included processes such as glycolysis, the tricarboxylic acid cycle, synaptic vesicle cycling, long-term potentiation, and cytoskeletal organization. Moreover, aging altered the daily expression rhythms of proteins implicated in hallmarks of aging and the pathogenesis of several age-related neurodegenerative brain disorders affecting the hippocampus. Notably, we identified age-related alterations in the rhythmicity of proteins involved in mitochondrial dysfunction and loss of proteostasis, as well as proteins involved in the pathogenesis of disorders such as Alzheimer's disease and Parkinson's disease. These insights into aging-induced changes in the hippocampal proteome provide a framework for understanding how the age-dependent circadian decline may contribute to cognitive impairment and the development of neurodegenerative diseases during aging.

Keywords: aging; circadian; hippocampus; mass spectrometry; neurodegenerative diseases; proteomics.

FIGURE 1

Aging disrupts the hippocampal circadian proteome. (A) Experimental design and workflow of the MS-based analysis of proteins extracted from hippocampal tissues of young (9–10 weeks old) and middle-aged (44–52 weeks old) C57BL/6J mice. Samples were collected every 4 h over 2 days, and proteins extracted from tissues of individual mice were digested with trypsin, fractionated, and analyzed by an Orbitrap Elite mass spectrometer. (B) Proteome coverage: Venn diagram displaying the number of proteins quantified in at least two biological replicates per time point in young or middle-aged mice and overlap between ages. (C) Circadian proteins detected in young or middle-aged mice using the Perseus periodicity algorithm (period = 23.6 h; q-value < 0.25). (D) Percent distribution of circadian proteins based on changes in rhythmicity during aging. (E) Heatmaps displaying z-score normalized abundances (log₁₀ LFQ intensities) of circadian proteins detected in young mice and their temporal expression profiles in young mice (left) and middle-aged mice (right). (F) Phase distribution of circadian proteins detected in young mice (green) or middle-aged mice (red). (G) Correlation heatmaps across 48 h in young mice (left) and middle-aged mice (right) for circadian proteins detected in young mice. Pearson correlation coefficients are shown as red (positive) or blue (negative).

FIGURE 2

Aging leads to loss of circadian oscillations in specific biological functions in the hippocampus. (A–C) Functional analysis of proteins displaying loss of circadian rhythmicity with aging. Enriched ontology terms corresponding to (A) KEGG pathways, (B) GO biological processes, and (C) GO cellular components are shown with colored bars (p ≤ 0.05, Fisher’s exact test relative to background of accurately quantified proteins in our dataset).

FIGURE 3

Aging disrupts the circadian regulation of proteins involved in energy metabolism in the hippocampus. (A) Schematic depiction of proteins involved in energy metabolism, including glycolysis, pyruvate metabolism, the TCA cycle, and oxidative phosphorylation. Proteins that displayed loss or gain of circadian rhythmicity in abundance during aging are highlighted in color. Proteins that were reliably quantified, but not detected as circadian at either age, are shown in gray. (B) Temporal abundance profiles of rhythmic proteins identified in young mice involved in energy metabolism.

FIGURE 4

Aging disrupts the circadian regulation of proteins involved in synaptic function in the hippocampus. (A) Schematic depiction of proteins involved in synaptic structure and function, including the synaptic vesicle cycle, LTP and AMPAR regulation, and the cytoskeleton. Proteins that displayed loss or gain of circadian rhythmicity in abundance during aging are highlighted in color. Proteins that were reliably quantified, but not detected as circadian at either age, are shown in gray. (B) Temporal abundance profiles of rhythmic proteins identified in young mice involved in synaptic structure and function.

FIGURE 5

Aging alters hippocampal circadian oscillations of proteins implicated in Alzheimer’s disease and hallmarks of aging. Proteins displaying changes in rhythmicity during aging and involved in hallmarks of aging and Alzheimer’s disease pathogenesis: genomic instability, epigenetic alterations, loss of proteostasis, glucose and mitochondrial dysfunction, synaptic dysfunction, and cytoskeletal abnormalities.