Loss of temporal coherence in the circadian metabolome across multiple tissues during ageing in mice.

Circadian clock function declines with ageing, which can aggravate ageing-related diseases such as type 2 diabetes and neurodegenerative disorders. Understanding age-related changes in the circadian system at a systemic level can contribute to the development of strategies to promote healthy ageing. The goal of this study was to investigate the impact of ageing on 24-h rhythms in amine metabolites across four tissues in young (2 months of age) and old (22-25 months of age) mice using a targeted metabolomics approach. Liver, plasma, the suprachiasmatic nucleus (SCN; the location of the central circadian clock in the hypothalamus) and the paraventricular nucleus (PVN; a downstream target of the SCN) were collected from young and old mice every 4 h during a 24-h period (n = 6-7 mice per group). Differential rhythmicity analysis revealed that ageing impacts 24-h rhythms in the amine metabolome in a tissue-specific manner. Most profound changes were observed in the liver, in which rhythmicity was lost in 60% of the metabolites in aged mice. Furthermore, we found strong correlations in metabolite levels between the liver and plasma and between the SCN and the PVN in young mice. These correlations were almost completely abolished in old mice. These results indicate that ageing is accompanied by a severe loss of the circadian coordination between tissues and by disturbed rhythmicity of metabolic processes. The tissue-specific impact of ageing may help to differentiate mechanisms of ageing-related disorders in the brain versus peripheral tissues and thereby contribute to the development of potential therapies for these disorders.

A multi-level assessment of the bidirectional relationship between aging and the circadian clock.

The daily temporal order of physiological processes and behavior contribute to the wellbeing of many organisms including humans. The central circadian clock, which coordinates the timing within our body, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Like in other parts of the brain, aging impairs the SCN function, which in turn promotes the development and progression of aging-related diseases. We here review the impact of aging on the different levels of the circadian clock machinery-from molecules to organs-with a focus on the role of the SCN. We find that the molecular clock is less effected by aging compared to other cellular components of the clock. Proper rhythmic regulation of intracellular signaling, ion channels and neuronal excitability of SCN neurons are greatly disturbed in aging. This suggests a disconnection between the molecular clock and the electrophysiology of these cells. The neuronal network of the SCN is able to compensate for some of these cellular deficits. However, it still results in a clear reduction in the amplitude of the SCN electrical rhythm, suggesting a weakening of the output timing signal. Consequently, other brain areas and organs not only show aging-related deficits in their own local clocks, but also receive a weaker systemic timing signal. The negative spiral completes with the weakening of positive feedback from the periphery to the SCN. Consequently, chronotherapeutic interventions should aim at strengthening overall synchrony in the circadian system using life-style and/or pharmacological approaches.

Uncovering functional signature in neural systems via random matrix theory.

Neural systems are organized in a modular way, serving multiple functionalities. This multiplicity requires that both positive (e.g. excitatory, phase-coherent) and negative (e.g. inhibitory, phase-opposing) interactions take place across brain modules. Unfortunately, most methods to detect modules from time series either neglect or convert to positive, any measured negative correlation. This may leave a significant part of the sign-dependent functional structure undetected. Here we present a novel method, based on random matrix theory, for the identification of sign-dependent modules in the brain. Our method filters out both local (unit-specific) noise and global (system-wide) dependencies that typically obfuscate the presence of such structure. The method is guaranteed to identify an optimally contrasted functional 'signature', i.e. a partition into modules that are positively correlated internally and negatively correlated across. The method is purely data-driven, does not use any arbitrary threshold or network projection, and outputs only statistically significant structure. In measurements of neuronal gene expression in the biological clock of mice, the method systematically uncovers two otherwise undetectable, negatively correlated modules whose relative size and mutual interaction strength are found to depend on photoperiod.

The influence of neuronal electrical activity on the mammalian central clock metabolome.

INTRODUCTION: Most organisms display circadian rhythms in physiology and behaviour. In mammals, these rhythms are orchestrated by the suprachiasmatic nucleus (SCN). Recently, several metabolites have emerged as important regulators of circadian timekeeping. Metabolomics approaches have aided in identifying some key metabolites in circadian processes in peripheral tissue, but methods to routinely measure metabolites in small brain areas are currently lacking. OBJECTIVE: The aim of the study was to establish a reliable method for metabolite quantifications in the central circadian clock and relate them to different states of neuronal excitability. METHODS: We developed a method to collect and process small brain tissue samples (0.2 mm3), suitable for liquid chromatography-mass spectrometry. Metabolites were analysed in the SCN and one of its main hypothalamic targets, the paraventricular nucleus (PVN). Tissue samples were taken at peak (midday) and trough (midnight) of the endogenous rhythm in SCN electrical activity. Additionally, neuronal activity was altered pharmacologically. RESULTS: We found a minor effect of day/night fluctuations in electrical activity or silencing activity during the day. In contrast, increasing electrical activity during the night significantly upregulated many metabolites in SCN and PVN. CONCLUSION: Our method has shown to produce reliable and physiologically relevant metabolite data from small brain samples. Inducing electrical activity at night mimics the effect of a light pulses in the SCN, producing phase shifts of the circadian rhythm. The upregulation of metabolites could have a functional role in this process, since they are not solely products of physiological states, they are significant parts of cellular signalling pathways.

Distinct temporal expression of 5-HT(1A) and 5-HT(2A) receptors on cerebellar granule cells in mice.

Serotonin plays an important role of controlling the physiology of the cerebellum. However, serotonin receptor expression has not been fully studied in the developing cerebellum. We have recently shown that cerebellar granule cells transiently express 5-HT3 receptors. In the present study, we investigate expression of 5-HT1 and 5-HT2 receptors in the mouse cerebellum both during postnatal development and in juvenile mice. Here, we show for the first time that 5-HT1A and 5-HT2A receptors are present on cerebellar granule cells with a distinct temporal expression pattern: 5-HT1A receptors are expressed only during the first 2 weeks, while 5-HT2A receptor expression persists until at least 8 weeks after birth. Because of its prolonged expression pattern, we investigated the electrophysiological properties of the 5-HT2A receptor. 5-HT2A receptors expressed by cerebellar granule cells promote stability by reducing variability of the synaptic response, and they modulate the paired-pulse ratio of the parallel fibre-Purkinje cell synapse. Furthermore, pharmacological block of 5-HT2A receptors enhances short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. We thus show a novel role for serotonin in controlling function of the cerebellum via 5-HT2A receptors expressed by cerebellar granule cells.

Serotonergic control of Purkinje cell maturation and climbing fibre elimination by 5-HT3 receptors in the juvenile mouse cerebellum.

Functional serotonin 3 (5-HT3) receptors are transiently expressed by cerebellar granule cells during early postnatal development, where they modulate short-term synaptic plasticity at the parallel fibre-Purkinje cell synapse. Here, we show that serotonin controls maturation of Purkinje cells in the mouse cerebellum. The 5-HT3 receptors regulate morphological maturation of Purkinje cells during early postnatal development, and this effect is mediated by the glycoprotein reelin. Using whole-cell patch-clamp recordings we also investigated physiological development of Purkinje cells in 5-HT3A receptor knockout mice during early postnatal development, and found abnormal physiological maturation, characterized by a more depolarized resting membrane potential, an increased input resistance and the ability to fire action potentials upon injection of a depolarizing current at an earlier age. Furthermore, short-term synaptic plasticity was impaired at both the parallel fibre-Purkinje cell and the climbing fibre-Purkinje cell synapses, and both the amplitude and the frequency of spontaneous miniature events recorded from Purkinje cells were increased. The expedited morphological and physiological maturation affects the whole cerebellar cortical network, as indicated by delayed climbing fibre elimination in 5-HT3A receptor knockout mice. There was no difference between wild-type and 5-HT3A receptor knockout mice in any of the morphological or physiological properties described above at later ages, indicating a specific time window during which serotonin regulates postnatal development of the cerebellum via 5-HT3 receptors expressed by granule cells.

Aging selectively dampens oscillation of lipid abundance in white and brown adipose tissue.

Lipid metabolism is under the control of the circadian system and circadian dysregulation has been linked to obesity and dyslipidemia. These factors and outcomes have also been associated to, or affected by, the process of aging. Here, we investigated whether murine white (WAT) and brown (BAT) adipose tissue lipids exhibit rhythmicity and if this is affected by aging. To this end, we have measured the 24 h lipid profiles of WAT and BAT using a global lipidomics analysis of > 1100 lipids. We observed rhythmicity in nearly all lipid classes including glycerolipids, glycerophospholipids, sterol lipids and sphingolipids. Overall, ~ 22% of the analyzed lipids were considered rhythmic in WAT and BAT. Despite a general accumulation of lipids upon aging the fraction of oscillating lipids decreased in both tissues to 14% and 18%, respectively. Diurnal profiles of lipids in BAT appeared to depend on the lipid acyl chain length and this specific regulation was lost in aged mice. Our study revealed how aging affects the rhythmicity of lipid metabolism and could contribute to the quest for targets that improve diurnal lipid homeostasis to maintain cardiometabolic health during aging.

Aging Affects the Capacity of Photoperiodic Adaptation Downstream from the Central Molecular Clock.

Aging impairs circadian clock function, leading to disrupted sleep-wake patterns and a reduced capability to adapt to changes in environmental light conditions. This makes shift work or the changing of time zones challenging for the elderly and, importantly, is associated with the development of age-related diseases. However, it is unclear what levels of the clock machinery are affected by aging, which is relevant for the development of targeted interventions. We found that naturally aged mice of >24 months had a reduced rhythm amplitude in behavior compared with young controls (3-6 months). Moreover, the old animals had a strongly reduced ability to adapt to short photoperiods. Recording PER2::LUC protein expression in the suprachiasmatic nucleus revealed no impairment of the rhythms in PER2 protein under the 3 different photoperiods tested (LD: 8:16, 12:12, and 16:8). Thus, we observed a discrepancy between the behavioral phenotype and the molecular clock, and we conclude that the aging-related deficits emerge downstream of the core molecular clock. Since it is known that aging affects several intracellular and membrane components of the central clock cells, it is likely that an impairment of the interaction between the molecular clock and these components is contributing to the deficits in photoperiod adaptation.

Evidence for Weakened Intercellular Coupling in the Mammalian Circadian Clock under Long Photoperiod.

For animals living in temperate latitudes, seasonal changes in day length are an important cue for adaptations of their physiology and behavior to the altered environmental conditions. The suprachiasmatic nucleus (SCN) is known as the central circadian clock in mammals, but may also play an important role in adaptations to different photoperiods. The SCN receives direct light input from the retina and is able to encode day-length by approximating the waveform of the electrical activity rhythm to the duration of daylight. Changing the overall waveform requires a reorganization of the neuronal network within the SCN with a change in the degree of synchrony between the neurons; however, the underlying mechanisms are yet unknown. In the present study we used PER2::LUC bioluminescence imaging in cultured SCN slices to characterize network dynamics on the single-cell level and we aimed to provide evidence for a role of modulations in coupling strength in the photoperiodic-induced phase dispersal. Exposure to long photoperiod (LP) induced a larger distribution of peak times of the single-cell PER2::LUC rhythms in the anterior SCN, compared to short photoperiod. Interestingly, the cycle-to-cycle variability in single-cell period of PER2::LUC rhythms is also higher in the anterior SCN in LP, and is positively correlated with peak time dispersal. Applying a new, impartial community detection method on the time series data of the PER2::LUC rhythm revealed two clusters of cells with a specific spatial distribution, which we define as dorsolateral and ventromedial SCN. Post hoc analysis of rhythm characteristics of these clusters showed larger cycle-to-cycle single-cell period variability in the dorsolateral compared to the ventromedial cluster in the anterior SCN. We conclude that a change in coupling strength within the SCN network is a plausible explanation to the observed changes in single-cell period variability, which can contribute to the photoperiod-induced phase distribution.

New Neurons in Aging Brains: Molecular Control by Small Non-Coding RNAs.

Adult neurogenesis generates functional neurons from neural stem cells present in specific brain regions. It is largely confined to two main regions: the subventricular zone of the lateral ventricle, and the subgranular zone of the dentate gyrus (DG), in the hippocampus. With age, the function of the hippocampus and particularly the DG is impaired. For instance, adult neurogenesis is decreased with aging, in both proliferating and differentiation of newborn cells, while in parallel an age-associated decline in cognitive performance is often seen. Surprisingly, the synaptogenic potential of adult-born neurons is only marginally influenced by aging. Therefore, although proliferation, differentiation, and synaptogenesis of adult-born new neurons in the DG are closely related to each other, they are differentially affected by aging. In this review we discuss the crucial roles of a novel class of recently discovered modulators of gene expression, the small non-coding RNAs, in the regulation of adult neurogenesis. Multiple small non-coding RNAs are differentially expressed in the hippocampus. In particular a subgroup of the small non-coding RNAs, the microRNAs, fine-tune the progression of adult neurogenesis. This makes small non-coding RNAs appealing candidates to orchestrate the functional alterations in adult neurogenesis and cognition associated with aging. Finally, we summarize observations that link changes in circulating levels of steroid hormones with alterations in adult neurogenesis, cognitive decline, and vulnerability to psychopathology in advanced age, and discuss a potential interplay between steroid hormone receptors and microRNAs in cognitive decline in aging individuals.