神經元強烈地依賴粒線體，因為粒線體對神經元的生長、生存和功能很重要。粒線體動力學的改變是各類神經退化性疾病常見的特徵。因此，研究粒線體動力學在分子基礎上如何調節至關重要。我們以綠色螢光蛋白標定線粒體的線蟲，探究軸突中線粒體的運輸情況。從反向基因篩選中，發現基因nhx-3降低ALM神經元近端軸突中的線粒體密度，線粒體形態則沒有改變。我們表明NHX-3在神經元組織中表達，並與粒線體共定位。從運動性分析發現，nhx-3（ok1049）突變體的逆行總運行長度減少，淨運行長度增加：然而，線粒體的順向和逆向速度沒有影響。除此之外，在nhx-3（ok1049）突變體中，線粒體呈現較低頻率的方向變化和更持久移動。對NHX-3表現量有正向調節作用的轉錄因子HBL-1確實在nhx-3突變中觀察到其影響力。在hbl-1（mg285）突變體中，粒線體的逆行速度和逆行總運行長度顯著減少，淨運行長度顯著增加。雙重突變分析表明，nhx-3不會進一步影響hbl-1基因的功能。基於這些結果，我們推測NHX-3在C. elegans中對粒線體逆行軸突運輸發揮關鍵作用。
　　由於STRING 分子互動組的數據揭示 NHX-3 和 KIF1 之間的關聯，我們分析 KIF1A/UNC-104 在 nhx-3 突變中的作用。的確，unc-104似乎在線粒體運輸中發揮作用。此外，觸感和扭動檢測揭示機械感知和運動的缺陷。趨向性分析證明纖毛在nhx-3突變體中也可能存在缺陷。最後，我們發現產卵缺陷和短化的體長。這些可量化的表現型可能有助於未來的抑制篩選，並開發一種疾病模型。

Neurons are strongly dependent on mitochondria, essential to neuronal growth, survival, and function. Alterations in mitochondrial dynamics are common hallmark in various types of neurodegenerative diseases. Thus, it is critical to study how mitochondrial dynamics is regulated on the molecular level. We investigated the transport of mitochondria in axons using worms expressing GFP-labeled mitochondria. From a reverse genetic screen, a gene nhx-3 was identified that reduces mitochondrial density in proximal axons of ALM neurons, while mitochondrial morphologies remain unaltered. We show that NHX-3 is expressed in neuronal tissues as well as co-localize with mitochondria. From motility analysis, we revealed that retrograde total run length decreased and that net run length increased; however, anterograde and retrograde velocity of mitochondria were not affected in nhx-3(ok1049) mutants. Nevertheless, mitochondria undergo less frequent reversals and move more persistent in nhx-3(ok1049) mutants. Furthermore, transcription factor HBL-1, known to positively regulates expression of NHX-3, indeed facilitated observed effects in nhx-3 mutant backgrounds: Retrograde velocity and total run length both were significantly decreased as well as net run length was significantly increased in hbl-1(mg285) mutants. Double mutant analysis suggests that nhx-3 does not further affect the epistasis function of the hbl-1 gene. Based on these results, we propose that NHX-3 may play a critical role in retrograde axonal transport of mitochondria in C. elegans.
Because data from STRING interactome tool revealed a link between NHX-3 and KIF1, we analyzed the role of KIF1A/UNC-104 on nhx-3 mutations. Indeed unc-104 seems to play a role in mitochondrial transport. Furthermore, touch and thrashing assays revealed defects in mechanosensation as well as in locomotion. Quadrant chemotaxis assay demonstrates that cilia may be defective in nhx-3 mutants as well. Lastly, we identified egg-laying defects and shortened body length in nhx-3 mutants. These quantifiable phenotypes may aid in future suppressor screens and to develop a disease model.

Acknowledgments ii
摘要 iii
Abstract iv
1 Introduction 3
1.1 C. elegans as a model organism 3
1.2 Transport of mitochondria in neurons 4
1.2.1 Importance of mitochondrial distribution in neurons 4
1.2.2 Machinery and regulation of mitochondrial transport 5
1.2.3 Dysfunctional mitochondria and neurodegenerative diseases 7
1.4 Kinesin-3 KIF1A/UNC-104 in C. elegans 9
1.5 Specific aim of this study 10
2 Materials & Methods 11
2.1 Worm maintenance 11
2.2 Reagents 11
2.2.1 M9 buffer 11
2.2.2 LB medium 11
2.2.3 Genomic DNA isolation buffer 11
2.2.4 Bleach solution 12
2.3 Plasmid constructs 12
2.4 C. elegans strains 12
2.5 RNA interference by feeding 13
2.6 Microinjection 14
2.7 Outcrossing, crossing, and genotyping 15
2.8 Worm imaging 16
2.9 Image analysis 16
2.9.1 Cluster analysis 16
2.9.2 Motility analysis 17
2.9.3 Colocalization analysis 17
2.9.4 Fluorescence intensity analysis 18
2.10 Behavioral assays 18
2.10.1 Thrashing assays 18
2.10.2 Touch response assays 18
2.10.3 Quadrant chemotaxis assay 19
2.10.4 Egg-laying assays 19
2.11 Body length analysis 19
2.12 Statistical analysis 19
3 Results 20
3.3 Expression patterns of NHX-3 22
3.4 Colocalization of NHX-3 and mitochondria 23
4 Discussion 28
4.1 The role of NHX-3 in axonal mitochondrial transport 28
4.2 Effects of HBL-1 on retrograde transport of mitochondria 30
4.3 Disrupting retrograde transport of mitochondria in axons 30
4.4 The relation of UNC-104 and NHX-3 in axonal mitochondrial transport 31
4.5 Defective phenotypes in nhx-3(ok1049) mutants 32
5 References 35
7 Figures 41
8 Appendix 57

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