化學遺傳學、光遺傳學和磁場遺傳學等多方技術的進步促進了神經科學研究的進展，這些基因工程工具分別利用改造過的配位基、光子和磁場來調控細胞的活性，進而解決神經科學上的重要問題並定位出作用在腦中的神經迴路。然而，這些技術皆有其無法避免的缺點：刺激源無法深入位於腦部深層的神經細胞。為了突破此限制，我們嘗試利用擁有非侵入性、聚焦性和高穿透力的超音波來達成我們的目標，其中一個重要的步驟為篩選出能夠幫助細胞感應超音波並引發下游反應的蛋白質，在我們近期的研究中發現Prestin蛋白能擔任此關鍵角色，其原為作用在哺乳類聽覺系統中的穿膜蛋白質，在運用超音波溝通的物種中，保守序列第7個和第308個氨基酸由天冬醯胺分別被置換成蘇氨酸和絲氨酸的比例相當高，將小鼠的Prestin蛋白質在兩位置做點突變並大量表現可以提高人類細胞對於超音波刺激的敏銳度。除了突變的Prestin(N7T, N308S)蛋白質，我們為了提升聲波遺傳學的技術也試圖找尋更有效率的超音波感應蛋白，因前人研究發現TRP4參與作用在線蟲對超音波的偵測，我們測試了2種哺乳類動物所屬的TRPC4蛋白質，同時另外研究了4種以回聲定位的蝙蝠身上的Prestin蛋白質，以及仿磷酸化、去磷酸化狀態的Prestin蛋白質以探討磷酸化的重要性。實驗結果顯示，小鼠的TRPC4蛋白、去磷酸化Prestin蛋白與人類的Prestin(N7T, N308S) 蛋白質能成功幫助細胞對超音波刺激做出反應，人類TRPC4蛋白、小鼠仿磷酸化Prestin蛋白和回聲物種Prestin蛋白則無法作為超音波感應蛋白。我們的研究成果證實了TRPC4蛋白及Prestin蛋白和細胞超音波感應的關係，並提供未來去進一步提升聲波遺傳學的可能研究方向。

The progression of neuroscience has been facilitated by the development of several approaches such as chemogenetics, optogenetics, and magnetogentics. These genetically encoded tools use engineering ligands, photon, and magnetic field, respectively, to manipulate cellular activities and have addressed a number of fundamental questions in neuroscience as well as have mapped the functional circuits in the brain. However, there are inevitable drawbacks to them: it is challenging for the stimuli to access to targeted neurons located in deeper brain regions. To circumvent these long-standing limitations, we attempt to use ultrasound, which is noninvasive, focused, and has great depth of penetration, to perturb neuronal activities. One strategy we make use of is the identification of the ultrasound-responsive proteins. Our recent results have demonstrated that the prestin, a transmembrane protein which resides in the mammalian auditory system, act as an ultrasound-responsive protein. Expression of prestin carried with N7T and N308S, two amino acid substitution frequently exist in echolocating species, endows transfected mammalian cells with ultrasound sensitivity. Besides the Prestin(N7T, N308S), one previous study also found that TRP4 is involved in ultrasound sensing of C. elegans. To strengthen the efficiency of our sonogenetic system, we here aimed to explore other ultrasound-responsive proteins with better ultrasound sensitivity. We have characterized two mammalian TRPC4 proteins, prestin proteins from four sonar bats and prestin variants with different phosphomimetic statuses. Our results confirmed that mouse TRPC4β, mouse Prestin(N7A, N308A), human Prestin(N7T, N308S) but not human TRPC4α, mouse Prestin(N7D, N308D), or prestin variants from four sonar bats act as ultrasound-responsive proteins to make cells excitable to ultrasound stimulation. The data obtained here provides new insights of how TRPC4 protein and prestin affect the ultrasound sensitivity and also gives clues to modify the Prestin(N7T, N308S) for further improving the efficiency of our sonogenetic system.

Abstract i
中文摘要 ii
致謝 iii
Table of Contents iv

Chapter 1. Introduction 1
1.1 The advantages and disadvantages of genetically-encoded tools for neuromodulation 1
1.2 Ultrasound, ultrasound-responsive proteins, and sonogenetics 3
1.3 Overview of prestin, a candidate of ultrasound-responsive proteins tested in our study 5
1.4 Overview of TRPC4, the other candidate of ultrasound-responsive proteins tested in our study 7

Chapter 2. Material and Methods 9
2.1 DNA constructs 9
2.2 Cell culture and transfection 9
2.3 Live cell-imaging 10
2.4 Ultrasound stimulation 11
2.5 Immunofluorescence staining 12
2.6 Statistical analysis 13

Chapter 3. Results 14
3.1 Our strategy for testing the ultrasound sensitivity of genetically-modified cells 14
3.2 Heterologous expression of human TRPC4α and mouse TRPC4β 15
3.3 The ultrasound sensitivity of cells expressing mammalian TRPC4 proteins 16
3.4 Characterization of prestin from four sonar species 17
3.5 Characterizing the prestin variants of different phosphomimetic statuses 20
3.6 Characterization of engineered human prestin 21

Chapter 4 Discussion 22
4.1 Summary of this thesis study 22
4.2 Why mTRPC4β-CFP but not hTRPC4α-CFP can response to 0.5 MHz ultrasound? 24
4.3 The poor ultrasound sensitivity of sonar prestin protein in HEK293T cells 26
4.4 Post-translational modifications of prestin may inhibit its ultrasound sensing 27

References 29
Figures 34

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