Glucagon-like peptide-1 (GLP-1) increases in plasma and colon tissue prior to estrus and circulating levels change with increasing age in reproductively competent Wistar rats

There is a well-documented association between cyclic changes to food intake and the changing ovarian hormone levels of the reproductive cycle in female mammals. Limited research on appetite-controlling gastrointestinal peptides has taken place in females, simply because regular reproductive changes in steroid hormones present additional experimental factors to account for. This study focussed directly on the roles that gastrointestinal-secreted peptides may have in these reported, naturally occurring, changes to food intake during the rodent estrous cycle and aimed to determine whether peripheral changes occurred in the anorexigenic (appetite-reducing) hormones peptide-YY (PYY) and glucagon-like peptide-1 (GLP-1) in female Wistar rats (32-44 weeks of age). Total forms of each peptide were measured in matched fed and fasted plasma and descending colon tissue samples for each animal during the dark (feeding) phase. PYY concentrations did not significantly change between defined cycle stages, in either plasma or tissue samples. GLP-1 concentrations in fed plasma and descending colon tissue were significantly increased during proestrus, just prior to a significant reduction in fasted stomach contents at estrus, suggesting increased satiety and reduced food intake at this stage of the cycle. Increased proestrus GLP-1 concentrations could contribute to the reported reduction in food intake during estrus and may also have biological importance in providing the optimal nutritional and metabolic environment for gametes at the potential point of conception. Additional analysis of the findings demonstrated significant interactions of ovarian cycle stage and fed/fasted status with age on GLP-1, but not PYY plasma concentrations. Slightly older females had reduced fed plasma GLP-1 suggesting that a relaxation of regulatory control of this incretin hormone may also take place with increasing age in reproductively competent females

Plasma Ghrelin Concentrations Were Altered with Oestrous Cycle Stage and Increasing Age in Reproductively Competent Wistar Females

Changes in appetite occur during the ovarian cycle in female mammals. Research on appetite-regulatory gastrointestinal peptides in females is limited, because reproductive changes in steroid hormones present additional experimental factors to control for. This study aimed to explore possible changes in the orexigenic (appetite-stimulating) gastrointestinal peptide hormone ghrelin during the rodent oestrous cycle. Fed and fasted plasma and stomach tissue samples were taken from female Wistar rats (32–44 weeks of age) at each stage of the oestrous cycle for total ghrelin quantification using radioimmunoassay. Sampling occurred during the dark phase when most eating takes place in rats. Statistical analysis was by paired-samples t-test, one-way ANOVA on normally distributed data, with Tukey post-hoc tests, or Kruskal-Wallis if not. GLM univariate analysis was used to assess main effects and interactions in ghrelin concentrations in the fed or fasted state and during different stages of the ovarian cycle, with age as a covariate. No consistent fed to fasted ghrelin increases were measured in matched plasma samples from the same animals, contrary to expectations. Total ghrelin concentrations did not significantly change between cycle stages with ANOVA, in either fed or fasted plasma or in stomach tissue. This was despite significantly decreased fasted stomach contents at oestrus (P = 0.028), suggesting decreased food intake. There was however a significant interaction in ghrelin plasma concentrations between fed and fasted proestrus rats and a direct effect of age with rats over 37 weeks old having lower circulating concentrations of ghrelin in both fed and fasted states. The biological implications of altered ghrelin plasma concentrations from 37 weeks of age are as yet unknown, but warrant further investigation. Exploring peripheral ghrelin regulatory factor changes with increasing age in reproductively competent females may bring to light potential effects on offspring development and nutritional metabolic programming

Interstitial cell network volume is reduced in the terminal bowel of ageing mice

Ageing is associated with impaired neuromuscular function of the terminal gastrointestinal (GI) tract, which can result in chronic constipation, faecal impaction and incontinence. Interstitial cells of cajal (ICC) play an important role in regulation of intestinal smooth muscle contraction. However, changes in ICC volume with age in the terminal GI tract (the anal canal including the anal sphincter region and rectum)have not been studied. Here, the distribution, morphology and network volume of ICC in the terminal GI tract of 3‐to 4‐month‐old and 26‐to 28‐month‐old C57BL/6mice were investigated. ICC were identified by immunofluorescence labelling of wholemount preparations with an antibody against c‐Kit. ICC network volume was measured by software‐based 3D volume rendering of confocal Z stacks. A significant reduction in ICC network volume per unit volume of muscle was measured in aged animals. No age‐associated change in ICC morphology was detected. The thickness of the circular muscle layer of the anal sphincter region and rectum increased with age, while that in the distal colon decreased. These results suggest that ageing is associated with a reduction in the network volume of ICC in the terminal GI tract, which may influence the normal function of these regions

Colostrinin™ alleviates amyloid-β induced toxicity in rat primary hippocampal cultures

Colostrinin™ (CLN), a complex mixture of proline-rich polypeptides derived from colostrums, can alleviate cognitive decline in early Alzheimer's disease patients. The molecular basis of the action of CLN has been studied in vitro using human neuroblastoma cell lines. The aim of the present study was to use quantitative immunocytochemistry and immunoblotting to investigate the ability of CLN to relieve amyloid-β (Aβ)-induced cytotoxicity in rat primary hippocampal neuronal cells. Our data confirm that CLN alleviates the effect of Aβ-induced cytotoxicity and causes a significant reduction in the elevated levels of the antioxidant enzyme SOD1

Differing effects of NT-3 and GDNF on dissociated enteric ganglion cells exposed to hydrogen peroxide in vitro

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and aging. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF) are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuoprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H2O2). H2O2 treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, Bis benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H2O2-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress

Loss of UCHL1 promotes age-related degenerative changes in the enteric nervous system.

UCHL1 (ubiquitin carboxyterminal hydrolase 1) is a deubiquitinating enzyme that is particularly abundant in neurons. From studies of a spontaneous mutation arising in a mouse line it is clear that loss of function of UCHL1 generates profound degenerative changes in the central nervous system, and it is likely that a proteolytic deficit contributes to the pathology. Here these effects were found to be recapitulated in mice in which the Uchl1 gene had been inactivated by homologous recombination. In addition to the previously documented neuropathology associated with loss of UCHL1 function, axonal swellings were detected in the striatum. In agreement with previously reported findings the loss of UCHL1 function was accompanied by perturbations in ubiquitin pools, but glutathione levels were also significantly depleted in the brains of the knockout mice, suggesting that oxidative defense mechanisms may be doubly compromised. To determine if, in addition to its role in the central nervous system, UCHL1 function is also required for homeostasis of the enteric nervous system the gastrointestinal tract was analyzed in UCHL1 knockout mice. The mice displayed functional changes and morphological changes in gut neurons that preceded degenerative changes in the brain. The changes were qualitatively and quantitatively similar to those observed in wild type mice of much greater age, and strongly resemble changes reported for elderly humans. UCHL1 knockout mice should therefore serve as a useful model of gut aging

Changes in neuromuscular structure and functions of human colon during ageing are region-dependent

Objective: To determine if human colonic neuromuscular functions decline with increasing age.Design: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35–91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35–60 years) groups.Results: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI ), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthaseimmunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/gtissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon.Conclusion: In ascending not descending colon, ageing impairs cholinergic function

Ageing of enteric neurons: oxidative stress, neurotrophic factors and antioxidant enzymes

Background: Ageing is associated with gastrointestinal dysfunction, which can have a major impact on quality of life of the elderly. A number of changes in the innervation of the gut during ageing have been reported, including neuronal loss and degenerative changes. Evidence indicates that reactive oxygen species (ROS) are elevated in ageing enteric neurons, but that neurotrophic factors may reduce generation of neuronal ROS. Two such factors, glial cell line derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3) have also been found to protect enteric neurons against oxidative stress induced cell death of enteric ganglion cells in vitro. We have investigated the possible roles of neurotrophic factors further, by examining their expression in the gut during ageing, and by analysing their effects on antioxidant enzyme production in cultures of enteric ganglion cells. Results: Analysis of the expression of GDNF and its receptors c-Ret and GFR α − 1 in rat gut by RT-PCR showed that expression continues throughout life and into ageing, in both ad libitum(AL) and calorically-restricted (CR) animals. Levels of expression of GDNF and GFR α − 1 were elevated in 24 month AL animals compared to 24 month CR animals, and to 24 CR and 6 month control animals respectively. The related factor Neurturin and its receptor GFR α − 2 were also expressed throughout life, the levels of the GFR – α-2(b) isoform were reduced in 24 m AL animals. Immunolabelling showed that c-Ret and GFR α − 1 proteins were expressed by myenteric neurons in ageing animals. GDNF, but not NT-3, was found to increase expression of Cu/Zn superoxide dismutase and catalase by cultured enteric ganglion cells. Conclusions: The neurotrophic factors GDNF and neurturin and their receptors continue to be expressed in the ageing gut. Changes in the levels of expression of GDNF , GFR α-1 and GFR α-2(b) isoform occurred in 24 m AL animals. GDNF, but not NT-3, increased the levels of antioxidant enzymes in cultured enteric ganglion cells, indicating a possible mechanism for the reported protective effect of GDNF against menadione-induced neuronal apoptosis in the ageing gut. Together these data suggest that GDNF family members may play a protective role in the gut throughout life, and support the suggestion that dysregulation of neurotrophic factor support could contribute to neuronal ageing in the gut

Postmitotic neurons develop a p21-dependent senescence-like phenotype driven by a DNA damage response

In senescent cells, a DNA damage response drives not only irreversible loss of replicative capacity but also production and secretion of reactive oxygen species (ROS) and bioactive peptides including pro-inflammatory cytokines. This makes senescent cells a potential cause of tissue functional decline in aging. To our knowledge, we show here for the first time evidence suggesting that DNA damage induces a senescence like state in mature postmitotic neurons in vivo. About 40 80% of Purkinje neurons and 20–40% of cortical, hippocampal and peripheral neurons in the myenteric plexus from old C57Bl ⁄ 6 mice showed severe DNA damage, activated p38MAPkinase, high ROS production and oxidative damage, interleukin IL-6 production, heterochromatinization and senescence-associated b-galactosidase activity. Frequencies of these senescence-like neurons increased with age. Short term caloric restriction tended to decrease frequencies of positive cells. The phenotype was aggravated in brains of late-generation TERC) ⁄ ) mice with dysfunctional telomeres. It was fully rescued by loss of p21(CDKN1A) function in late- generation TERC) ⁄ )CDKN1A) ⁄ ) mice, indicating p21 as the necessary signal transducer between DNA damage response and senescence-like phenotype in neurons, as in senescing fibroblasts and other proliferation competent cells. We conclude that a senescence-like phenotype is possibly not restricted to proliferation-competent cells. Rather, dysfunctional telomeres and ⁄ or accumulated DNA damage can induce a DNA damage response leading to a phenotype in postmitotic neurons that resembles cell senescence in multiple features. Senescence-like neurons might be a source of oxidative and inflammatory stress and a contributor to brain aging

Age-Associated mRNA and miRNA Expression Changes in the Blood-Brain Barrier

Functional and structural age-associated changes in the blood-brain barrier (BBB) may affect the neurovascular unit and contribute to the onset and progression of age-associated neurodegenerative pathologies, including Alzheimer’s disease. The current study interrogated the RNA profile of the BBB in an ageing human autopsy brain cohort and an ageing mouse model using combined laser capture microdissection and expression profiling. Only 12 overlapping genes were altered in the same direction in the BBB of both ageing human and mouse cohorts. These included genes with roles in regulating vascular tone, tight junction protein expression and cell adhesion, all processes prone to dysregulation with advancing age. Integrated mRNA and miRNA network and pathway enrichment analysis of the datasets identified 15 overlapping miRNAs that showed altered expression. In addition to targeting genes related to DNA binding and/or autophagy, many of the miRNAs identified play a role in age-relevant processes, including BBB dysfunction and regulating the neuroinflammatory response. Future studies have the potential to develop targeted therapeutic approaches against these candidates to prevent vascular dysfunction in the ageing brain