ECM (Enlargement and clustering mitochondria)是個具有鐵硫簇結合結構(CDGSH iron-sulfur domain)的蛋白，且具有高度的演化保留性。於小鼠實驗中發現缺乏ECM蛋白會導致粒線體失能和細胞死亡。而後我們比對了ECM之基因序列發現於果蠅中的同源基因。首先，透過果蠅細胞、果蠅的肌肉組織、幼蟲之唾腺細胞之免疫染色結果發現ECM坐落於粒線體上。先前研究結果中得知，在果蠅肌肉組織中過量表現ECM會造成粒線體膨大且聚集之現象，顯示出粒線體的恆定性出現問題，而粒線體的恆定可透過粒線體的自噬作用來調控。因此我們首先探討ECM與粒線體自噬之關聯性。其中，蛋白激酶磷酸化蛋白(PINK1)和泛素連結酶(Parkin)在粒線體自噬的機制中扮演著重要的角色，便藉由探討ECM與Parkin 和PINK1之關係，再進一步探討ECM是否會影響mitophagy的進行。PINK1或Parkin突變會導致ECM蛋白量累積，且ECM過量表現也可造成Parkin及PINK1的mRNA表現量下降。當ECM過量表現時會導致粒線體膨大且聚集、肌肉組織退化、壽命減短、爬行能力降低等，而此些現象也皆能夠藉由同時表現Parkin而恢復，由以上結果可顯示出ECM可能藉由調控Parkin和PINK1來影響mitophagy的進行，進而產生粒線體膨大卻無法代謝之現象。而進一步探討粒線體自噬各步驟之指標，當ECM過量表達會使泛素化(ubitquitination, FK2)和自噬體銜接蛋白(autophagic adaptor, Ref(2)p)以及自噬體蛋白(autophagosome marker, LC3)積聚在肌肉組織中，由此實驗結果指出ECM過量表達會抑制粒線體自噬過程中自噬體與溶酶體的融合，導致粒線體失常卻沒有被清除，粒線體因此而膨大且堆積於肌肉組織中。Parkin和PINK1突變是造成隱性的帕金森氏症(Parkinson's disease,PD)之致病原因之一，其病理特徵包含神經及肌肉之退化。而於果蠅之多巴胺神經中過量表現ECM會造成細胞數下降且粒線體膨大之現象；藉由網膜電氣生理試驗測試於成蟲眼睛中過量表現ECM之果蠅，其接收光刺激與光適應力皆降低；而將ECM過量表現於果蠅之蕈狀體中，會引起神經元退化且進一步降低果蠅學習及記憶之能力。由上述結果表明，ECM可能與神經退化有關。總結，我們於果蠅的肌肉與神經組織中，發現ECM可能是透過影響Parkin和PINK1所調控之粒線體自噬機制來造成粒線體之形態上的改變。而由實驗結果所指出由ECM造成的神經退化會隨著老化而趨於嚴重，可能與PINK1、Parkin 的mRNA會隨著年老而下降有關。而接下來我們將進一步探討ECM於粒線體自噬機制中所扮演之角色及機制。

The Enlargement and Clustering Mitochondria (ECM) gene is evolutionarily conserved, and encodes an iron-sulfur domain-containing protein. Previously, we identified a single ortholog of ECM in Drosophila based on genomic sequence alignment. In Drosophila, we found that ECM localizes to the mitochondria in the Drosophila S2 cell line and in the muscle of adult flies and salivary gland cells of larvae. The PTEN-induced putative protein kinase 1 (PINK1) and E3 ubiquitin ligase (Parkin) genes play central roles in the clearance of damaged or dysfunctional mitochondria in mitophagy, and mutations in Parkin and PINK1 are associated with an autosomal recessive form of parkinsonism, which suggests that these genes are involved in muscle and neuron degeneration. We investigated whether ECM is involved in Parkin/PINK1-mediated mitochondrial processes in Drosophila. The Parkin or PINK1 mutants resulted in an increase in the level of ECM protein and mRNA. On the other hand, Parkin and PINK1 mRNA level were reduced by ECM overexpression. The overexpression of ECM caused mitochondrial enlargement and revealed distinct vacuoles within the muscle mass that was rescued by coexpression of wild-type Parkin. When ECM overexpression were caused the ubiquitylation and autophagosome adaptor proteins and autophagosome marker were accumulated in muscle. These results suggested that the overexpression of ECM inhibited mitophagy. The overexpression of ECM in eyes of adult flies caused early neurodegeneration, as determined by electroretinogram assay. The overexpression of ECM in the mushroom body caused neuron degeneration, and diminished learning and memory performance. These results suggest that ECM may be involved in neurodegeneration. In summary, we found that ECM plays important roles in mitochondria homeostasis in the muscle cells and neurons of Drosophila through its effects on Parkin/PINK1-mediated signaling in mitophagy, and we propose that ECM links mitochondrial homeostasis in neuron degeneration with the aging process. Future investigations of the molecular mechanism underlying the effect of ECM on mitophagy are warranted.

中文摘要 I
Abstract II
Table of Contents III
Figure List V
Table List VI

Introduction 1
1. CDGSH iron-sulfur domain-containing (CISD) protein family 1
2. Mitochondria .3
3. Mitochondrial quality control 5
4. Molecular mechanism of PINK1- and Parkin- mediated mitophagy 6
5. Mitophagy and Parkinson's disease (PD)－ PINK1 and Parkin. 7
6. Mitochondrial dysfunciton 8
7.The aims of thesis 10

Materials and Methods 11
1. Drosophila genetics and stains 11
2. Generation of Transgenic flies 11
3. Behavior assay - Climbing assay 11
4. Lifespan 11
5. Western Blot 12
6. Real-time PCR (qPCR) 14
7. Adult Drosophila muscle dissection and immuonofluorescence 16
8. Salivary glands of larvae dissection and immunofluorescence 16
9. Drosophila Brain dissection and immunofluorescence 17
10. Paraffin section of muscle 18
11. Electroretinogram (ERG) 18



Results 19
The isoform protein of ECM was a dimer form 19
ECM is a mitochondria -associated protein which was more abundance in muscle of flies 19
Overexpression of ECM effected the distribution and structure of endoplasmic reticulum (ER) 20
ECM protein level was under Parkin- and PINK1- mediated degradation 20
The Mitochondrial enlargement and clusters phenotype could be rescued by PINK1 and Parkin 22
ECM overexpression induced muscle degeneration and enlarged muscle endo/ perimysium structure 22
ECM overexpression leading the climbing ability to defect have no obviously different in ECM co-express PINK1 or Parkin 23
Parkin or PINK1 prolong the lifespan of the ECM-expressed fly 23
Progressive accumulation of polyubiquitinated protein and autophagy marker aggregates is associated with ECM overexpression 24
Autophagic flux was blocked by ECM overexpression and stopped at lysosome fuse with autophagosome 24
ECM overexpression caused neurodegeneration in dopaminergic neuron, which was decreased cell number and the mitochondrial morphology enlargement 25
ECM overexpression in fly eyes caused neurodegeneration 26
OK107-gal4 drive ECM in mushroom body causes γ neuron axonal loss 26
γ neuron axonal loss induced by ECM become severe during the aging 27
Mitochondrial Mislocalization Underlies ECM-Induced Neuronal Dysfunction in Mushroom body 27
Summary 28

Discussions and Conclusion 29
References 34
Figures 40
Tables 64

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