S-Space College of Medicine/School of Medicine (의과대학/대학원) Dept. of Biomedical Sciences (대학원 의과학과) Theses (Ph.D. / Sc.D._의과학과)
The role of SCAMP5 in modulating synaptic vesicle endocytosis during neuronal activity
신경활성상태에서 SCAMP5을 통한 시냅스낭 순환과정에 대한 조절기전
- 의과대학 의과학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2015. 2. 장성호.
- Neurotransmitter release is the key process to initiate synaptic transmission at the presynaptic nerve terminal. Upon stimulation, synaptic vesicles (SVs) that contain neurotransmitters exocytose and fuse with the plasma membrane to release neurotransmitters. To maintain continuous synaptic transmission, once fused SVs should be recycled with endocytosis. Although several synaptic vesicle proteins have been implicated in these events, their precise function in SV trafficking remain largely elusive.
Secretory carrier membrane proteins (SCAMPs) are tetraspan vesicle membrane proteins. SCAMPs are comprised of an N-terminal tail (N-term), four transmembrane regions (TM1-4), the loop region between TM2 and TM3 (2/3 loop) and C-terminal cytoplasmic tail (C-term). Among five currently known isoforms of SCAMPs (SCAMP1-5), SCAMP1-4 are ubiquitously expressed while SCAMP5 is mainly expressed in the brain and is highly abundant in SVs. SCAMPs 1-3 are known to play not only in regulation of fusion pore formation during dense-core vesicle (DCV) secretion in PC12 cells at the plasma membrane but also in trafficking events in the trans-golgi network (TGN) and endosomal recycling compartment, suggesting their fundamental function in vesicular traffic. Recently SCAMP5 is identified as a candidate gene for autism and its expression is found to be reduced to < 40% in a patient with idiopathic, sporadic autism. Moreover, SCAMP5 expression is also found to be markedly increased in the striatum of Huntingtons disease. Therefore, the change in the expression of SCAMP5 may be related to the synaptic dysfunction observed in these diseases, but none is known for SCAMP5 role at the synapse so far.
The aim of the present study is to reveal the role of SCAMP5 in presynaptic function. For this purpose, I used SCAMP5-specific shRNAs to knock down (KD) endogenous expression of SCAMP5 in cultured rat hippocampal neurons. Here, I found that SCAMP5 KD induced reduction in total SVs pool size concomitant with decreases in both recycling and resting pool size but the ratio of recycling/resting pool was significantly increased. I also found that although exocytotic kinetics was unaffected by SCAMP5 KD, SCAMP5 KD slowed down endocytosis after stimulation but the most severely impaired was SV endocytosis during stimulation.The endocytic defects were independently verified using FM1-43. I further found that SCAMP5 KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during stimulation. The endocytosis defect in SCAMP5 KD was rescued by co-expression of shRNA-resistant SCAMP5. To find out which domain(s) of SCAMP5 is responsible for endocytosis regulation, I overexpressed N-term, 2/3 loop, or C-term and found that overexpression SCAMP5 N-term led to slower endocytosis during strong stimulation which is similar to the endocytosis defects found in SCAMP5 KD. In addition, I found that SCAMP5 binds to the AP-2 through its N-terminal YXXΦ motif. Accordingly, overexpression of N-term YXXΦ point mutant (Y22A) failed to induce endocytic defects during stimulation.
In summary, my results suggest that SCAMP5 mainly functions during high neuronal activity when a heavy load is imposed on endocytosis. Considering recent findings in which SCAMP5 expression is either increased or decreased in Huntington disease or autism patient, my data also raise the possibility that a reduction or an increase of SCAMP5 expression in Huntington or autism patient may be related to synaptic dysfunction observed in each disease.