Adenosine A1R/A3R agonist AST-004 reduces brain infarction in mouse and rat models of acute ischemic stroke.

Acute ischemic stroke (AIS) is the second leading cause of death globally. No Food and Drug Administration (FDA) approved therapies exist that target cerebroprotection following stroke. Our group recently reported significant cerebroprotection with the adenosine A1/A3 receptor agonist, AST-004, in a transient stroke model in non-human primates (NHP) and in a preclinical mouse model of traumatic brain injury (TBI). However, the specific receptor pathway activated was only inferred based on in vitro binding studies. The current study investigated the underlying mechanism of AST-004 cerebroprotection in two independent models of AIS: permanent photothrombotic stroke in mice and transient middle cerebral artery occlusion (MCAO) in rats. AST-004 treatments across a range of doses were cerebroprotective and efficacy could be blocked by A3R antagonism, indicating a mechanism of action that does not require A1R agonism. The high affinity A3R agonist MRS5698 was also cerebroprotective following stroke, but not the A3R agonist Cl-IB-MECA under our experimental conditions. AST-004 efficacy was blocked by the astrocyte specific mitochondrial toxin fluoroacetate, confirming an underlying mechanism of cerebroprotection that was dependent on astrocyte mitochondrial metabolism. An increase in A3R mRNA levels following stroke suggested an intrinsic cerebroprotective response that was mediated by A3R signaling. Together, these studies confirm that certain A3R agonists, such as AST-004, may be exciting new therapeutic avenues to develop for AIS.

The Circadian Protein Nocturnin Regulates Metabolic Adaptation in Brown Adipose Tissue.

Fine-tuning of transcriptional responses can be critical for long-term outcomes in response to an environmental challenge. The circadian protein Nocturnin belongs to a family of proteins that include exonucleases, endonucleases, and phosphatases and is most closely related to the CCR4 family of deadenylases that regulate the cellular transcriptome via control of poly(A) tail length of RNA transcripts. In this study, we investigate the role of Nocturnin in regulating the transcriptional response and downstream metabolic adaptations during cold exposure in brown adipose tissue. We find that Nocturnin exhibits dual localization within the cytosol and mitochondria, and loss of Nocturnin causes changes in expression of networks of mRNAs involved in mitochondrial function. Furthermore, Nocturnin-/- animals display significantly elevated levels of tricarboxylic acid cycle intermediates, indicating that they have distinct metabolic adaptations during a prolonged cold exposure. We conclude that cold-induced stimulation of Nocturnin levels can regulate long-term metabolic adaptations to environmental challenges.

Time to Target Stroke: Examining the Circadian System in Stroke.

Stroke is the 5th leading cause of death in the United States and a leading cause of long-term disability. Ischemic strokes account for 87 percent of total stroke cases, yet the only FDA-approved treatments involve disruption of the blood clot to restore blood flow. New treatments aimed at saving or protecting neural tissue have largely failed in clinical trials and so new methodology or targets must be found. The occurrence of strokes significantly increases between 6 AM and 12 PM, implicating the circadian system in the onset of this debilitating brain injury. But it is not known whether or how the circadian system may regulate the response to and recovery from stroke. New strategies to identify treatments for stroke are beginning to look at cell types other than neurons as therapeutic targets, including astrocytes. In this review, we present links between the astrocyte circadian clock, the molecular response to stroke, and the damage caused by ischemia. We highlight aspects of astrocyte circadian function that could dictate new methodologies for stroke treatment, including the potential of chronotherapy.

A novel mouse model overexpressing Nocturnin results in decreased fat mass in male mice.

Nocturnin (NOCT) belongs to the Mg2+ dependent Exonucleases, Endonucleases, Phosphatase (EEP) family of enzymes that exhibit various functions in vitro and in vivo. NOCT is known to function as a deadenylase, cleaving poly-A tails from mRNA (messenger RNA) transcripts. Previously, we reported a role for NOCT in regulating bone marrow stromal cell differentiation through its interactions with PPARγ. In this study, we characterized the skeletal and adipose tissue phenotype when we globally overexpressed Noct in vivo. After 12 weeks of Noct overexpression, transgenic male mice had lower fat mass compared to controls, with no significant differences in the skeleton. Based on the presence of a mitochondrial target sequence in NOCT, we determined that mouse NOCT protein localizes to the mitochondria; subsequently, we found that NOCT overexpression led to a significant increase in the preadipocytes ability to utilize oxidative phosphorylation for ATP (adenosine triphosphate) generation. In summary, the effects of NOCT on adipocytes are likely through its novel role as a mediator of mitochondrial function.

Temporal Control of Metabolic Amplitude by Nocturnin.

The timing of food intake and nutrient utilization is critical to health and regulated partly by the circadian clock. Increased amplitude of circadian oscillations and metabolic output has been found to improve health in diabetic and obesity mouse models. Here, we report a function for the circadian deadenylase Nocturnin as a regulator of metabolic amplitude across the day/night cycle and in response to nutrient challenge. We show that mice lacking Nocturnin (Noct-/-) display significantly increased amplitudes of mRNA expression of hepatic genes encoding key metabolic enzymes regulating lipid and cholesterol synthesis, both over the daily circadian cycle and in response to fasting and refeeding. Noct-/- mice have increased plasma triglyceride throughout the night and increased amplitude of hepatic cholesterol levels. Therefore, posttranscriptional control by Nocturnin regulates the amplitude of these critical metabolic pathways, and loss of this activity results in increased metabolic flux and reduced obesity.

Diurnal Oscillations in Liver Mass and Cell Size Accompany Ribosome Assembly Cycles.

The liver plays a pivotal role in metabolism and xenobiotic detoxification, processes that must be particularly efficient when animals are active and feed. A major question is how the liver adapts to these diurnal changes in physiology. Here, we show that, in mice, liver mass, hepatocyte size, and protein levels follow a daily rhythm, whose amplitude depends on both feeding-fasting and light-dark cycles. Correlative evidence suggests that the daily oscillation in global protein accumulation depends on a similar fluctuation in ribosome number. Whereas rRNA genes are transcribed at similar rates throughout the day, some newly synthesized rRNAs are polyadenylated and degraded in the nucleus in a robustly diurnal fashion with a phase opposite to that of ribosomal protein synthesis. Based on studies with cultured fibroblasts, we propose that rRNAs not packaged into complete ribosomal subunits are polyadenylated by the poly(A) polymerase PAPD5 and degraded by the nuclear exosome.

Circadian rhythms and metabolism: from the brain to the gut and back again.

This paper focuses on the relationship between the circadian system and glucose metabolism. Research across the translational spectrum confirms the importance of the circadian system for glucose metabolism and offers promising clues as to when and why these systems go awry. In particular, basic research has started to clarify the molecular and genetic mechanisms through which the circadian system regulates metabolism. The study of human behavior, especially in the context of psychiatric disorders, such as bipolar disorder and major depression, forces us to see how inextricably linked mental health and metabolic health are. We also emphasize the remarkable opportunities for advancing circadian science through big data and advanced analytics. Advances in circadian research have translated into environmental and pharmacological interventions with tremendous therapeutic potential.

Activity-dependent FUS dysregulation disrupts synaptic homeostasis.

The RNA-binding protein fused-in-sarcoma (FUS) has been associated with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two neurodegenerative disorders that share similar clinical and pathological features. Both missense mutations and overexpression of wild-type FUS protein can be pathogenic in human patients. To study the molecular and cellular basis by which FUS mutations and overexpression cause disease, we generated novel transgenic mice globally expressing low levels of human wild-type protein (FUS(WT)) and a pathological mutation (FUS(R521G)). FUS(WT) and FUS(R521G) mice that develop severe motor deficits also show neuroinflammation, denervated neuromuscular junctions, and premature death, phenocopying the human diseases. A portion of FUS(R521G) mice escape early lethality; these escapers have modest motor impairments and altered sociability, which correspond with a reduction of dendritic arbors and mature spines. Remarkably, only FUS(R521G) mice show dendritic defects; FUS(WT) mice do not. Activation of metabotropic glutamate receptors 1/5 in neocortical slices and isolated synaptoneurosomes increases endogenous mouse FUS and FUS(WT) protein levels but decreases the FUS(R521G) protein, providing a potential biochemical basis for the dendritic spine differences between FUS(WT) and FUS(R521G) mice.

TH17 cell differentiation is regulated by the circadian clock.

Circadian clocks regulate numerous physiological processes that vary across the day-night (diurnal) cycle, but if and how the circadian clock regulates the adaptive immune system is mostly unclear. Interleukin-17-producing CD4(+) T helper (T(H)17) cells are proinflammatory immune cells that protect against bacterial and fungal infections at mucosal surfaces. Their lineage specification is regulated by the orphan nuclear receptor RORγt. We show that the transcription factor NFIL3 suppresses T(H)17 cell development by directly binding and repressing the Rorγt promoter. NFIL3 links T(H)17 cell development to the circadian clock network through the transcription factor REV-ERBα. Accordingly, TH17 lineage specification varies diurnally and is altered in Rev-erbα(-/-) mice. Light-cycle disruption elevated intestinal T(H)17 cell frequencies and increased susceptibility to inflammatory disease. Thus, lineage specification of a key immune cell is under direct circadian control.

Nocturnin: at the crossroads of clocks and metabolism.

Many aspects of metabolism exhibit daily rhythmicity under the control of endogenous circadian clocks, and disruptions in circadian timing result in dysfunctions associated with the metabolic syndrome. Nocturnin (Noc) is a robustly rhythmic gene that encodes a deadenylase thought to be involved in the removal of polyA tails from mRNAs. Mice lacking the Noc gene display resistance to diet-induced obesity and hepatic steatosis, due in part to reduced lipid trafficking in the small intestine. In addition, Noc appears to play important roles in other tissues and has been implicated in lipid metabolism, adipogenesis, glucose homeostasis, inflammation and osteogenesis. Therefore, Noc is a potential key post-transcriptional mediator in the circadian control of many metabolic processes.

Photoperiod alters phase difference between activity onset in vivo and mPer2::luc peak in vitro.

Photoperiod is a significant modulator of behavior and physiology for many organisms. In rodents changes in photoperiod are associated with changes in circadian period and photic resetting of circadian pacemakers. Utilizing rhythms of in vivo behavior and in vitro mPer2::luc expression, we investigated whether different entrainment photoperiods [light:dark (L:D) 16:8 and L:D 8:16] alter the period or phase relationships between these rhythms and the entraining light cycle in Per2::luc C57BL/6J mice. We also tested whether mPer2::luc rhythms differs in anterior and posterior suprachiasmatic nucleus (SCN) slices. Our results demonstrate that photoperiod significantly changes the timing of the mPer2::luc peak relative to the time of light offset and the activity onset in vivo. In both L:D 8:16 and L:D 16:8 the mPer2::luc peak maintained a more stable phase relationship to activity offset, while altering the phase relationship to activity onset. After the initial cycle in culture, the period, phase, and peaks per cycle were not significantly different for anterior vs. posterior SCN slices taken from animals within one photoperiod. After short-photoperiod treatment, anterior SCN slices showed increased-amplitude Per2::luc waveforms and posterior SCN slices showed shorter-duration peak width. Finally, the SCN tissue in vitro did not demonstrate differences in period attributable to photoperiod pretreatment, indicating that period aftereffects observed in behavioral rhythms after long- and short-day photoperiods are not sustained in Per2::luc rhythms in vitro. The change in phase relationship to activity onset suggests that Per2::luc rhythms in the SCN may track activity offset rather than activity onset. The reduced amplitude rhythms following long-photoperiod treatment may represent a loss of coupling of component oscillators.