Masterson, Mitchell
(2021)
An investigation of the role of Neuronal PAS Domain Protein 2 (NPAS2) in peripheral circadian clocks.
PhD thesis, University of Nottingham.
Abstract
Circadian cycles are 24-hour biological oscillations, observable in an array of physiological and behavioural systems including the sleep wake cycle, metabolism, cardiovascular activity, and cognition. The core circadian clock, located in the suprachiasmatic nucleus (SCN) of the brain, is entrained by environmental light and is responsible for entrainment of oscillators in other tissues, known as peripheral clocks. Light entrainment via the SCN is not the only signal that can entrain circadian cycles, they can also be entrained by other cues such as time of feeding (reflecting environmental food availability), which acts to entrain peripheral cycles without requirement of the SCN. The molecular underpinnings of these peripheral clocks, responsible for this entrainment and various tissue specific circadian effects, are incompletely understood. In these studies, we focused on investigation of the functional role of the transcription factor NPAS2 in liver and fibroblast peripheral clocks.
NPAS2 is a bHLH-PAS domain transcription factor and heterodimeric partner of BMAL1 that forms part of the positive arm of the core circadian feedback loop. Expression of either NPAS2 or its more well characterised paralog CLOCK have been shown in mouse gene KO studies to be required for functional circadian cycles. Constitutive KO of the NPAS2 gene in mice has also been found to severely delay circadian entrainment to time of feeding and enhance the entrainment to light but the precise role of NPAS2 in this entrainment is unclear. As feeding entrainment of behaviour is closely associated with the entrainment of peripheral clocks it is unknown if this effect is due to the loss of NPAS2 in the peripheral tissues or in the core clock in the constitutive KO. We aimed to investigate the function of NPAS2 in the molecular feedback cycle in peripheral clocks, and how it contributes to circadian behavioural entrainment. As loss of NPAS2 expression has also been found to impair long term fear conditioning performance we investigated the potential contribution of the liver peripheral clock to this, and latent inhibition of fear conditioning.
To investigate NPAS2 function we utilised both cellular and mouse models. For cellular studies in NIH3T3 fibroblasts, NPAS2 or CLOCK genes were targeted using CRISPR-Cas9 editing. Clonal lines were selected that showed loss of expression/function as confirmed by allele genotyping, western blots and RT-qPCR. It was observed that ablation of NPAS2 in NIH3T3 cells resulted in alterations in rhythmic expression of other core clock genes, including CRY2 and both REV ERBα and REV ERBβ genes of the negative arm of the feedback loop. Indeed, robust increases of the endogenous REV ERBα and REV ERBβ genes were observed, suggesting that NPAS2 may negatively regulate these genes.
A liver specific NPAS2 cKO mouse model was used to investigate expression of select core clock genes in liver and brain tissues, and also correlate this with behavioural effects of the loss of NPAS2 in the liver. In this mouse model we observed modulated expression levels of the REV ERB genes in the liver across circadian time points. Interestingly however, we also observed significant changes in rhythmic expression of selected core clock genes in brain samples that included tissue from the prefrontal cortex and from other cortical (including motor cortical) and subcortical regions of the liver specific cKO mice. We observed the appearance of variance in expression of BMAL1 at different times of day and the loss of similar time variance in the expression of REV ERBβ. This raises the novel suggestion that ablation of NPAS2 in the liver clock can influence rhythmic gene expression in the brain.
In contrast to the constitutive KO model, we found no effect of loss of liver NPAS2 on light entrainment but observed an enhancement of restricted feeding entrainment, although this effect was restricted to female mice. This suggests a sex-specific mechanism is involved in feeding entrainment and that loss of NPAS2 in the liver is beneficial for food entrainment. Investigation of fear conditioning and latent inhibition identified no effect of the loss of NPAS2 in the liver although a genotype independent sex effect on fear conditioning supports further sex specific investigation.
These findings suggest a systemic effect of loss of NPAS2 expression within the liver, influencing behavioural cycles and gene expression in separate tissues. This suggests the liver peripheral clock contributes to circadian behavioural cycles and entrainment of other tissues and that NPAS2 contributes to these activities of the liver clock. The mechanism behind this effect is unknown but it may be through effects on endocrine or metabolite signalling. The potential exists to breed mice with a brain specific NPAS2 KO in the future to complement these findings and investigate the specific effect of loss of NPAS2 from the central clock.
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