The cerebellum modulates thirst.

The cerebellum, a phylogenetically ancient brain region, has long been considered strictly a motor control structure. Recent studies have implicated the cerebellum in cognition, sensation, emotion and autonomic function, making it an important target for further investigation. Here, we show that cerebellar Purkinje neurons in mice are activated by the hormone asprosin, leading to enhanced thirst, and that optogenetic or chemogenetic activation of Purkinje neurons induces rapid manifestation of water drinking. Purkinje neuron-specific asprosin receptor (Ptprd) deletion results in reduced water intake without affecting food intake and abolishes asprosin's dipsogenic effect. Purkinje neuron-mediated motor learning and coordination were unaffected by these manipulations, indicating independent control of two divergent functions by Purkinje neurons. Our results show that the cerebellum is a thirst-modulating brain area and that asprosin-Ptprd signaling may be a potential therapeutic target for the management of thirst disorders.

Asprosin promotes feeding through SK channel-dependent activation of AgRP neurons.

Asprosin, a recently identified adipokine, activates agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus (ARH) via binding to protein tyrosine phosphatase receptor δ (Ptprd) to increase food intake. However, the intracellular mechanisms responsible for asprosin/Ptprd-mediated activation of AgRPARH neurons remain unknown. Here, we demonstrate that the small-conductance calcium-activated potassium (SK) channel is required for the stimulatory effects of asprosin/Ptprd on AgRPARH neurons. Specifically, we found that deficiency or elevation of circulating asprosin increased or decreased the SK current in AgRPARH neurons, respectively. AgRPARH-specific deletion of SK3 (an SK channel subtype highly expressed in AgRPARH neurons) blocked asprosin-induced AgRPARH activation and overeating. Furthermore, pharmacological blockade, genetic knockdown, or knockout of Ptprd abolished asprosin's effects on the SK current and AgRPARH neuronal activity. Therefore, our results demonstrated an essential asprosin-Ptprd-SK3 mechanism in asprosin-induced AgRPARH activation and hyperphagia, which is a potential therapeutic target for the treatment of obesity.

Overexpression and ELISA-based detection of asprosin in cultured cells and mice.

The unreliability of commercial recombinant asprosin preparations and variability between asprosin detection assays have proven to be a bottleneck in experimental interpretation. This protocol describes the use of viral vectors and expression plasmid for overexpression and secretion of human asprosin to achieve sustained elevation of asprosin protein in mice and HEK293T cells without using recombinant proteins. This protocol also includes a sandwich ELISA using anti-asprosin monoclonal antibodies for detection of asprosin in media from cultured cells and in plasma of mice. For complete details on the use and execution of this protocol, please refer to Duerrschmid et al. (2017), Mishra et al. (2021), and Mishra et al. (2022).

Peripheral Sympathectomy Alters Neuroinflammatory and Microglial Responses to Sleep Fragmentation in Female Mice.

Sleep loss, either induced by obstructive sleep apnea or other forms of sleep dysfunction, induces an inflammatory response, as commonly measured by increased circulating levels of pro-inflammatory cytokines. Increased catecholamines from sympathetic nervous system (SNS) activation regulates this peripheral inflammation. However, the role that catecholamines play in mediating neuroinflammation from sleep perturbations is undescribed. The aims of this study were to determine (i) the effect of peripheral SNS inhibition upon neuroinflammatory responses to sleep fragmentation (SF) and (ii) whether homeostasis can be restored after 1 week of recovery sleep. We measured gene expression levels of pro- and anti-inflammatory cytokines and microglial activity in brain (prefrontal cortex, hippocampus and hypothalamus) of female mice that were subjected to acute SF for 24 hours, chronic SF for 8 weeks, or 7 days of recovery after chronic SF. In each experiment, SF and control mice were peripherally sympathectomized with 6-hydroxydopamine (6-OHDA) or injected with vehicle. SF elevated cytokine mRNA expression in brain and increased microglial density and cell area in some regions. In addition, chronic SF promoted hyper-ramification in resting microglia upon exposure to chronic, but not acute, SF. Effects of chronic SF were more pronounced than acute SF, and 1 week of recovery was not sufficient to alleviate neuroinflammation. Importantly, 6-OHDA treatment significantly alleviated SF-induced inflammation and microglial responses. This study provides evidence of SNS regulation of neural inflammation from SF, suggesting a potential role for therapeutics that could mitigate neuroinflammatory responses to sleep dysfunction.

Protein tyrosine phosphatase receptor δ serves as the orexigenic asprosin receptor.

Asprosin is a fasting-induced glucogenic and centrally acting orexigenic hormone. The olfactory receptor Olfr734 is known to be the hepatic receptor for asprosin that mediates its effects on glucose production, but the receptor for asprosin's orexigenic function has been unclear. Here, we have identified protein tyrosine phosphatase receptor δ (Ptprd) as the orexigenic receptor for asprosin. Asprosin functions as a high-affinity Ptprd ligand in hypothalamic AgRP neurons, regulating the activity of this circuit in a cell-autonomous manner. Genetic ablation of Ptprd results in a strong loss of appetite, leanness, and an inability to respond to the orexigenic effects of asprosin. Ablation of Ptprd specifically in AgRP neurons causes resistance to diet-induced obesity. Introduction of the soluble Ptprd ligand-binding domain in the circulation of mice suppresses appetite and blood glucose levels by sequestering plasma asprosin. Identification of Ptprd as the orexigenic asprosin receptor creates a new avenue for the development of anti-obesity therapeutics.

Asprosin-neutralizing antibodies as a treatment for metabolic syndrome.

Background: Recently, we discovered a new glucogenic and centrally acting orexigenic hormone - asprosin. Asprosin is elevated in metabolic syndrome (MS) patients, and its genetic loss results in reduced appetite, leanness, and blood glucose burden, leading to protection from MS. Methods: We generated three independent monoclonal antibodies (mAbs) that recognize unique asprosin epitopes and investigated their preclinical efficacy and tolerability in the treatment of MS. Results: Anti-asprosin mAbs from three distinct species lowered appetite and body weight, and reduced blood glucose in a dose-dependent and epitope-agnostic fashion in three independent MS mouse models, with an IC50 of ~1.5 mg/kg. The mAbs displayed a half-life of over 3days in vivo, with equilibrium dissociation-constants in picomolar to low nanomolar range. Conclusions: We demonstrate that anti-asprosin mAbs are dual-effect pharmacologic therapy that targets two key pillars of MS - over-nutrition and hyperglycemia. This evidence paves the way for further development towards an investigational new drug application and subsequent human trials for treatment of MS, a defining physical ailment of our time. Funding: DK118290 and DK125403 (R01; National Institute of Diabetes and Digestive and Kidney Diseases), DK102529 (K08; National Institute of Diabetes and Digestive and Kidney Diseases), Caroline Wiess Law Scholarship (Baylor College of Medicine, Harrington Investigatorship Harrington Discovery Institute at University Hospitals, Cleveland); Chao Physician Scientist Award (Baylor College of Medicine); RP150551 and RP190561 (Cancer Prevention and Research Institute of Texas [CPRIT]).

Chemical sympathectomy reduces peripheral inflammatory responses to acute and chronic sleep fragmentation.

Sleep loss contributes to the development of cardiovascular, metabolic, and neurological disorders by promoting a systemic proinflammatory phenotype. The neuroendocrine-immune mechanisms contributing to such pathologies are poorly understood. The sympathetic nervous system (SNS) regulates immunity and is often activated following sleep disturbances. The aims of this study were to determine 1) the effect of SNS inhibition on inflammatory responses to sleep fragmentation (SF) and 2) whether homeostasis can be restored after 1 wk of recovery sleep. We measured stress responses (norepinephrine and corticosterone), gene expression levels of pro- and anti-inflammatory cytokines in peripheral (heart, liver, and spleen) tissues, and protein levels of cytokines and chemokines in serum of female mice that were subjected to acute SF for 24 h, chronic SF for 8 wk, or 7 days of recovery after chronic SF. In each experiment, SF and control mice were chemically sympathectomized with 6-hydroxydopamine (6-OHDA) or injected with vehicle. Both acute and chronic SF elevated mRNA and protein levels of cytokines in peripheral tissues. Changes in inflammatory responses mirrored stress-axes activation, with increased corticosterone and norepinephrine in SF mice. 6-OHDA treatment significantly alleviated SF-induced inflammation, thus providing evidence of SNS regulation of peripheral inflammation from SF. Effects of chronic SF were more severe than acute SF, and 1 wk of recovery from SF sufficiently alleviated peripheral inflammatory responses but not NE responses.

Changes in brain peptides associated with reproduction and energy homeostasis: Putative roles of gonadotrophin-releasing hormone-II and tyrosine hydroxylase in determining reproductive performance in response to daily food availability times in diurnal zebra finches.

Previous studies have demonstrated 'quality-quantity' trade-offs with daily food availability times in zebra finches. Compared with food access ad lib., zebra finch pairs with restricted food access for 4 hours in the morning produced poor quality offspring, whereas those with the same food access in the evening produced fewer but better quality offspring. The present study investigated whether food-time-dependent differential effects on reproductive performance involved brain peptides associated with reproduction and energy homeostasis in zebra finches. We measured peptide/protein expression of gonadotrophin-releasing hormone (GnRH)-I, GnRH-II, gonadotrophin-inhibitory hormone (GnIH), tyrosine hydroxylase (TH), neuropeptide Y (NPY), cocaine- and amphetamine regulated transcript (CART) and ZENK (a neuronal activation marker) by immunohistochemistry and mRNA expression of genes coding for the type 2 (DIO2) and type 3 (DIO3) deiodinase by a quantitative polymerase chain reaction in male and female zebra finches that were paired and kept under a 12:12 hour light/dark photocycle at 24 ± 2°C temperature for > 12 months with access to food ad lib., or for only 4 hours in the morning or evening. In both sexes, GnRH-I, DIO2 and DIO3 expression did not differ significantly between the three feeding conditions, although levels showed an overall food effect. However, in males, GnIH expression was significantly higher in evening-fed birds compared to ad lib. fed birds. Interestingly, GnRH-II and TH levels were significantly lower in restricted feeding compared to the ad lib. group and, importantly, GnRH-II and TH-immunoreactivity levels were negatively and positively correlated with egg laying latency and reproductive success (offspring/brood/pair), respectively. At the same time, we found no effect on the hypothalamic expression of orexigenic (NPY) and anorexigenic (CART) peptides, or ZENK protein (ie, the neuronal activity marker). These results suggest the involvement of reproductive neuropeptides, with putative roles for GnRH-II and TH, in the food-time-dependent effect on reproductive performance, albeit with subtle sex differences, in diurnal zebra finches, which possess the ability to reproduce year-round, in a manner similar to other continuously breeding vertebrates.

Developmental effects of daily food availability times on song behaviour and neuronal plasticity of song-control system in male zebra finches.

Food availability is a major ecological factor and affects body condition and sexual traits. Here, we investigated whether males' song behaviour, a trait for female mate choice, was sensitive to the food availability period and its timing in songbirds. We manipulated daily food availability to 4 h in the morning or evening, with controls on food ad libitum, and assessed its effects on song behaviour and forebrain song control system in male zebra finches that were held as adult (parent) or offspring (since birth) at 24 ± 2 °C under 12 h daily photoperiod. Food restriction significantly affected both temporal and spectral features of daily song in offspring, not the parent. In offspring, we found reduced mesor (mean 24-h levels), attenuated amplitude (daily maxima relative to mesor) and altered acrophase (estimated time of daily maxima) of 24-h rhythm, and reduced motif length (in morning-fed), per motif unique syllables and an enhanced song pitch (in evening-fed). There was also a positive correlation of motif length with cheek patch and plasma testosterone levels, and of per motif syllables with cheek patch and daily activity levels in offspring. Among main song controlling forebrain nuclei, LMAN and HVC were reduced in size, and Area X and HVC showed decreased neuronal recruitment in offspring on food restrictions. These results demonstrate the importance of daily food availability and its timing in determining males' sexual signals, and support growing evidence that among vertebrates well-fed males contain reproductive traits that females use for its mate choice.

Changes in the expression of genes involved in DNA methylation and histone modification in response to daily food availability times in zebra finches: epigenetic implications.

We hypothesised that daily food availability times serve as an 'epigenetic' factor and affect reproductive physiology in continuously reproducing species. This we tested by measuring mRNA expression of genes coding for enzymes involved in DNA methylation-demethylation (dnmt, tet) and histone modification (hat1, hdac) in the hypothalamus, liver and gonads of male and female zebra finches that were paired for a year under 12 h light:12 h dark conditions with food availability restricted to 4 h in the morning (morning FA group) or evening (evening FA group), with controls provided with food ad libitum The overall hypothalamic and hepatic expression patterns of hat1 and hdac were similar but those of dnmt and tet were different between males and females. Irrespective of the timing of food availability, both hat1 and hdac mRNA levels were increased in the hypothalamus, but not in the liver, in which hat1 mRNA levels were increased in the morning FA group. While hypothalamic tet levels were higher in evening FA males, hepatic tet levels were higher in morning FA birds (tet1, only males). Gonadal expression levels similarly varied and showed sex differences. Histone-modifying genes did not show food availability effects, except for elevated testicular hdac3 levels. Similarly, testicular dnmt3b and tet2 mRNA levels were increased and decreased in morning and evening FA groups, respectively, whereas ovarian dnmt1 and tet2 levels were reduced in morning FA and tet1 levels were reduced in evening FA groups. The present results suggest that an enforced daily feeding schedule in the long term could serve as a conditioning environment that shapes overall hypothalamic regulation, and liver and gonadal function at the epigenetic level in diurnal vertebrates.