Mark Yeoman

Insights into CNS ageing from animal models of senescence

In recent years, novel model systems have made significant contributions to our understanding of the processes that control the ageing of whole organisms. However, there are limited data to show that the mechanisms that gerontologists have identified as having a role in organismal ageing contribute significantly to the ageing of the central nervous system. Two recent discoveries illustrate this particularly well. The first is the consistent failure of researchers to demonstrate a simple relationship between organismal ageing and oxidative stress — a mechanism often assumed to have a primary role in brain ageing. The second is the demonstration that senescent cells play a causal part in organismal ageing but remain essentially unstudied in a CNS context. We argue that the animal models now available (including rodents, flies, molluscs and worms), if properly applied, will allow a paradigm shift in our current understanding of the normal processes of brain ageing.

Renin- angiotensin- system gene polymorphisms and depression

Given the abundance of the renin-angiotensin system (RAS) components in the brain, their importance in behavior and cognition, and the data that implicates them in the etiology and treatment of depression, it is possible that those RAS gene polymorphisms associated with increased RAS activity may also be associated with depression. The frequencies of common polymorphisms of genes encoding for components of the RAS, namely angiotensinogen (M235T), angiotensin converting enzyme (ACE) (insertion, I; deletion, D), angiotensin receptor type I (A1166C), and angiotensin receptor type II (C3123A) were determined in DNA extracted from buccal cells from a Lebanese population of 132 depressed patients and their first-degree relative case-controls. The angiotensin receptor type 1 (A1166C) CC genotype was significantly associated with depression (p=0.036). None of the other common RAS-associated polymorphisms were significantly associated. The results support the hypothesis that increased RAS activity may increase relative risk of depression in that the angiotensin receptor type 1 (A1166C) CC genotype is associated with increased responsiveness to angiotensin II.

Matrix-assisted laser desorption/ionization time of flight mass spectrometric analysis of the pattern of peptide expression in single neurons resulting from alternative mRNA splicing of the FMRFamide gene

MALDI‐ToF MS (matrix‐assisted laser desorption/ionization time of flight mass spectrometry) has become a fast, reliable and sensitive technique for the identification of neuropeptides in biological tissues. Here, we applied this technique to identified neurons of the cardioregulatory network in the snail Lymnaea that express the FMRFamide gene. This enabled us to study the complex processing of the FMRFamide gene at the level of single identified neurons. In the CNS of Lymnaea, FMRFamide‐like and additional peptides are encoded by a common, multiexon gene. Alternate mRNA splicing of the FMRFamide gene leads to the production of two different mRNAs. Type 1 mRNA (exon II) encodes for the tetrapeptides (FLRF/FMRFamide), whereas Type 2 (exons III–V) encodes for the heptapeptides (SDPFLRFamide/GDPFLRFamide). Previous in situ hybridization and immunocytochemical studies indicated that these two transcripts are expressed in the CNS neurons of Lymnaea in a differential and mutually exclusive manner. Two single identified neurons of the cardiorespiratory network, the Ehe neuron and the visceral white interneuron (VWI), were known to express the FMRFamide gene (Ehe, type 1 mRNA; VWI, type 2 mRNA). MALDI‐ToF MS analysis of these neurons and other neurons expressing the FMRFamide gene confirmed the mutually exclusive expression of the distinct sets of peptides encoded on the two transcripts and revealed the pattern of post‐translational processing of both protein precursors. From the gene sequence it was predicted that 16 final peptide products from the two precursor proteins could possibly exist. We showed that most of these peptides were indeed present in the identified neurons (13) while others were not (three), suggesting that not all of the potential cleavage sites within the two precursors are utilized. In this way, the neuronal expression of the full range of the peptide products resulting from alternative mRNA splicing was revealed for the first time.

Processing of the FMRFamide Precursor Protein in the Snail Lymnaea stagnalis: Characterization and Neuronal Localization of a Novel Peptide, ‘SEEPLY’

In the pulmonate snail Lymnaea stagnalis, FMRFamide‐like neuropeptides are encoded by a multi‐exon genomic locus which is subject to regulation at the level of mRNA splicing. We aim to understand the post‐translational processing of one resulting protein precursor encoding the tetrapeptide FMRFamide and a number of other putative peptides, and determine the distribution of the final peptide products in the central nervous system (CNS) and periphery of Lymnaea. We focused on two previously unknown peptide sequences predicted by molecular cloning to be encoded in the tetrapeptide protein precursor consecutively, separated by the tetrabasic cleavage site RKRR. Here we report the isolation and structural characterization of a novel non‐FMRFamide‐like peptide, the 22 amino acid peptide SEQPDVDDYLRDWLQSEEPLY. The novel peptide is colocalized with FMRFamide in the CNS in a number of identified neuronal systems and their peripheral motor targets, as determined by in situ hybridization and immunocytochemistry. Its detection in heart excitatory motoneurons and in nerve fibres of the heart indicated that the novel peptide may play a role, together with FMRFamide, in heart regulation in the snail. The second predicted peptide, STEAGGQSEEMTHRTA (16 amino acids), was at very low abundance in the CNS and was only occasionally detected. Our current findings, suggestive of a distinct pattern of post‐translational processing, allowed the reassessment of a previously proposed hypothesis that the two equivalent sequences in the Aplysia FMRFamide gene constitute a molluscan homologue of vertebrate corticotrophin releasing factor‐like peptides.

Myenteric neuron numbers are maintained in aging mouse distal colon

Age‐associated myenteric neuronal loss has been described in several species. In some studies, cholinergic neurons have been reported to be selectively vulnerable, whereas nitrergic neurons are spared. Aging of the mouse enteric nervous system (ENS) and the subtypes of mouse myenteric neurons that may be lost have been little studied. We therefore investigated changes in the numbers of total neurons and two neuronal subpopulations in the mouse distal colon during aging.

Ageing and the nervous system: insights from studies on invertebrates.

Ageing can have profound effects on the post-mitotic organ ofbehaviour, the brain. As yet the precise causes of thesedeleterious effects are unknown. However, clear insights intothe putative mechanisms and consequences of ageing in the CNShave been achieved through the use of invertebrate models. It isnow clear that ageing alters the endogenous properties ofneurones, their morphology, the efficacy of the connections thatthe neurones make with their targets and may even lead to neuroneloss. While the precise mechanisms underlying these changes arepresently unclear clues from post-mitotic organisms such as C.elegans have provided putative targets which are currently beinginvestigated. It is clear to date that the age-induced changesin CNS function observed in invertebrates are conserved inmammalian species and that further work on invertebrates mayprovide informative insights in to the mechanisms of neuronalageing.

Identification, distribution and physiological activity of three novel neuropeptides of Lymnaea: EFLRlamide and pQFYRlamide encoded by the FMRFamide gene, and a related peptide.

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamideand QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif –FXRlamide. In this paper we adopt a novel approach to first identify and characterize –FXRlamide‐like peptides in extracts from the central nervous system of Lymnaea. By a combination of high‐performance liquid chromatography (HPLC) and continuous‐flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post‐translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immuno reactive to all three peptides, revealed EFLRlamide‐like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post‐translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardio excitatory motoneurons, the Ehe cells (E heart excitors of the visceral ganglion) and penialmotoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that the serespective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide‐like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many centralneurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart‐beat: an initial inhibitory response was followed by a long‐lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post‐translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.

Age-related changes in melatonin release in the murine distal colon

Constipation and fecal impaction are conditions of the bowel whose prevalence increases with age. Limited information is known about how these conditions manifest; however, functional deficits are likely to be due to changes in signaling within the bowel. This study investigated the effects of age on colonic mucosal melatonin (MEL) release and the consequences this had on colonic motility. Electrochemical measurements of MEL overflow demonstrated that both basal and mechanically stimulated MEL release decreased with age. The MEL/serotonin also decreased with increasing age, and the trend was similar to that of MEL overflow, suggestive that age-related changes were primarily due to a reduction in MEL levels. Levels of N-acetylserotonin and the N-acetylserotonin/serotonin ratio were reduced with age, providing an explanation for the reduction in MEL release. Decreases in colonic motility were observed in animals between 3 and 24 months old. Exogenous application of MEL could reverse this deficit in aged colon. In summary, we propose that the age-related decline in MEL release may be due to either decreases or alterations in mechanosensory channels and/or a loss in levels/activity of the N-acetyltransferase enzyme responsible for the synthesis of N-acetylserotonin. Decreases in MEL release may explain the decreases in colonic motility observed in 24 month old animals and could offer a new potential therapeutic treatment for age-related constipation.

Synapse‐specific changes in serotonin signalling contribute to age‐related changes in the feeding behaviour of the pond snail, Lymnaea

This study utilised the pond snail, Lymnaea to examine the contribution that alterations in serotonergic signalling make to age‐related changes in feeding. Age‐related decreases in 5‐HIAA levels in feeding ganglia were positively correlated with a decrease in the number of sucrose‐evoked bites and negatively correlated with an increase in inter‐bite interval, implicating alterations in serotonergic signalling in the aged phenotype. Analysis of the serotonergic cerebral giant cell (CGC) input to the protraction motor neurone (B1) demonstrated that fluoxetine (10–100 nM) increased the amplitude/duration of the evoked EPSP in both young and middle aged but not in old neurones, suggesting an age‐related attenuation of the serotonin transporter. 5‐HT evoked a concentration‐dependent increase in the amplitude/duration of B1 EPSP, which was greater in old neurones compared to both young and middle aged. Conversely, the 5‐HT‐evoked depolarisation and conditional bursting of the swallow motor neurone (B4) were attenuated in old neurones, functions critical for a full feeding rhythm. The CGCs’ ability to excite B1 was blocked by cinanserin but not by methysergide. Conversely, the CGC to B4 connection was completely blocked by methysergide and only partially by cinanserin suggesting that age‐related changes may be receptor‐specific. In summary, synapse‐specific attenuation of the CGC‐B4 connection and enhancement of the CGC‐B1 connection would slow the swallow phase and maintain protraction, consistent with behavioural observations.

Impaired colonic motility and reduction in tachykinin signalling in the aged mouse

Ageing is associated with an increased incidence of constipation in humans. The contribution that the ageing process makes to this condition is unclear. The aim of this study was to determine the effects of age on faecal output and colonic motility in male C57BL/6J mice and to determine the role that altered tachykinin signalling plays in this process. Total faecal output recorded over a 24h period decreased with age due to a reduction in the number of pellets produced and their water content. These changes occurred in the absence of any significant change in food and water intake. There was an increase in the amount of faecal matter stored in the isolated colon with age which caused a proportional increase in colonic length. Analysis of colonic motility using an artificial pellet demonstrated that pellets moved in a stepwise fashion through the colon. There was an age-related increase in pellet transit time due to decreases in the step distance, velocity, and frequency of stepwise movements. These changes were reversed using the neurokinin 2 (NK2) receptor agonist neurokinin A. Addition of the NK2receptor antagonist GR159897 significantly increased transit time in the young animals by decreasing step distance, velocity and frequency, but was without effect in the aged colon. In summary, the ageing C57BL/6J mouse shows an impaired motility phenotype. These effects appear, at least in part, to be due to an attenuation of tachykinin signalling via NK2 receptors.