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postgraduate thesis: A functional study of human synaptogyrin 3 : implications in the pathogenesis of Parkinson's disease
Title | A functional study of human synaptogyrin 3 : implications in the pathogenesis of Parkinson's disease |
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Authors | |
Advisors | |
Issue Date | 2019 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Li, L. [李凌菲]. (2019). A functional study of human synaptogyrin 3 : implications in the pathogenesis of Parkinson's disease. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Parkinson’s disease (PD) is a chronic neurodegenerative movement disorder
characterized by progressive loss of nigrostriatal neurons. Striatal presynaptic
dysfunction causes dopamine (DA) imbalance and nigrostriatal neurodegeneration in
PD. Striatal synaptosomes from mice with LRRK2 R1441G knockin (KI) mutation
were found to be more susceptible to dopamine uptake and locomotor deficits induced
by reserpine and rotenone compared with age- and sex- matched wildtype (WT) mice.
A subsequent study by members of our laboratory found that synaptogyrin 3
(SYNGR3) mRNA concentration was reduced in DA neurons isolated from the
substantia nigra of aged LRRK2 R1441G KI mice compared with levels in WT
controls, using Mouse Parkinson's Disease PCR Arrays. SYNGR3 is a synaptic
vesicular protein reported to be involved in DA uptake. The interrelationship among
DA uptake, SYNGR3 and LRRK2 in WT mice is unclear. Furthermore, how the
LRRK2 R1441G mutation affects this interrelationship is unknown.
iii
Hence, in this study, I explored: a) the expression of SYNGR3 protein in WT and
LRRK2 R1441G KI mice, b) how expression of a synaptic vesicle protein, SYNGR3,
affects DA uptake in neuronal cells, c) the possible regulatory mechanisms controlling
SYNGR3 expression by promoter analysis of its 5'-flanking region.
Firstly, SDS-PAGE/western blotting and ELISA of mouse whole striatal lysates
were performed to explore genotype and aging effects on striatal SYNGR3 protein
expression. Total SYNGR3 protein levels in striatum were reduced in LRRK2
R1441G KI mice compared with WT controls. Secondly, the overexpression of
SYNGR3 in dopaminergic SH-SY5Y cells increased cellular DA uptake. Lastly, gene
promoter studies of human SYNGR3 gene revealed a core promoter element specific
to most TATA-less gene promoters, X core promoter element 1 (XCPE1), which is
critical for SYNGR3 gene transcription. Moreover, three consensus-like sequences
‘AGGTCA’, NGFI-B response element-like (NBRE-like) sites were identified in the
5'-flanking region of SYNGR3 gene, indicating Nurr1 may regulate SYNGR3 gene
expression. Site-directed mutagenesis assay found that Nurr1 had a weak, but
significant effect in terms of transactivation of SYNGR3 promoter in human
neuroblastoma SH-SY5Y cell line, but not in non-neuronal HEK293 cell line. The
specificity of Nurr1 binding to NBRE-like sites was confirmed by electrophoretic
mobility shift assay. Treatment of Nurr1 activator, C-DIM12, in SH-SY5Y cells
significantly increased total cellular SYNGR3 promoter activity and protein
expression.
iv
These findings suggest that reduced SYNGR3 expression may be a reason of
higher susceptibility to synaptic dysfunction in LRRK2 R1441G KI mice. Inducing
SYNGR3 expression by Nurr1 activators may be a novel therapeutic strategy to
attenuate synaptic dysfunction in PD. |
Degree | Doctor of Philosophy |
Subject | Membrane proteins Parkinson's disease - Animal models Parkinson's disease - Pathogenesis Synaptic vesicles |
Dept/Program | Medicine |
Persistent Identifier | http://hdl.handle.net/10722/281297 |
DC Field | Value | Language |
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dc.contributor.advisor | Ho, SL | - |
dc.contributor.advisor | Ho, WL | - |
dc.contributor.author | Li, Lingfei | - |
dc.contributor.author | 李凌菲 | - |
dc.date.accessioned | 2020-03-10T08:46:33Z | - |
dc.date.available | 2020-03-10T08:46:33Z | - |
dc.date.issued | 2019 | - |
dc.identifier.citation | Li, L. [李凌菲]. (2019). A functional study of human synaptogyrin 3 : implications in the pathogenesis of Parkinson's disease. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/281297 | - |
dc.description.abstract | Parkinson’s disease (PD) is a chronic neurodegenerative movement disorder characterized by progressive loss of nigrostriatal neurons. Striatal presynaptic dysfunction causes dopamine (DA) imbalance and nigrostriatal neurodegeneration in PD. Striatal synaptosomes from mice with LRRK2 R1441G knockin (KI) mutation were found to be more susceptible to dopamine uptake and locomotor deficits induced by reserpine and rotenone compared with age- and sex- matched wildtype (WT) mice. A subsequent study by members of our laboratory found that synaptogyrin 3 (SYNGR3) mRNA concentration was reduced in DA neurons isolated from the substantia nigra of aged LRRK2 R1441G KI mice compared with levels in WT controls, using Mouse Parkinson's Disease PCR Arrays. SYNGR3 is a synaptic vesicular protein reported to be involved in DA uptake. The interrelationship among DA uptake, SYNGR3 and LRRK2 in WT mice is unclear. Furthermore, how the LRRK2 R1441G mutation affects this interrelationship is unknown. iii Hence, in this study, I explored: a) the expression of SYNGR3 protein in WT and LRRK2 R1441G KI mice, b) how expression of a synaptic vesicle protein, SYNGR3, affects DA uptake in neuronal cells, c) the possible regulatory mechanisms controlling SYNGR3 expression by promoter analysis of its 5'-flanking region. Firstly, SDS-PAGE/western blotting and ELISA of mouse whole striatal lysates were performed to explore genotype and aging effects on striatal SYNGR3 protein expression. Total SYNGR3 protein levels in striatum were reduced in LRRK2 R1441G KI mice compared with WT controls. Secondly, the overexpression of SYNGR3 in dopaminergic SH-SY5Y cells increased cellular DA uptake. Lastly, gene promoter studies of human SYNGR3 gene revealed a core promoter element specific to most TATA-less gene promoters, X core promoter element 1 (XCPE1), which is critical for SYNGR3 gene transcription. Moreover, three consensus-like sequences ‘AGGTCA’, NGFI-B response element-like (NBRE-like) sites were identified in the 5'-flanking region of SYNGR3 gene, indicating Nurr1 may regulate SYNGR3 gene expression. Site-directed mutagenesis assay found that Nurr1 had a weak, but significant effect in terms of transactivation of SYNGR3 promoter in human neuroblastoma SH-SY5Y cell line, but not in non-neuronal HEK293 cell line. The specificity of Nurr1 binding to NBRE-like sites was confirmed by electrophoretic mobility shift assay. Treatment of Nurr1 activator, C-DIM12, in SH-SY5Y cells significantly increased total cellular SYNGR3 promoter activity and protein expression. iv These findings suggest that reduced SYNGR3 expression may be a reason of higher susceptibility to synaptic dysfunction in LRRK2 R1441G KI mice. Inducing SYNGR3 expression by Nurr1 activators may be a novel therapeutic strategy to attenuate synaptic dysfunction in PD. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | Membrane proteins | - |
dc.subject.lcsh | Parkinson's disease - Animal models | - |
dc.subject.lcsh | Parkinson's disease - Pathogenesis | - |
dc.subject.lcsh | Synaptic vesicles | - |
dc.title | A functional study of human synaptogyrin 3 : implications in the pathogenesis of Parkinson's disease | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Medicine | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_991044104147603414 | - |
dc.date.hkucongregation | 2019 | - |
dc.identifier.mmsid | 991044104147603414 | - |