清大生醫學院分生所王歐力教授（左二）與研究團隊成員

Associate Professor Dr. Oliver I. Wagner of the Institute of Molecular and Cellular Biology, College of Life Sciences and Medicine, National Tsing Hua University, with research team member.

論文標題Paper Title

UNC-10/SYD-2 links kinesin-3 to RAB-3-containing vesicles in the absence of the motor's PH domain.

學術期刊刊名Journal Name

疾病神經生物學Neurobiology of Disease

研究團隊 Research Team

主持人Principle Investigator：

王歐力 教授Oliver I. Wagner, Professor

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, College of Life Science and Medicine (CLSM), National Tsing Hua University (NTHU).

參與者Participants:：

Odvogmed Bayansan

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, Department of Life Science, NTHU

Prerana Bhan

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, Department of Life Science, NTHU

Chien-Yu Chang

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, Department of Life Science, NTHU

Syed Nooruzuha Barmaver

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, Department of Life Science, NTHU

Che-Piao Shen

國立清華大學生命科學暨醫學院 分子與細胞生物研究所

Institute of Molecular and Cellular Biology, Department of Life Science, NTHU

摘要

Abstract

KIF1A/UNC-104 是突觸囊泡的主要轉運體。此分子馬達的突變與 KIF1A 相關神經系統疾病 (KAND) 有關。 UNC-104 透過其 PH 結構域與突觸囊泡僅進行微弱的結合，我們假設存在其他相互作用方案。已知 UNC-104 調節劑 SYD-2 與 UNC-10 相互作用，並且 UNC-10 本身與結合突觸囊泡的 RAB-3 相互作用。 RT-PCR 和 Western blot 實驗揭示了 unc-10 和 syd-2 之間的遺傳關係，但沒有揭示 unc-10 和 rab-3 之間的遺傳關係。 unc-10 和 rab-3 均不影響 UNC-104 表現。然而，共免疫沉澱和 BiFC 分析揭示了 UNC-104、SYD-2、UNC-10 和 RAB-3 之間的功能性相互作用。儘管 SNB-1 和 RAB-3 都由 UNC-104 主動運輸，但在 SYD-2 和 UNC-10 存在的情況下，RAB-3 的運動性得到促進。刪除 UNC-104 的 PH 結構域不會影響 UNC-104/RAB-3 共定位，但會顯著影響 UNC-104/SNB-1 共定位。類似地，當 PH 結構域發生點突變時，RAB-3 標記囊泡的運動性僅發生輕微改變，而 SNB-1 的運動性則降低。純化的突觸囊泡的蛋白質印跡表明，rab-3/unc-10 雙突變體中的 UNC-104 大幅減少。我們的研究結果表明，UNC-10/SYD-2 複合物可作為一種新型連接體，將 UNC-104 與含有 RAB-3 的囊泡連接起來，有助於實現馬達/貨物相互作用的特異性。

KIF1A/UNC-104 is the major transporter of synaptic vesicles. Mutations in this molecular motor are linked to KIF1A-associated neurological disorders (KAND). UNC-104 binds only weakly to synaptic vesicles via its PH domain and we hypothesize that other interaction schemes exist. It is known that UNC-104 regulator SYD-2 interacts with UNC-10 and that UNC-10 itself interacts with RAB-3 bound to synaptic vesicles. RT-PCR and Western blot experiments expose genetic relationships between unc-10 and syd-2, but not between unc-10 and rab-3. Neither unc-10 nor rab-3 affects UNC-104 expression. However, co-immunoprecipitation and BiFC assays reveal functional interactions between UNC-104, SYD-2, UNC-10 and RAB-3. Though both SNB-1 and RAB-3 are actively transported by UNC-104, motility of RAB-3 is facilitated in the presence of SYD-2 and UNC-10. Deletion of UNC-104's PH domain did not affect UNC-104/RAB-3 colocalization, but significantly affected UNC-104/SNB-1 colocalization. Similarly, motility of RAB-3-labeled vesicles is only slightly altered when carrying a point mutation in the PH domain, whereas movement of SNB-1 is reduced. Western blots of purified synaptic vesicles reveal strong reduction of UNC-104 in rab-3/unc-10 double mutants. Our findings suggest that the UNC-10/SYD-2 complex acts as a novel linker to connect UNC-104 to RAB-3-containing vesicles contributing to the specificity of motor/cargo interactions.

研究成果Result/Contributions

KIF1A 相關神經系統疾病 (KAND) 是一種罕見的遺傳疾病，由 KIF1A 基因突變引起，該基因編碼突觸小泡的主要軸突轉運體。這種疾病主要影響兒童，其常見症狀包括發育遲緩、智力障礙、肌張力低下（肌張力低下）發展為肌張力亢進（僵硬）、痙攣性截癱、共濟失調（協調問題）和癲癇。嚴重程度取決於突變的類型和位置，有些病例會導致危及生命的併發症。目前尚無治癒方法。王歐力教授實驗室認為，了解這種轉運蛋白調控的潛在分子機制將有助於開發新型藥物來延緩或治癒KAND疾病。

KIF1A 在秀麗隱桿線蟲中被稱為 UNC-104，由於其高度同源性，這種模型生物為研究 KAND 疾病提供了絕佳的平台。 Oliver Wagner 博士的實驗室發表了十幾篇關於 UNC-104 調控的論文，深入揭示了這種突觸囊泡轉運體在分子層面的控制方式。例如，實驗室發現 SYD-2（liprin-α）是一種銜接蛋白，可控制 UNC-104 的速度調節，並可作為長神經元中該馬達的支架。另一種蛋白質 PTP-3（LAR）根據 SYD-2 的分子內折疊來調節其磷酸化。該實驗室還發現了一種負責 UNC-104 長距離移動的蛋白質 LIN-2（CASK）。此外，研究發現，UNC-104 運輸 tau 蛋白，而 tau 的突變會影響該馬達的運動性。與微管平行運行的神經絲似乎也能促進長距離運動。

至關重要的是，UNC-104 透過其 PH（pleckstrin 同源性）結構域與突觸小泡僅發生微弱的相互作用。因此，該實驗室想知道運動細胞和突觸小泡之間是否存在其他更具體、更強的相互作用。該實驗室利用大量分子生物學技術發現 UNC-104 特異性地與結合 RAB-3 的突觸囊泡相互作用。在這裡，馬達採用了由兩種蛋白質 UNC-10（Rims1）和 SYD-2 組成的新型連接器。有趣的是，這種相互作用似乎具有高度特異性，例如 SNB-1（突觸小泡蛋白）的運輸。

KIF1A-associated neurological disorder (KAND) is a rare genetic condition caused by mutations in the KIF1A gene, which encodes for the major axonal transporter of synaptic vesicles. Mostly children are affected by this disorder and symptoms commonly include developmental delays, intellectual disability, hypotonia (low muscle tone) progressing to hypertonia (stiffness), spastic paraplegia, ataxia (coordination issues), and epilepsy. Severity depends on the mutation type and location, with some cases leading to life-threatening complications. There is currently no cure. Dr. Wagner’s (王歐力教授) lab believes that understanding the underlying molecular mechanisms how this transporter is regulated will lead to the development of novel drugs to delay or cure KAND disease. KIF1A is termed UNC-104 in C. elegans and due to the its high homology, this model organism provides an excellent platform to study KAND disease. Dr. Oliver Wagner’s lab has published a dozen papers on the regulation of UNC-104 unraveling deep insights how this synaptic vesicle transporter is controlled on the molecular level. For example, the lab found that SYD-2 (liprin-α) is an adaptor protein that controls speed regulation of UNC-104 as well as acting as a scaffold for this motor along the lengthy neurons. Another protein PTP-3 (LAR) regulates phosphorylation of SYD-2 depending on its intramolecular folding. The lab also identified a protein LIN-2 (CASK) that is responsible for UNC-104’s long-range travels. Moreover, it was found that UNC-104 transports tau protein and mutations in tau affect the motility of this motor. Neurofilaments running in parallel to microtubules seem to promote long motor runs as well.

Critically, UNC-104 interacts with synaptic vesicles only weakly via its PH (pleckstrin homology) domain. Therefore, the lab wanted to know whether perhaps other, more specific and stronger interactions between motor and synaptic vesicle exist. Using a large set of molecular biology techniques, the lab identified that UNC-104 specifically interacts with synaptic vesicles that bind RAB-3. Here, the motor employs a novel linker composed of two proteins UNC-10 (Rims1) and SYD-2. Interestingly, this interaction seems highly specific such as the transport of SNB-1 (Synaptobrevin).

論文連結 Paper Link

https://www.sciencedirect.com/science/article/pii/S0969996124003681

https://www.childrenshospital.org/conditions/kif1a-associated-neurological-disorder-kand

https://www.kif1a.org/this-is-kif1a/