Translational and Posttranslational Dynamics in a Model Peptidergic System.

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus, whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses to neuronal stimulation are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganization of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and are hyperphosphorylated following stimulation. By multiomic integration of transcriptome and proteomic data, we identify changes to proteins present in afferent inputs to this nucleus.

Immunohistochemical evidence of P2X7R, P2X4R and CaMKK2 in pyramidal neurons of frontal cortex does not align with Alzheimer's disease.

Alzheimer's disease (AD) is an incurable neurodegenerative condition resulting in progressive cognitive decline. Pathological features include Aß plaques, neurofibrillary tangles, neuroinflammation and neuronal death. Purinergic receptors 7 and 4 (P2X7R and P2X4R) and calcium/calmodulin-dependent kinase kinase 2 (CaMKK2) are implicated in neuronal death. We used immunohistochemistry to investigate the distribution of these proteins in neurones from frontal cortex of donors (n = 3/group; aged 79-83 years) who died with and without AD. Neurones were identified morphologically and immunoperoxidase staining was achieved using commercial antibodies. Immunoreactive neurones were counted for each protein by 2-3 raters blinded to the diagnoses. We observed no differences in percentages of P2X7R, P2X4R or CaMKK2 positive neurones (p = 0.2-0.99), but sections from individuals with AD had marginally fewer neurones (p = 0.10). Hence P2X7R, P2X4R or CaMKK2 appear to be expressed in neurones from older donors, but expression does not associate with AD.

Rosemary extract increases neuronal cell glucose uptake and activates AMPK.

Glucose is the primary metabolic substrate of neurons and is responsible for supporting many vital functions including neuronal signalling. Decreases in glucose uptake and utilization are common characteristics of dementia, particularly Alzheimer's disease, and thus agents that can restore neuronal glucose availability may be especially valuable to the field. Diets rich in antioxidants and polyphenols have been associated with reductions in the risk of chronic disease that are associated with aging. In previous studies, rosemary extract (RE) has been reported to have antioxidant, anti-inflammatory, anticancer, and antidiabetic properties. The purpose of the present study was to explore the effects of RE on neuronal glucose uptake. Human SH-SY5Y neuroblastoma cells exposed to varied concentrations of RE showed a dose-dependent increase in glucose uptake, with a significant increase observed following treatment with 5 µg/mL RE for 2 h (159% ± 20.81% of control) that was comparable to maximum insulin stimulation (135.6% ± 3.2% of control). This increase in glucose uptake was paralleled by increases in AMP-activated protein kinase (AMPK), but not Akt, phosphorylation/activation. The present study is the first to report that treatment with rosemary extract can stimulate glucose uptake in a neuronal cell line. These results demonstrate the potential of RE to be used as an agent to regulate neuronal glucose homeostasis. Novelty: RE increases neuronal glucose uptake. RE activates AMPK in neurons. RE increases neuronal glucose uptake independently of insulin signalling.

Morphological plasticity of the tuberoinfundibular dopaminergic neurones in the rat during the oestrous cycle and lactation.

The hypothalamic tuberoinfundibular dopaminergic (TIDA) neurones are critical with respect to regulating prolactin secretion from the anterior pituitary. Under most physiological conditions, they are stimulated by prolactin to release dopamine into the median eminence which subsequently suppresses further prolactin secretion from the lactotrophs. During lactation, the TIDA neurones are known to undergo both electrophysiological and neurochemical changes that alleviate this negative-feedback, thus allowing circulating prolactin levels to rise. The present study aimed to determine whether TIDA neurone morphology, most notably spine density, is also modified during lactation. This was achieved by stereotaxically injecting the arcuate nucleus of female, tyrosine hydroxylase-promoter driven Cre-recombinase transgenic rats with Cre-dependent adeno-associated virus-expressing Brainbow. This resulted in the highly specifici transfection of between 10% and 30% of the TIDA neurones, thus allowing the morphologies on multiple individual neurones to be examined in a single hypothalamic slice. The transfected neurones exhibited a range of complex forms, including a diversity of soma and location of axonal origin. Neuronal spine counting showed that the density of somatic, but not dendritic, spines was significantly higher during lactation than at any other reproductive stage. There was also a significant fall in somatic spine density across the oestrous cycle from dioestrus to oestrus. Although the functional characteristics of the additional somatic spines have not been determined, if, as might be expected, they represent an increased excitatory input to the TIDA neurones, this could have important physiological implications by perhaps supporting altered neurotransmitter release at their neuroendocrine terminals. Enhanced excitatory input may, for example, favour the release of the opioid peptide enkephalin rather than dopamine, which is potentially significant because the expression of the peptide is known to increase in the TIDA neurones during lactation and, in contrast to dopamine, it stimulates rather than inhibits prolactin secretion from the pituitary.

Ghrelin receptor in agouti-related peptide neurones regulates metabolic adaptation to calorie restriction.

Ghrelin is a gut hormone that signals to the hypothalamus to stimulate growth hormone release, increase food intake and promote fat deposition. The ghrelin receptor, also known as growth hormone secretagogue receptor (GHS-R), is highly expressed in the brain, with the highest expression in agouti-related peptide (AgRP) neurones in the hypothalamus. Compelling evidence indicates that ghrelin serves as a survival hormone with respect to maintaining blood glucose and body weight during nutritional deficiencies. Recent studies have demonstrated that AgRP neurones are involved in metabolic and behavioural adaptation to an energy deficit to improve survival. In the present study, we used a neuronal subtype-specific GHS-R knockout mouse (AgRP-Cre;Ghsrf/f ) to investigate the role of GHS-R in hypothalamic AgRP neurones in metabolic and behavioural adaptation to hypocaloric restricted feeding. We subjected the mice to a restricted feeding regimen of 40% mild calorie restriction (CR), with one-quarter of food allotment given in the beginning of the light cycle and three-quarters given at the beginning of the dark cycle, to mimic normal mouse intake pattern. The CR-fed AgRP-Cre;Ghsrf/f mice exhibited reductions in body weight, fat mass and blood glucose. Metabolic profiling of these CR-fed AgRP-Cre;Ghsrf/f mice showed a trend toward reduced basal metabolic rate, significantly reduced core body temperature and a decreased expression of thermogenic genes in brown adipose tissue. This suggests a metabolic reset to a lower threshold. Significantly increased physical activity, a trend toward increased food anticipatory behaviour and altered fuel preferences were also observed in these mice. In addition, these CR-fed AgRP-Cre;Ghsrf/f mice exhibited a decreased counter-regulatory response, showing impaired hepatic glucose production. Lastly, hypothalamic gene expression in AgRP-Cre;Ghsrf/f mice revealed increased AgRP expression and a decreased expression of genes in ß-oxidation pathways. In summary, our data suggest that GHS-R in AgRP neurones is a key component of the neurocircuitry involved in metabolic adaptation to calorie restriction.

Hypothalamic epigenetics driving female puberty.

Puberty involves a series of morphological, physiological and behavioural changes during the last part of the juvenile period that culminates in the attainment of fertility. The activation of the pituitary-gonadal axis by increased hypothalamic secretion of gonadotrophin-releasing hormone (GnRH) is an essential step in the process. The current hypothesis postulates that a loss of transsynaptic inhibition and a rise in excitatory inputs are responsible for the activation of GnRH release. Similarly, a shift in the balance in the expression of puberty activating and puberty inhibitory genes exists during the pubertal transition. In addition, recent evidence suggests that the epigenetic machinery controls this genetic balance, giving rise to the tantalising possibility that epigenetics serves as a relay of environmental signals known for many years to modulate pubertal development. Here, we review the contribution of epigenetics as a regulatory mechanism in the hypothalamic control of female puberty.

A novel role of the corticotrophin-releasing hormone regulating peptide, teneurin C-terminal associated peptide 1, on glucose uptake into the brain.

Teneurin C-terminal associated peptide (TCAP) is an ancient paracrine signalling agent that evolved via lateral gene transfer from prokaryotes into an early metazoan ancestor. Although it bears structural similarity to corticotrophin-releasing hormone (CRH), it inhibits the in vivo actions of CRH. The TCAPs are highly expressed in neurones, where they induce rapid cytoskeletal rearrangement and are neuroprotective. Because these processes are highly energy-dependent, this suggests that TCAP has the potential to regulate glucose uptake because glucose is the primary energy substrate in brain, and neurones require a steady supply to meet the high metabolic demands of neuronal communication. Therefore, the objective of the present study was to assess the effect of TCAP-mediated glucose uptake in the brain and in neuronal cell models. TCAP-mediated 18 F-deoxyglucose (FDG) uptake into brain tissue was assessed in male wild-type Wistar rats by functional positron emission tomography. TCAP-1 increased FDG uptake by over 40% into cortical regions of the brain, demonstrating that TCAP-1 can significantly enhance glucose supply. Importantly, a single nanomolar injection of TCAP-1 increased brain glucose after 3 days and decreased blood glucose after 1 week. This is corroborated by a decreased serum concentration of insulin and an increased serum concentration of glucagon. In immortalised hypothalamic neurones, TCAP-1 increased ATP production and enhanced glucose uptake by increasing glucose transporter recruitment to the plasma membrane likely via AKT and mitogen-activated protein kinase/ERK phosphorylation events. Taken together, these data demonstrate that TCAP-1 increases glucose metabolism in neurones, and may represent a peptide signalling agent that regulated glucose uptake before insulin and related peptides.

Electroacupuncture Promotes Central Nervous System-Dependent Release of Mesenchymal Stem Cells.

Electroacupuncture (EA) performed in rats and humans using limb acupuncture sites, LI-4 and LI-11, and GV-14 and GV-20 (humans) and Bai-hui (rats) increased functional connectivity between the anterior hypothalamus and the amygdala and mobilized mesenchymal stem cells (MSCs) into the systemic circulation. In human subjects, the source of the MSC was found to be primarily adipose tissue, whereas in rodents the tissue sources were considered more heterogeneous. Pharmacological disinhibition of rat hypothalamus enhanced sympathetic nervous system (SNS) activation and similarly resulted in a release of MSC into the circulation. EA-mediated SNS activation was further supported by browning of white adipose tissue in rats. EA treatment of rats undergoing partial rupture of the Achilles tendon resulted in reduced mechanical hyperalgesia, increased serum interleukin-10 levels and tendon remodeling, effects blocked in propranolol-treated rodents. To distinguish the afferent role of the peripheral nervous system, phosphoinositide-interacting regulator of transient receptor potential channels (Pirt)-GCaMP3 (genetically encoded calcium sensor) mice were treated with EA acupuncture points, ST-36 and LIV-3, and GV-14 and Bai-hui and resulted in a rapid activation of primary sensory neurons. EA activated sensory ganglia and SNS centers to mediate the release of MSC that can enhance tissue repair, increase anti-inflammatory cytokine production and provide pronounced analgesic relief. Stem Cells 2017;35:1303-1315.