Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.695769
Title: Examining CLN7 function in Drosophila
Author: O'Hare, Megan Beatrice
ISNI:       0000 0004 5991 0623
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2016
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Abstract:
My thesis aims to establish the normal functional role of the transmembrane protein CLN7; mutations in Cln7 cause a debilitating neurodegenerative disorder in infants known as Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). NCL are a collection of early-onset inherited neurodegenerative disorders characterised by abnormal accumulations of autofluorescent storage material within lysosomal organelles. Mutations in thirteen genes have so far been identified in patients across all forms of NCL and disease models established, however no model has been generated for late infantile CLN7 disease. I propose to generate a knock out mutant in order to establish Drosophila as a model to study the cellular role of the hitherto functionally unknown CLN7. Initially I determined a potential shared conservational functionality between Drosophila CLN7 homologue, CG8596, and human MFSD8/CLN7 by establishing a similar subcellular localisation pattern within the endo-lysosomal pathway in vitro. In order to study the functional role of CLN7 I designed an imprecise P-element excision screen, which yielded three genetically null mutants for Cln7. Characterisation of the mutants revealed loss of Cln7 in Drosophila did not recapitulate the hallmark intralysosomal storage material accumulation whilst also causing a profound lifespan extension. However, studying the developing synapse in late stage larvae revealed loss of Cln7 to have a significant effect on a specific motoneuron innervation at the NMJ. Behavioural assays showed Cln7-/- larvae displayed altered larval locomotion; mutant animals were unable to maintain consistent movement and showed impaired complex movement. Defects in tonic motoneuron innervation were observed in Cln7 loss of function animals suggesting their intrinsic properties make them more vulnerable to loss of Cln7 compared to the unaffected phasic motoneurons. As a consequence of reduced synaptic input from tonic motoneurons, physiological studies revealed Cln7-/- animals display characteristic phasic motor input in the form of synaptic depression and larger recorded EPSPs. Examination of model systems and patient autopsy tissue for other NCL forms have revealed severe CNS regional-specific neuronal death underpins the disorder, whilst cellular studies reveal a multitude of mechanisms of pathology including cellular pathways such as autophagy and oxidative stress. I carried out genetic interaction studies focussing on the NMJ, these suggest CLN7 functions downstream of autophagy but upstream of mTORC2 to regulate synapse development.
Supervisor: Tear, Guy Justin Clive Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.695769  DOI: Not available
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