Targeting RTN4/NoGo-Receptor reduces levels of ALS protein ataxin-2.

Gene-based therapeutic strategies to lower ataxin-2 levels are emerging for the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and spinocerebellar ataxia type 2 (SCA2). Additional strategies to lower levels of ataxin-2 could be beneficial. Here, we perform a genome-wide arrayed small interfering RNA (siRNA) screen in human cells and identify RTN4R, the gene encoding the RTN4/NoGo-Receptor, as a potent modifier of ataxin-2 levels. RTN4R knockdown, or treatment with a peptide inhibitor, is sufficient to lower ataxin-2 protein levels in mouse and human neurons in vitro, and Rtn4r knockout mice have reduced ataxin-2 levels in vivo. We provide evidence that ataxin-2 shares a role with the RTN4/NoGo-Receptor in limiting axonal regeneration. Reduction of either protein increases axonal regrowth following axotomy. These data define the RTN4/NoGo-Receptor as a novel therapeutic target for ALS and SCA2 and implicate the targeting of ataxin-2 as a potential treatment following nerve injury.

Hypertension alters the function and expression profile of the peptide cotransporters PEPT1 and PEPT2 in the rodent renal proximal tubule.

Hypertension is a major risk factor for kidney and cardiovascular disease. The treatment of hypertensive individuals by selected ACE inhibitors and certain di-and tripeptides halts the progression of renal deterioration and extends life-span. Renal reabsorption of these low molecular weight substrates are mediated by the PEPT1 and PEPT2 cotransporters. This study aims to investigate whether hypertension and ageing affects renal PEPT cotransporters at gene, protein expression and distribution as well as function in the superficial cortex and the outer medulla of the kidney. Membrane vesicles from the brush border (BBMV) and outer medulla (OMMV) were isolated from the kidneys of young Wistar Kyoto (Y-WKY), young spontaneously hypertensive (Y-SHR), and middle aged SHR (M-SHR) rats. Transport activity was measured using the substrate, ß-Ala-Lys (AMCA). Gene expression levels of PEPT genes were assessed with qRT-PCR while renal localisation of PEPT cotransporters was examined by immunohistochemistry with Western Blot validation. The Km and Vmax of renal PEPT1 were decreased significantly in SHR compared to WKY BBMV, whilst the Vmax of PEPT2 showed differences between SHR and WKY. By contrast to the reported cortical distribution of PEPT1, PEPT1-staining was detected in the outer medulla, whilst PEPT2 was expressed primarily in the cortex of all SHR; PEPT1 was significantly upregulated in the cortex of Y-SHR. These outcomes are indicative of a redistribution of PEPT1 and PEPT2 in the kidney proximal tubule under hypertensive conditions that has potential repercussions for nutrient handling and the therapeutic use of ACE inhibitors in hypertensive individuals.

The Diversity of Volatile Compounds in Australia's Semi-Desert Genus Eremophila (Scrophulariaceae).

Australia's endemic desert shrubs are commonly aromatic, with chemically diverse terpenes and phenylpropanoids in their headspace profiles. Species from the genus Eremophila (Scrophulariaceae ex. Myoporaceae) are the most common, with 215 recognised taxa and many more that have not yet been described, widely spread across the arid parts of the Australian continent. Over the years, our research team has collected multiple specimens as part of a survey to investigate the chemical diversity of the genus and create leads for further scientific enquiry. In the current study, the diversity of volatile compounds is studied using hydrodistilled essential oils and leaf solvent extracts from 30 taxa. Several rare terpenes and iridoids were detected in chemical profiles widely across the genus, and three previously undescribed sesquiterpenes were isolated and are assigned by 2D NMR-E-11(12)-dehydroisodendrolasin, Z-11-hydroxyisodendrolasin and 10-hydroxydihydro-α-humulene acetate. Multiple sampling from Eremophila longifolia, Eremophila arbuscular, Eremophila latrobei, Eremophila deserti, Eremophila sturtii, Eremophila oppositifolia and Eremophila alternifolia coneys that species in Eremophila are highly chemovariable. However, taxa are generally grouped according to the expression of (1) furanosesquiterpenes, (2) iridoids or oxides, (3) mixtures of 1 and 2, (4) phenylpropanoids, (5) non-furanoid terpenes, (6) mixtures of 4 and 5, and less commonly (7) mixtures of 1 and 5. Furthermore, GC-MS analysis of solvent-extracted leaves taken from cultivated specimens conveys that many heavier 'volatiles' with lower vapour pressure are not detected in hydrodistilled essential oils and have therefore been neglected in past chemical studies. Hence, our data reiterate that chemical studies of the genus Eremophila will continue to describe new metabolites and that taxon determination has limited predictive value for the chemical composition.

Effect of ageing and hypertension on the expression and activity of PEPT2 in normal and hypertrophic hearts.

Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether the expression and activity of the cardiac-type PEPT2 cotransporter could be affected by ageing and/or hypertension. Sarcolemmal vesicles (SV) were isolated from the hearts of all rat groups using a standard procedure to investigate the transport activity and protein abundance by fluorescence spectroscopy and Western blot, respectively. SLC15A2 "PEPT2" gene expression was relatively quantified by RT-qPCR. In the Wistar rat groups, the protein and gene expression of PEPT2 were upregulated with ageing. These changes were accompanied by corresponding increases in the competitive inhibition and the transport rate (Vmax) of ß-Ala-Lys (AMCA) into SV isolated from middle-aged hearts. Although, the transport rate of ß-Ala-Lys (AMCA) into SV isolated from old hearts was significantly the lowest compared to middle-aged and young adult hearts, the inhibition percentage of ß-Ala-Lys (AMCA) transport by Gly-Gln was the highest. In the WKY and SHR rat groups, Y-SHR hypertrophied hearts showed an increase in PEPT2 gene expression accompanied by a significant decrease in protein expression and activity. With advanced age, however, M-SHR hypertrophied hearts revealed significantly lower gene expression, but higher protein expression and activity than Y-SHR hearts. These findings suggest that increased expression of PEPT2 cotransporter in all types of middle-aged hearts could be exploited to facilitate di-and tripeptide transport by PEPT2 in these hearts, which subsequently could result in improved myocardial protection in these populations.

Prostanthera (Lamiaceae) as a 'Cradle of Incense': Chemophenetics of Rare Essential Oils from Both New and Forgotten Australian 'Mint Bush' Species.

The highly aromatic Australian mint bushes from the genus Prostanthera Labill. produce a high yield of essential oil on hydrodistillation. Together with its rich history, horticultural potential, iconic flowers, and aromatic leaves, it achieves high ornamental and culinary value. Species in the genus express highly diverse and chemically unique essential oils that demonstrate intra- and inter-specific patterns that have inspired taxonomic reinterpretation for over a hundred years. Previous studies have conveyed that phenoplastic expression of volatiles creates chemotypes within taxa, adding complexity to chemophenetic exploration. The current study chemically characterised essential oils from 64 highly aromatic specimens, representative of 25 taxa, giving yields as high as >2% g/g. The chemical profiles of essential oils are diverse, but generally include 1,8-cineole and signatory compounds such as sesquiterpene oxides, caryophyllene oxide, kessane and cis-dihydroagarofuran; sesquiterpene alcohols, globulol, epiglobulol, maaliol, prostantherol, spathulenol and ledol; and monoterpene derivatives of common scaffolds, borneol, bornyl acetate, bornanone, linalool and linalyl acetate. As in previous studies, analysis of chemical data confirms that the chemistry strongly agrees with taxonomic classifications. Importantly, as in classical taxonomy, the current chemical study complemented morphological analysis but conveys chemovariation, obscuring the taxonomic agreement. Nevertheless, variation within taxa may be due to environmental factors, meaning that cultivation of species in gardens will create different chemical profiles as compared to those published here.

Hypothalamic POMC deficiency increases circulating adiponectin despite obesity.

OBJECTIVE: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc-/-) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc-/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels. METHODS: Circulating adiponectin was measured in obese ArcPomc-/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc-/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc-/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc-/- mice. Finally, we generated compound Adiopoq-/-:ArcPomc-/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls. RESULTS: Obese ArcPomc-/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc-/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc-/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc-/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc-/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc-/- mice did not exacerbate their metabolic abnormalities. CONCLUSION: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc-/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Expression of a hypomorphic Pomc allele alters leptin dynamics during late pregnancy.

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

Selective Restoration of Pomc Expression in Glutamatergic POMC Neurons: Evidence for a Dynamic Hypothalamic Neurotransmitter Network.

Hypothalamic POMC deficiency leads to obesity and metabolic deficiencies, largely due to the loss of melanocortin peptides. However, POMC neurons in the arcuate nucleus (ARC) are comprised of glutamatergic and GABAergic subpopulations. The developmental program, relative proportion and function of these two subpopulations are unresolved. To test whether glutamatergic POMC neurons serve a distinct role in maintaining energy homeostasis, we activated Pomc expression Cre- dependently in Vglut2-expressing neurons of mice with conditionally silenced Pomc alleles. The Vglut2-Pomc restored mice had normal ARC Pomc mRNA levels, POMC immunoreactivity, as well as body weight and body composition at age 12 weeks. Unexpectedly, the cumulative total of Vglut2+ glutamatergic- and Gad67+ GABAergic-Pomc neurons detected by in situ hybridization (ISH) exceeded 100% in both Vglut2- Pomc restored and control mice, indicating that a subpopulation of Pomc neurons must express both neuronal markers. Consistent with this hypothesis, triple ISH of C57BL/6J hypothalami revealed that 35% of ARC Pomc neurons were selectively Gad67+, 21% were selectively Vglut2+, and 38% expressed both Gad67 and Vglut2. The single Gad67+ and Vglut2+Pomc neurons were most prevalent in the rostral ARC, while the Vglut2/Gad67+ dual-phenotype cells predominated in the caudal ARC. A lineage trace using Ai9-tdTomato reporter mice to label fluorescently all Vglut2-expressing neurons showed equal numbers of tdTomato+ and tdTomato- POMC immunoreactive neurons. Together, these data suggest that POMC neurons exhibit developmental plasticity in their expression of glutamatergic and GABAergic markers, enabling re-establishment of normal energy homeostasis in the Vglut2-Pomc restored mice.

Hypothalamic POMC or MC4R deficiency impairs counterregulatory responses to hypoglycemia in mice.

OBJECTIVE: Life-threatening hypoglycemia is a major limiting factor in the management of diabetes. While it is known that counterregulatory responses to hypoglycemia are impaired in diabetes, molecular mechanisms underlying the reduced responses remain unclear. Given the established roles of the hypothalamic proopiomelanocortin (POMC)/melanocortin 4 receptor (MC4R) circuit in regulating sympathetic nervous system (SNS) activity and the SNS in stimulating counterregulatory responses to hypoglycemia, we hypothesized that hypothalamic POMC as well as MC4R, a receptor for POMC derived melanocyte stimulating hormones, is required for normal hypoglycemia counterregulation. METHODS: To test the hypothesis, we induced hypoglycemia or glucopenia in separate cohorts of mice deficient in either POMC or MC4R in the arcuate nucleus (ARC) or the paraventricular nucleus of the hypothalamus (PVH), respectively, and measured their circulating counterregulatory hormones. In addition, we performed a hyperinsulinemic-hypoglycemic clamp study to further validate the function of MC4R in hypoglycemia counterregulation. We also measured Pomc and Mc4r mRNA levels in the ARC and PVH, respectively, in the streptozotocin-induced type 1 diabetes mouse model and non-obese diabetic (NOD) mice to delineate molecular mechanisms by which diabetes deteriorates the defense systems against hypoglycemia. Finally, we treated diabetic mice with the MC4R agonist MTII, administered stereotaxically into the PVH, to determine its potential for restoring the counterregulatory response to hypoglycemia in diabetes. RESULTS: Stimulation of epinephrine and glucagon release in response to hypoglycemia or glucopenia was diminished in both POMC- and MC4R-deficient mice, relative to their littermate controls. Similarly, the counterregulatory response was impaired in association with decreased hypothalamic Pomc and Mc4r expression in the diabetic mice, a phenotype that was not reversed by insulin treatment which normalized glycemia. In contrast, infusion of an MC4R agonist in the PVH restored the counterregulatory response in diabetic mice. CONCLUSION: In conclusion, hypothalamic Pomc as well as Mc4r, both of which are reduced in type 1 diabetic mice, are required for normal counterregulatory responses to hypoglycemia. Therefore, enhancing MC4R function may improve hypoglycemia counterregulation in diabetes.

Molecular changes to the rat renal cotransporters PEPT1 and PEPT2 due to ageing.

Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and selected peptidomimetic drugs. We aim to comprehensively characterise age-related changes of the renal PEPT cotransporters at the gene, protein, and functional level. Brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) were isolated from the kidneys of young, middle-aged and old rats. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group. PEPT2 protein expression was not only increased with ageing, but PEPT2 also was found in BBMV from middle-aged and old groups. SLC15A2 gene expression in the renal outer medulla increased in the old group. These changes in the expressions and localisations of PEPT1 and PEPT2 could explain the changes to transport activity in BBMV and OMMV. These findings provide novel insights that would be useful for maintaining protein nutrition and optimising the delivery of some peptidomimetic drugs in elderly individuals.