Genomic Convergence among ERRα, PROX1, and BMAL1 in the Control of Metabolic Clock Outputs

Metabolic homeostasis and circadian rhythms are closely intertwined biological processes. Nuclear receptors, as sensors of hormonal and nutrient status, are actively implicated in maintaining this physiological relationship. Although the orphan nuclear receptor estrogen-related receptor α (ERRα, NR3B1) plays a central role in the control of energy metabolism and its expression is known to be cyclic in the liver, its role in temporal control of metabolic networks is unknown. Here we report that ERRα directly regulates all major components of the molecular clock. ERRα-null mice also display deregulated locomotor activity rhythms and circadian period lengths under free-running conditions, as well as altered circulating diurnal bile acid and lipid profiles. In addition, the ERRα-null mice exhibit time-dependent hypoglycemia and hypoinsulinemia, suggesting a role for ERRα in modulating insulin sensitivity and glucose handling during the 24-hour light/dark cycle. We also provide evidence that the newly identified ERRα corepressor PROX1 is implicated in rhythmic control of metabolic outputs. To help uncover the molecular basis of these phenotypes, we performed genome-wide location analyses of binding events by ERRα, PROX1, and BMAL1, an integral component of the molecular clock. These studies revealed the existence of transcriptional regulatory loops among ERRα, PROX1, and BMAL1, as well as extensive overlaps in their target genes, implicating these three factors in the control of clock and metabolic gene networks in the liver. Genomic convergence of ERRα, PROX1, and BMAL1 transcriptional activity thus identified a novel node in the molecular circuitry controlling the daily timing of metabolic processes

Axonal mRNA Translation: An Unexpected Link to Axon Survival and the Mitochondrion

Localized mRNA translation plays roles in dendrites and axons, but the regulatory mechanisms and downstream pathways are not well understood. An article in Cell by Yoon et al. (2012) shows that lamin B2, well known as a nuclear protein, undergoes regulated synthesis in axons, promoting mitochondrial function and axon survival

Analysis of circadian liver gene expression by ADDER, a highly sensitive method for the display of differentially expressed mRNAs

We describe a novel and highly sensitive method for the differential display of mRNAs, called ADDER (Amplification of Double-stranded cDNA End Restriction fragments). The technique involves the construction and PCR amplification of double-stranded cDNA restriction fragments complementary to 3′-terminal mRNA sequences. Aliquots of these cDNA fragments are then amplified by touchdown PCR with 192 pairs of display primers (16 upstream primers and 12 downstream primers) that differ in their ultimate and penultimate nucleotides and the PCR products are compared by size-fractionation on urea–polyacrylamide sequencing gels. By using the ADDER technology for the comparison of liver RNAs harvested at different times around the clock we detected nearly 300 cDNA fragments complementary to mRNAs with circadian accumulation profiles and sequenced 51 of them. The majority of these cDNAs correspond to genes which were not previously known to be rhythmically expressed. A large fraction of the identified genes encoded factors involved in the processing and detoxification of nutrients. This suggests that a primary purpose of circadian transcription in the liver is the anticipation of food processing and detoxification. Several genes involved in human disease were also identified, including the one encoding presenilin II, a protein implicated in the development of Alzheimer’s Disease

Rhythms of Mammalian Body Temperature Can Sustain Peripheral Circadian Clocks

Background: Low-amplitude temperature oscillations can entrain the phase of circadian rhythms in several unicellular and multicellular organisms, including Neurospora and Drosophila. Because mammalian body temperature is subject to circadian variations of 1°C–4°C, we wished to determine whether these temperature cycles could serve as a Zeitgeber for circadian gene expression in peripheral cell types. Results: In RAT1 fibroblasts cultured in vitro, circadian gene expression could be established by a square wave temperature rhythm with a ΔT of 4°C (12 hr 37°C/12 hr 33°C). To examine whether natural body temperature rhythms can also affect circadian gene expression, we first measured core body temperature cycles in the peritoneal cavities of mice by radiotelemetry. We then reproduced these rhythms with high precision in the liquid medium of cultured fibroblasts for several days by means of a homemade computer-driven incubator. While these “in vivo” temperature rhythms were incapable of establishing circadian gene expression de novo, they could maintain previously induced rhythms for multiple days; by contrast, the rhythms of control cells kept at constant temperature rapidly dampened. Moreover, circadian oscillations of environmental temperature could reentrain circadian clocks in the livers of mice, probably via the changes they imposed upon both body temperature and feeding behavior. Interestingly, these changes in ambient temperature did not affect the phase of the central circadian pacemaker in the suprachiasmatic nucleus (SCN) of the hypothalamus. Conclusions: We postulate that both endogenous and environmental temperature cycles can participate in the synchronization of peripheral clocks in mammals

IMP2 axonal localization, RNA interactome, and function in the development of axon trajectories

RNA-based regulatory mechanisms play important roles in the development and plasticity of neural circuits and neurological disease. Developing axons provide a model well suited to the study of RNA-based regulation, and contain specific subsets of mRNAs that are locally translated and have roles in axon pathfinding. However, the RNA-binding proteins involved in axon pathfinding, and their corresponding mRNA targets, are still largely unknown. Here we find that the RNA-binding protein IMP2 (Igf2bp2) is strikingly enriched in developing axon tracts, including in spinal commissural axons. We used the HITS-CLIP approach to perform a genome-wide identification of RNAs that interact directly with IMP2 in the native context of developing mouse brain. This IMP2 interactome was highly enriched for mRNA targets related to axon guidance. Accordingly, IMP2 knockdown in the developing spinal cord led to strong defects in commissural axon trajectories at the midline intermediate target. These results reveal a highly distinctive axonal enrichment of IMP2, show that it interacts with a network of axon guidance-related mRNAs, and reveal that it is required for normal axon pathfinding during vertebrate development

Restricted feeding uncouples circadian oscillators in peripheral tissues from the central pacemaker in the suprachiasmatic nucleus

In mammals, circadian oscillators exist not only in the suprachiasmatic nucleus, which harbors the central pacemaker, but also in most peripheral tissues. It is believed that the SCN clock entrains the phase of peripheral clocks via chemical cues, such as rhythmically secreted hormones. Here we show that temporal feeding restriction under light–dark or dark–dark conditions can change the phase of circadian gene expression in peripheral cell types by up to 12 h while leaving the phase of cyclic gene expression in the SCN unaffected. Hence, changes in metabolism can lead to an uncoupling of peripheral oscillators from the central pacemaker. Sudden large changes in feeding time, similar to abrupt changes in the photoperiod, reset the phase of rhythmic gene expression gradually and are thus likely to act through a clock-dependent mechanism. Food-induced phase resetting proceeds faster in liver than in kidney, heart, or pancreas, but after 1 wk of daytime feeding, the phases of circadian gene expression are similar in all examined peripheral tissues

In vitro assessment of dietary bioactives for TFEB activation as a possible target to support cognitive and emotional wellbeing

Aging and chronic stress are associated with altered brain plasticity and an increased risk of developing cognitive and emotional health problems. A key cellular mechanism involved is autophagy, which is known to be important for memory encoding and information processing, and is also thought to be important for psychiatric and emotional health. Activation of the autophagic system can be established by the transcription factor EB (TFEB), which regulates both autophagosome formation and lysosomal biogenesis and function. As well-known activators of autophagy like caloric restriction, fasting, or regular exercise require intervention programs that are hard to adhere to, the search for nutritional solutions with similar effects, is increasing. We therefore evaluated pro-autophagic dietary bioactives for effects specifically through TFEB activation and assessed whether combining bioactives can lead to synergistic or additive effects. We also discuss current evidence supporting the use of these natural dietary compounds to promote cognitive and emotional wellbeing. Methods: Primary cortical astrocytes were treated with 13 different ingredients and analyzed for their effect on TFEB nuclear translocation by high content imaging. Effective ingredients were then evaluated at different concentrations for their effect on TFEB downstream signaling by analyzing mRNA levels of Beclin1, LC3, Lamp-1 and Cathepsin D. Furthermore, the synergistic effects of sub-optimal concentrations of different ingredient combinations were evaluated on the same TFEB downstream signaling markers. Potential signs of toxicity of all ingredients and their combinations were evaluated by analysis of ATP production and nuclear counts. Results: DHA, EPA, 7,8-DHF, ellagic acid, curcumin and spermidine increased TFEB nuclear localization. DHA and spermidine exhibited the strongest effects when measuring mRNA levels of TFEB downstream signaling markers. The most significant synergies were observed when combining spermidine with ellagic acid or when combining DHA with curcumin. None of the ingredients alone or in combination presented any sign of toxicity at the concentrations tested. Discussion/Conclusion: The results of our study show for the first time a synergistic effect of spermidine and ellagic acid and of DHA and curcumin on TFEB signaling and thus its ability to act on the autophagic system in brain cells. These combinations may hold potential as dietary promoters of cognitive and emotional wellbeing

Therapeutic potential of glutathione-enhancers in stress-related psychopathologies

The mammalian brain has high energy demands, which may become higher in response to environmental challenges such as psychogenic stress exposure. Therefore, efficient neutralization of reactive oxygen species that are produced as a by-product of ATP synthesis is crucial for preventing oxidative damage and ensuring normal energy supply and brain function. Glutathione (GSH) is arguably the most important endogenous antioxidant in the brain. In recent years, aberrant GSH levels have been implicated in different psychiatric disorders, including stress-related psychopathologies. In this review, we examine the available data supporting a role for GSH levels and antioxidant function in the brain in relation to anxiety and stress-related psychopathologies. Additionally, we identify several promising compounds that could raise GSH levels in the brain by either increasing the availability of its precursors or the expression of GSH-regulating enzymes through activation of Nuclear factor erythroid-2-related factor 2 (Nrf2). Given the high tolerability and safety profile of these compounds, they may represent attractive new opportunities to complement existing therapeutic manipulations against stress-related psychopathologies