This study characterized the previously unknown impacts of RCAN1 abolition and overexpression on the entrained diurnal as well as circadian periodicity, intensity, active versus inactive phase distribution, and rhythmicity of wheel running in light-entrained and free-running young versus aged mice. Using the Dp16 mouse model for DS, we recapitulated our findings that RCAN1 modulates light-entrained diurnal and circadian locomotor activity profiles by demonstrating Rcan1 dosage correction improved or normalized wheel running in Dp16 mice. We also generated novel data showing that RCAN1 expression is normally arrhythmic in the young light-entrained brain, implying that RCAN1 expression is tightly regulated to maintain constant levels throughout early adulthood. Perturbation of RCAN1 levels early during aging, as shown using young Rcan1 KO and RCAN1 TG mice or Rcan1 triplication in Dp16 mice, disrupted light-entrained diurnal as well as circadian wheel running behavior in manners reminiscent of DS, AD, and aging. Therefore, these findings demonstrate that balanced expression of RCAN1 is necessary for normal diurnal and circadian regulation of locomotor activity in mice and suggest that changes to RCAN1 levels observed in DS, AD, and normal aging (Wu and Song, 2013; Wong et al., 2015) may mediate the diurnal and circadian dysfunctions associated with these conditions (Bonanni et al., 2005; Fernandez et al., 2017; Duncan, 2019; Leng et al., 2019).
In the DS population and DS mouse models, diurnal rest-activity and circadian-related disturbances are common, but the contribution of different loci on HSA21 has been unclear. Using RCAN1-overxpressing transgenic and Dp16 mice, we endeavored to elucidate the contribution of RCAN1. In light-entrained young RCAN1 TG and Dp16 mice, we found that daily wheel running in the dark phase was reduced and could be normalized in Dp16 mice by restoring Rcan1 to two copies. These data strongly suggest that RCAN1 overexpression may underlie the hypoactivity in dark-phase wheel running reported for the Tc1 mouse model of DS (Heise et al., 2015). While Tc1 mice engaged in less wheel-running activity during the dark phase, they were simultaneously more active in other locomotor behaviors, such as walking, climbing, feeding, and grooming (Heise et al., 2015). Additionally, they showed increased wheel running during the light phase (Heise et al., 2015), differing from our findings in young RCAN1 TG and Dp16 mice. Our data also differed from previous studies that reported hyperactivity in the Ts65Dn mouse model for DS during the dark phase (Stewart et al., 2007; Ruby et al., 2010) and hyperactivity of Dp16 mice measured by the distance moved in the home cage during the light phase (Levenga et al., 2018). These differences from our findings may be explained by the activity measurement used (e.g., wheel running versus other locomotor behaviors) or suggest different interaction effects of the DS-related genes overexpressed in each mouse model. A hyperactive phenotype is more consistent with the hyperactivity characteristic of DS (Vicari, 2006; Ekstein et al., 2011). However, an accelerated senescence phenotype is also characteristic of DS (Lott & Head, 2001; Lott, 2012), and general activity levels are well-known to decrease with aging (Banks et al., 2015; Musiek et al., 2018). Congruent with this, we found that decreased daily wheel running in aged mice was not further reduced by RCAN1 overexpression, suggesting that the hypoactivity in young RCAN1 TG and Dp16 mice reflects premature aging. RCAN1 overexpression is further implicated by the observation that Dp16/Rcan12N mice displayed a significant normalization of both light-entrained diurnal and circadian wheel running phenotypes compared to Dp16 mice (Fig. 7). It remains possible that RCAN1 TG and Dp16 mice display hyperactivity by other measures or at earlier ages, which would be interesting to investigate in future studies.
The present study also detected attenuated rhythmicity of light-entrained diurnal wheel running as indicated by a reduced amplitude in young RCAN1 TG and Dp16 mice, comparable to Tc1 (Heise et al., 2015) and Ts65Dn (Stewart et al., 2007) mice. Taken together with the reduced amplitudes of diurnal rest-activity rhythms reported in DS (Fernandez et al., 2017) and the reduced impact of the DS genotype in Dp16/Rcan12N mice, these data suggest a primary role for RCAN1 overexpression in DS-linked dampening of diurnal activity rhythms. Consistent with premature aging in young RCAN1 TG and Dp16 mice, flattened rest-activity rhythmicity occurs with aging as well (Banks et al., 2015; Musiek et al., 2018). Our data also indicated reduced amplitudes of light-entrained diurnal wheel running rhythms in aged mice relative to young mice. Activity rhythm amplitudes were not further reduced in aged RCAN1 TG mice compared with NTG littermates, suggesting RCAN1 overexpression early in life as found in DS (Sun et al., 2011; Wu and Song, 2013) accelerates the aging-associated attenuation of diurnal rest-activity rhythm amplitudes. Moreover, RCAN1 TG mice exhibited a lengthened circadian period of wheel activity, which has similarly been observed in normally aging mice (Banks et al., 2015). This finding reveals aging-associated circadian activity rhythm dysfunction in RCAN1 TG mice, providing additional evidence that RCAN1 overexpression promotes senescence-related phenotypes. As RCAN1 levels are elevated with age independent of DS (Cook et al., 2005; Wong et al., 2015), RCAN1 may also mediate the lengthened circadian period that manifests with normal aging. In young Dp16 mice, interestingly, we detected no change in length of the light-entrained diurnal or circadian wheel running period whether or not RCAN1 was restored to disomic levels. This may suggest other genes triplicated in the Dp16 model interact with RCAN1 overexpression effects to regulate diurnal and circadian rest-activity rhythms in DS. More studies testing the contribution of different loci on HSA21 to behavioral rhythm phenotypes in DS will be important. Collectively, these results suggest that RCAN1 overexpression contributes to aging-associated diurnal and circadian alterations that are accelerated in DS.
A major feature of the accelerated senescence phenotype in DS is the nearly ubiquitous early-age onset of AD, which is similarly characterized by circadian disruptions (Leng et al., 2019). Since RCAN1 is also elevated in AD (Harris et al., 2007; Sun et al., 2011; Wu and Song, 2013; Wong et al., 2015), RCAN1 overexpression may mediate DS-AD comorbidity and link diurnal rest-activity and circadian abnormalities in both disorders. Supporting this notion, the generalized hypoactivity of daily wheel running detected in young RCAN1 TG and Dp16 mice mimics the increased daytime sleepiness and hypoactivity documented in AD patients (Satlin et al., 1995; Bonanni et al., 2005; Weissová et al., 2016). Furthermore, the attenuated intensity and amplitude of wheel running rhythms in young RCAN1 TG and Dp16 mice are analogous to the fragmentation and reduced amplitude of daily rest-activity rhythms in both preclinical (Musiek et al., 2018) and clinical (Satlin et al., 1995) AD. The lengthened circadian period of wheel running identified in young RCAN1 TG is similarly observed in mouse models of AD (Wisor et al., 2005; Stevanovic et al., 2017). Acrophase estimates for light-entrained diurnal and circadian wheel running rhythms did not differ between young RCAN1 TG or Dp16 mice and WT controls, indicating that elevated RCAN1 levels do not contribute to the circadian phase shifts observed in DS, AD, aging individuals, and animal models thereof (Satlin et al., 1995; Stewart et al., 2007; Duffy et al., 2015; Fernandez et al., 2017; Duncan, 2019). In aggregate, these findings support the idea that RCAN1 overexpression mediates overlapping age-associated disturbances of light-entrained diurnal and circadian rest-activity rhythms in DS and AD.
Importantly, circadian disruptions precede the appearance of AD-linked pathology and neurodegeneration in RCAN1 TG mice (Wong et al., 2015), mirroring the progression of disease in AD (Musiek et al., 2018; Duncan, 2019; Leng et al., 2019; Van Egroo et al., 2019). In a previous study, we found AD-like hippocampal mitochondrial dysfunction, oxidative stress, synaptic plasticity failures, and memory impairments in aged, but not young, RCAN1 TG mice (Wong et al., 2015). However, young RCAN1 TG mice showed AD-like increases in tau hyperphosphorylation that reached the levels of aged NTG mice. This tau hyperphosphorylation was not further increased in aged RCAN1 TG mice (Wong et al., 2015), suggesting RCAN1 overexpression accelerates tau pathology that may feedforward and contribute to AD-like phenotypes in aged mice. In the present study, we found diurnal and circadian activity rhythm alterations reminiscent of aged phenotypes in young RCAN1 TG mice. Thus, both tau pathology and diurnal as well as circadian rhythm dysfunction manifested before the development of other AD-related phenotypes in these mice, which models the preclinical, clinical, and pathophysiological characteristics of AD (Musiek et al., 2018; Duncan, 2019; Leng et al., 2019; Van Egroo et al., 2019). Mounting evidence points to tau pathology as a more robust biomarker of AD risk than Aβ accumulation, correlating more strongly with the onset of early cognitive symptoms and eventual clinical presentation of AD (Brier et al., 2016). Diurnal and circadian dysfunction are also emerging as risk factors for AD, based on data demonstrating that altered behavioral rhythms precede cognitive deficits in AD (Tranah et al., 2011; Lim et al., 2013; Musiek et al., 2018) and that disrupting the circadian clockwork can drive aging- and AD-related cognitive and pathological features (Yin et al., 2017; Zhao et al., 2017). Our data suggest that RCAN1 upregulation can promote or mediate the consequences of tau pathology and circadian dysfunction. Interestingly, the presence of tauopathy can disrupt biological rhythms (Stevanovic et al., 2017; Buhl et al., 2019), suggesting that RCAN1 overexpression may additively or synergistically perturb diurnal and circadian rhythmicity through upregulating tau pathology. Given the influences of biological clock function on memory performance (Smarr et al., 2014), these findings together imply that RCAN1 overexpression causes diurnal and circadian activity disruptions that may induce or exacerbate AD-related neurodegeneration.
RCAN1 deficiency also altered wheel running phenotypes in the same directions as RCAN1 overexpression for some parameters but in opposite directions for others. Neither removal nor overexpression of RCAN1 affected the light-entrained periodicity of wheel running. However, the circadian wheel running periods in young Rcan1 KO and RCAN1 TG mice were comparably lengthened, indicating that optimal levels of RCAN1 are necessary to maintain the circadian periodicty of activity. With photic entrainment, RCAN1 removal and overexpression reduced daily total and dark phase wheel running and attenuated the oscillatory mean (MESOR) and oscillatory range (amplitude) of diurnal wheel running rhythms in young mice. These phenotypes resemble aging, suggesting that both loss and aberrant gain of RCAN1 may accelerate aging. In contrast to RCAN1 overexpression, RCAN1 abolition in young mice increased wheel running during the light (inactive) phase when mice are typically resting, reminiscent of increased nighttime awakenings/activity in DS and AD (Bonanni et al., 2005; Fernandez et al., 2017). Free-running Rcan1 KO mice also showed divergent behavior from RCAN1 TG mice. Whereas daily total and active-phase wheel running and parameters of activity rhythms including MESOR and amplitude were reduced in young RCAN1 TG mice consistently across LD12:12 and DD conditions, these measures were increased in young free-running Rcan1 KO mice, which differed from their light-entrained counterparts. These bidirectional effects of RCAN1 downregulation and upregulation indicate that RCAN1 titrates diurnal and circadian activity levels and rhythms, which aligns with prior studies demonstrating dose-dependent regulation of locomotor activity rhythms by the Drosophilia RCAN1 homolog sra (Kweon et al., 2018). The convergent effects of RCAN1 downregulation and upregulation on wheel running profiles also mirror previous findings that deletion of either sra, which disinhibited CaN activity, or CanA-14F, which encodes a catalytic subunit of CaN in Drosophila, both led to hyperactivity, short sleep, and arrhythmic clocks (Nakai et al., 2011; Kweon et al., 2018). Altogether, these data suggest that balanced RCAN1 expression is required for normative light-entrained diurnal as well as circadian activity patterns and rhythms, and deviations of RCAN1 levels confer DS-, AD-, and aging-like aberrations thereof.
Our western blot analyses revealed that RCAN1 levels are stable over a 24-h cycle in the hippocampi of young light-entrained WT mice, implying that increases to RCAN1 levels as found in RCAN1 TG and Dp16 mice or with aging (Wong et al., 2015) and decreases to RCAN1 levels as seen in Rcan1 KO mice tilt the balance of RCAN1 signaling that regulates light-entrained diurnal as well as circadian functionality in early adulthood. Consistent with this result, levels of the RCAN1.1L isoform and CaN do not fluctuate in the mouse heart (Rotter et al., 2014). CaN levels in the hamster suprachiasmatic nucleus (SCN) and chick retina are similarly stable (Katz et al., 2008; Huang et al., 2012). By contrast, the RCAN1.4 isoform exhibits circadian oscillations in the mouse heart and skeletal muscle (Rotter et al., 2014; Dyar et al., 2015), demonstrating isoform-specific roles of RCAN1. Although our data suggest all RCAN1 isoforms are arrhythmic in the mouse hippocampus, it nevertheless remains feasible that fluctuations in RCAN1.4 levels are masked by RCAN1.1S or the converse, since these isoforms share the same molecular weight. Interestingly, in cases where RCAN1 or CaN levels do not show daily fluctuations, the phosphatase activity of CaN exhibits rhythmic oscillations (Katz et al., 2008; Huang et al., 2012; Rotter et al., 2014). Therefore, RCAN1 may regulate diurnal and circadian activity rhythms in part by modulating the rhythmicity of CaN activity. Since RCAN1 is known to both inhibit and facilitate CaN function (Vega et al., 2003; Liu et al., 2009; Wong et al., 2015) and to act independently of CaN (Keating et al., 2008), future studies are needed to determine how CaN participates in RCAN1-mediated daily activity rhythm disruptions associated with DS, AD, and aging. Moreover, it will be informative to assess rhythmic changes in hippocampal CaN activity, considering that the hippocampus contains an autonomous molecular clock that has been linked to memory performance (Kondratova et al., 2010; Smarr et al., 2014; Kwapis et al., 2018), and since hippocampus-dependent memory deficits were previously observed in both Rcan1 KO (Hoeffer et al., 2007) and RCAN1 TG (Wong et al., 2015) mice. Furthermore, profiling the rhythmicity of RCAN1 levels and RCAN1-dependent modulation of CaN activity in other brain regions, such as the SCN, will be essential to establish if RCAN1 differentially regulates rhythmicity throughout the brain and to delineate the mechanisms whereby RCAN1 regulates diurnal and circadian activity patterns and rhythms.