14-3-3 proteins are a ubiquitous family known for their ability to regulate myriad cellular processes including lifespan, neuronal signalling and transduction, protein trafficking and transmitter production and release. Previous work has shown that 14-3-3 proteins are linked to a variety of pathological neurodegenerative diseases although their role in healthy brain ageing is currently unclear. This study utilised the pond snail, Lymnaea stagnalis to examine the contribution made by 14-3-3 to the decrease in feeding rate that is seen in this model system with age. Interrogation of a Lymnaea CNS cDNA library identified four putative 14-3-3 isoforms, one highly similar to the mammalian 14-3-3ε (14-3-3Lyml), while the other three (14-3- 3 Lym2, 14-3-3Lym3 and 14-3-3Lym3var) were more distinct. Expression, localisation and function of these proteins were studied using a range of biochemical and analytical techniques, in three different animal age groups (3, 6-7 and 10-12 months). Western blot (W8) showed a significant decrease in the overall level of 14-3-3Lym3 in the cerebro-buccal ganglia that correlated with feeding rate. There was no overall change in the expression of 14-3-3Lyml and 2. 14-3-3Lym3var was not detected. CNS 14-3-3 expression was seen in all 11 ganglia including the cerebral and buccal ganglia which are important for regulating feeding. 14-3-3Lyml expression was limited to the neuronal cell cytoplasm and plasma membrane, whereas the remaining isoforms appeared to be distributed throughout the cell, including the nucleus. Expression was shown in key neurones that regulate feeding including identified dopaminergic and serotonergic neurones. 14-3-3 proteins have previously been shown to regulate the synthesis of both dopamine (DA) and serotonin (S-HT) through actions on tyrosine and tryptophan hydroxylase (TH and TPH respectively), the rate-limiting enzymes in their production. HPLC analysis demonstrated that antagonism of 14-3-3 proteins with R18 significantly reduced the production of L-DOPA and S-HTP in the cerebral and buccal ganglia, suggesting that 14-3-3 proteins can regulate DA and S-HT production in these areas. In summary, the 14-3-3Lym proteins are capable of regulating the activity of TH and TPH and the change in expression pattern of these proteins with age may explain the noted age-related changes in S-HT and DA signalling in the cerebro-buccal ganglia and the consequential decrease in feeding rate seen with age.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589945 |
| Date | January 2012 |
| Creators | Morgan, Lindsay Dawn |
| Publisher | University of Brighton |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://research.brighton.ac.uk/en/studentTheses/e356047e-62f2-4905-bcef-c995044ceb44 |
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