File Download
Supplementary
-
Citations:
- Appears in Collections:
postgraduate thesis: Cloning and characterization of heparanase 2 in rat brain
Title | Cloning and characterization of heparanase 2 in rat brain |
---|---|
Authors | |
Issue Date | 2016 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Lau, W. [劉泳欣]. (2016). Cloning and characterization of heparanase 2 in rat brain. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5736654. |
Abstract | Synaptic activity modulates the density of synaptic AMPARs, thereby affecting synaptic function, learning and memory. The molecular mechanisms regulating AMPAR trafficking is under intense interest. Heparan sulfates (HS) of perineuronal proteoglycans are implicated in controlling the open-state of AMPA receptor-ion channels for maintaining synaptic strength. Heparanase 1 (Hpa1) is an endoglycosidase that cleaves heparan sulfate (HS) polysaccharides on HS proteoglycans (HSPGs). Its pro-form which is however enzymatically inactive induces internalization of HSPGs and AMPARs at post-synaptic terminal in neurons, resulting in decrease in synaptic strength and long-term potentiation (LTP). Its homolog, heparanase 2 (Hpa2), with about 40% homology is found expressed in the human brain, were found interacting with heparanase 1. We hypothesize that Hpa2 is expressed in rat brain and secreted from the hippocampal neurons, interact with hpa1 and regulate synaptic plasticity.
In this project the expression, localization and secretion of Hpa2 were studied in rat brain. It is found heparanase 2 expression is highest among different distinct brain region. Hippocampal DIV14-16 neurons in culture from embryonic (E)17-18 rats and adult rat brain slices are adopted for localization analysis. Immunohistochemical staining on brain slice demonstrated that Hpa2 was localized at the CA1, CA3 and DG region in the hippocampus and Fix cell immunocytochemistry showed Hpa2 was co-localized with syndecan-3 in the nucleus, while in live cell staining hpa2 were found on cell surface co-localized with unknown HSPG. RT-PCR and Western blot analysis showed the presence of Hpa2 in hippocampal neurons at in In our hippocampal neuron culture model, Hpa2 is secreted from hippocampal neurons culture spontaneously. We pursue to study the signaling that regulates the neuronal production/secretion of Hpa2. Hpa2 exocytosis by the hippocampal neurons was enhanced by protein kinase C (PKC) activation and protein kinase A (PKA) inhibition.
To figure out if hpa2 release is activity dependent, TTX and BIC were added to the hippocampal neuron cultures to induce synaptic scaling. Yet, hpa2 release and synthesis is not affected.
In order to fully understand the function of hpa2, we aimed to produce recombinant hpa2. We discovered a new isoform of rat heparanase 2 which has 98% of similarity with human hpa2. The hpa2 isoform is successfully cloned and a stable expression cell line of hpa2 in HEK293 cell is developed.
Taken together, the findings from the experiments support the hypothesis to show Hpa2 is expressed, localized and secreted in the rat hippocampal and hippocampal neurons. However, the function of heparanase 2 in hippocampus, especial its role in regulation of synaptic plasticity remain to be revealed. |
Degree | Master of Philosophy |
Subject | Heparin Brain |
Dept/Program | Biomedical Sciences |
Persistent Identifier | http://hdl.handle.net/10722/238186 |
HKU Library Item ID | b5736654 |
DC Field | Value | Language |
---|---|---|
dc.contributor.author | Lau, Wing-yan | - |
dc.contributor.author | 劉泳欣 | - |
dc.date.accessioned | 2017-02-04T00:18:15Z | - |
dc.date.available | 2017-02-04T00:18:15Z | - |
dc.date.issued | 2016 | - |
dc.identifier.citation | Lau, W. [劉泳欣]. (2016). Cloning and characterization of heparanase 2 in rat brain. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b5736654. | - |
dc.identifier.uri | http://hdl.handle.net/10722/238186 | - |
dc.description.abstract | Synaptic activity modulates the density of synaptic AMPARs, thereby affecting synaptic function, learning and memory. The molecular mechanisms regulating AMPAR trafficking is under intense interest. Heparan sulfates (HS) of perineuronal proteoglycans are implicated in controlling the open-state of AMPA receptor-ion channels for maintaining synaptic strength. Heparanase 1 (Hpa1) is an endoglycosidase that cleaves heparan sulfate (HS) polysaccharides on HS proteoglycans (HSPGs). Its pro-form which is however enzymatically inactive induces internalization of HSPGs and AMPARs at post-synaptic terminal in neurons, resulting in decrease in synaptic strength and long-term potentiation (LTP). Its homolog, heparanase 2 (Hpa2), with about 40% homology is found expressed in the human brain, were found interacting with heparanase 1. We hypothesize that Hpa2 is expressed in rat brain and secreted from the hippocampal neurons, interact with hpa1 and regulate synaptic plasticity. In this project the expression, localization and secretion of Hpa2 were studied in rat brain. It is found heparanase 2 expression is highest among different distinct brain region. Hippocampal DIV14-16 neurons in culture from embryonic (E)17-18 rats and adult rat brain slices are adopted for localization analysis. Immunohistochemical staining on brain slice demonstrated that Hpa2 was localized at the CA1, CA3 and DG region in the hippocampus and Fix cell immunocytochemistry showed Hpa2 was co-localized with syndecan-3 in the nucleus, while in live cell staining hpa2 were found on cell surface co-localized with unknown HSPG. RT-PCR and Western blot analysis showed the presence of Hpa2 in hippocampal neurons at in In our hippocampal neuron culture model, Hpa2 is secreted from hippocampal neurons culture spontaneously. We pursue to study the signaling that regulates the neuronal production/secretion of Hpa2. Hpa2 exocytosis by the hippocampal neurons was enhanced by protein kinase C (PKC) activation and protein kinase A (PKA) inhibition. To figure out if hpa2 release is activity dependent, TTX and BIC were added to the hippocampal neuron cultures to induce synaptic scaling. Yet, hpa2 release and synthesis is not affected. In order to fully understand the function of hpa2, we aimed to produce recombinant hpa2. We discovered a new isoform of rat heparanase 2 which has 98% of similarity with human hpa2. The hpa2 isoform is successfully cloned and a stable expression cell line of hpa2 in HEK293 cell is developed. Taken together, the findings from the experiments support the hypothesis to show Hpa2 is expressed, localized and secreted in the rat hippocampal and hippocampal neurons. However, the function of heparanase 2 in hippocampus, especial its role in regulation of synaptic plasticity remain to be revealed. | - |
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 | Heparin | - |
dc.subject.lcsh | Brain | - |
dc.title | Cloning and characterization of heparanase 2 in rat brain | - |
dc.type | PG_Thesis | - |
dc.identifier.hkul | b5736654 | - |
dc.description.thesisname | Master of Philosophy | - |
dc.description.thesislevel | Master | - |
dc.description.thesisdiscipline | Biomedical Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.identifier.doi | 10.5353/th_b5736654 | - |
dc.identifier.mmsid | 991019345289703414 | - |