Endoplasmic reticulum stress in wake-active neurons progresses with aging

Abstract

Fragmentation of wakefulness and sleep are expected outcomes of advanced aging. We hypothesize that wake neurons develop endoplasmic reticulum dyshomeostasis with aging, in parallel with impaired wakefulness. In this series of experiments, we sought to more fully characterize age-related changes in wakefulness and then, in relevant wake neuronal populations, explore functionality and endoplasmic reticulum homeostasis. We report that old mice show greater sleep/wake transitions in the active period with markedly shortened wake periods, shortened latencies to sleep, and less wake time in the subjective day in response to a novel social encounter. Consistent with sleep/wake instability and reduced social encounter wakefulness, orexinergic and noradrenergic wake neurons in aged mice show reduced c-fos response to wakefulness and endoplasmic reticulum dyshomeostasis with increased nuclear translocation of CHOP and GADD34. We have identified an age-related unfolded protein response injury to and dysfunction of wake neurons. It is anticipated that these changes contribute to sleep/wake fragmentation and cognitive impairment in aging.

Fig. 1. Age-dependent characteristics of baseline wakefulness

A: hourly percentiles ± standard error of wakefulness in young (2 mos, blue line) and old (24 mos, red line) mice. An insert of the average wake time for the lights on and lights off periods for both age groups are presented. No significant differences are observed in these parameters. B: sleep wake histograms for a representative young (upper) and old (lower) mouse across the mid dark period, the time of greatest age difference in wake time. Red lines highlight wake bouts. C: Wake bout numbers and duration are present as mean ± standard error for the lights on and lights off periods. Lines delineate age-dependent differences, * = p<0.05 and ** = p<0.01.

Fig. 2. Murine multiple sleep latency responses and wake response to novel social encounter

A. Mean sleep latency across four nap opportunities at the end of the rest period in young (black) and old (gray) mice for conditions of undisturbed sleep (baseline) and after 6 hours of EEG monitored enforced wakefulness. Lines denote differences, *=p<0.05, **=p<0.01. B. Average percent wakefulness across a 12 hour (lights off period) novel social encounter, where a non littermate male mouse was placed in the cage. Young mice (black bars) showed an increase in percentage of time awake from baseline (control) to novel encounter, p=0.001. No difference in wake time was observed in older mice (gray bars) across baseline and social encounter. Young mice, relative to older mice, showed increased wake time, p<0.001.

Fig 3. C-fos response to wakefulness

A. Representative images of c-fos responses in orexinergic neurons (green) with c-fos (red). Arrows highlight some of the c-fos positive nuclei. Rare weakly positive c-fos (arrows) nuclei are observed in aged mice. B. Average percentiles of c-fos positive nuclei in orexinergic (left) and noradrenergic locus coeruleus (right) neurons in mice left undisturbed (control) and those with enforced wakefulness. Young mice (black bars, n=5) showed marked increases in c-fos immunoreactivity in both wake groups in response to enforced wakefulness, paired t, p<0.001. In contrast old mice showed no difference in either group in response to wakefulness. In old vs. young mice, the percentage of orexinergic and noradrenergic neurons c-fos + with enforced wakefulness was markedly diminished, non-paired t, p<0.05.

Fig. 4. PERK activation in wake neurons

Phosphorylation of PERK (p-PERK), indicative of an unfolded protein response, is evident in both orexinergic lateral hypothalamic and noradrenergic locus coeruleus neurons. A. Representative image of p-PERK (red) in orexinergic (green) neurons. Left image, 2 month (mo) old mouse; right 24 mo old mouse. Black arrows highlight neurons deemed positive with p-PERK labeling. B. Average percentages of orexinergic neurons p-PERK positive ± SE in young (black columns) and old (gray columns), * denotes p<0.05.

Fig 5. Uncompensated ER response in wake neurons

Upper panel: representative images of CHOP, (GADD153), a pro-apoptotic protein in orexinergic neurons and mean ± SE immunodensity data. Arrows delineate nuclei with CHOP translocation. Lower panel: GADD34 (red), a marker of CHOP activation co-localizes in orexinergic (green) neurons. Orexin is predominantly in the ER and golgi (centralized in neurons, while GADD34 is evident throughout the cytoplasm. Lower histogram: percentage of orexinergic neurons with GADD34 is presented, *=p<0.05. Increased CHOP and GADD34 are also evident in non-orexinergic cells in the lateral hypothalamus in aged mice.