Phosphoproteome Profiling Reveals Circadian Clock Regulation of Posttranslational Modifications in the Murine Hippocampus

Abstract

The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior, and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by posttranslational modifications, in particular, phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short-to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry (MS). Of the 3,052 proteins and 2,868 phosphosites on 1,368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphorylation events exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative MS analysis of the circadian phosphoproteome and proteome of the murine hippocampus and highlights the significance of rhythmic regulation at the posttranslational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.

Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology

“…Moreover, several recent phospho-proteomics studies indicate daily variation in the phosphorylation of WNK isoforms in several tissues, including cultured fibroblasts 44 , mouse liver 12 and forebrain 27 ( Supplementary Fig. 4b-d), as well as rhythms in the phosphorylation of NKCC and KCC isoforms 27,31 , suggesting that similar mechanisms operate in vivo.…”

“…The activity of OXSR1 itself is regulated upstream through phosphorylation by lysine deficient protein kinases family members (WNKs), which are stimulated by molecular crowding and low cytosolic Cl - [28][29][30] . Daily variation in the phosphorylation of WNK isoforms has been reported in mouse liver 12 and forebrain 27 , as has the phosphorylation of several members of the SLC12A family 27,31 .…”

Physiological and cognitive consequences of a daily 26h photoperiod in a primate(M. murinus)

“…It is well documented that a strong link exists between circadian clock and cerebral performances. Through its link with the hippocampus (Borgs et al, 2009; Chiang et al, 2017; Schnell et al, 2014, Snider et al, 2018), the circadian system influences mood, learning, time-place association and memory in laboratory mice (Albrecht, 2017; Cain et al, 2004; Legates et al, 2012; Ruby et al, 2008), and the involvement of clock genes is well established (Snider et al, 2016; Van der Zee et al, 2008; Wang et al, 2009). Furthermore, numerous data indicate that circadian disorganization (jet-lag, phase shifts, aging alterations, sleep impairments, shift work…) invariably leads to impaired cognitive performances which suggests that clock resynchronization indirectly impacts cognitive capacities (Antoniadis et al, 2000; Chellappa et al, 2018; Gibson et al, 2010; Krishnan and Lyons, 2015; Loh et al, 2010; Rouch et al, 2005).…”

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

“…Several molecular and cellular mechanisms underlying synaptic plasticity and hippocampal function, as well as hippocampal-dependent memory itself, have previously been shown to be clock-regulated and disrupted by clock dysregulation (Smarr et al, 2014;Hannou et al, 2018;Snider et al, 2018). Circadian clocks modulate the expression or activity of diverse proteins involved in processes contributing to hippocampal function, such as neurotransmission, long-term potentiation (LTP), and energy metabolism (Chiang et al, 2017;Hannou et al, 2018;Greco and Sassone-Corsi, 2019). These include proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase 3β (GSK3β), the GluA1 AMPA receptor (AMPAR) subunit, synaptic vesicle glycoprotein 2A (SV2A), and isocitrate dehydrogenase (IDH) (Chiang et al, 2014(Chiang et al, , 2017Neufeld-Cohen et al, 2016;Hannou et al, 2018;Snider et al, 2018).…”

“…Circadian clocks modulate the expression or activity of diverse proteins involved in processes contributing to hippocampal function, such as neurotransmission, long-term potentiation (LTP), and energy metabolism (Chiang et al, 2017;Hannou et al, 2018;Greco and Sassone-Corsi, 2019). These include proteins such as calcium/calmodulin-dependent protein kinase II (CaMKII), glycogen synthase kinase 3β (GSK3β), the GluA1 AMPA receptor (AMPAR) subunit, synaptic vesicle glycoprotein 2A (SV2A), and isocitrate dehydrogenase (IDH) (Chiang et al, 2014(Chiang et al, , 2017Neufeld-Cohen et al, 2016;Hannou et al, 2018;Snider et al, 2018). Moreover, circadian dysfunction and cognitive decline are common features of aging and multiple agerelated neurodegenerative disorders, and accumulating evidence suggests that aging-and disease-related circadian disruption contributes to memory impairment (Kondratova and Kondratov, 2012;Musiek and Holtzman, 2016).…”