Specific Aims: Syntaphilin (SNPH) is a neuron-specific and axon-targeted protein initially identified in our lab as a candidate inhibitor of presynaptic function (1). Our effort in generating SNPH KO mice has led to the discovery of a novel role for SNPH in the control of axonal mitochondrial motility (2). Our study reveals that SNPH is required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule (MT)-based cytoskeleton. Three lines of evidence support this view. First, the mitochondria that associated with exogenously expressed GFP-SNPH were almost entirely immobile. This occurs through interactions with the cytoskeleton because SNPH contains a MT-binding domain, which is necessary and sufficient for SNPH-mediated immobilization of axonal mitochondria. Second, by recording mitochondrial movement in living neurons followed by retrospective immunostaining for endogenous SNPH, we demonstrate that the immobility of axonal mitochondria depends on their association with endogenous SNPH, and further reveal a binomial distribution with a strong correlation between the endogenous SNPH-tagged mitochondria (62%) and stationary mitochondria (65%). Finally, deletion of the snph gene in mice resulted in a substantially higher proportion of axonal mitochondria in the mobile state than that found in wild-type neurons, and reduced the densities of total and inter-bouton mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. Thus, SNPH acts as a static anchor for docking/retaining mitochondria in axons and at synapses. These findings reveal for the first time a neuron-specific protein capable of docking axonal mitochondria and regulating their densities within axons. Mitochondrial balance between the motile and stationary phases is a target for regulating mitochondrial redistribution. How are motile mitochondria recruited to the stationary pool? In particular, the mechanisms regulating SNPH-mediated mitochondrial anchoring at axons remain elusive. By applying a proteomic approach combined with time-lapse imaging in live snph (+/+) and (-/-) neurons, we revealed that dynein light chain LC8 enhances the docking efficiency by binding to SNPH (3). Four lines of evidence support this view. First, SNPH interacts with LC8 via its 7-residue motif (ERAIQTD);the SNPH-LC8 complex is detected by immunoprecipitation of brain homogenates;the interaction is independent of the dynein motor complex. Second, SNPH recruits LC8 to axonal mitochondria via its 7-residue LC8-binding motif. Deleting this motif reduces the SNPH capacity in docking axonal mitochondria. Third, elevated LC8 expression in snph (+/+) neurons inhibits the mobility of axonal mitochondria. In contrast, this effect is not observed in snph null neurons, suggesting that the role of LC8 is depending on its interaction with SNPH. Forthermore, CD spectrum analysis revealed that LC8 enhances docking by stabilizing an α−helical coiled-coil within the MT-binding domain of SNPH against thermal unfolding. Altogether, our studies provide new mechanistic insights into how SNPH and LC8 coordinately immobilize mitochondria through enhanced interaction of SNPH and MTs. In summary, using genetic mouse models combined with time-lapse imaging in live neurons, we elucidate molecular mechanism underlying the complex mobility patterns of axonal mitochondria. Such a mechanism enables neurons to maintain proper mitochondrial densities within axons and near synapses. We further provide the physiological evidence that the mobility and density of axonal mitochondria play a critical role in short-term synaptic plasticity. It is expected that defective mitochondrial docking/anchoring could affect neuronal functions. Dysfunction and defective trafficking of axonal mitochondria have been implicated in the pathologic processes of neurodegenerative diseases such as Alzheimers and Huntingtons and amyotrophic lateral sclerosis. The continued application of live cell imaging in combination with a multi-disciplinary analysis of genetically crossed mouse models will improve our understanding as how the changes in mitochondrial mobility affect axonal neurodegeneration. Papers published from the lab related to the project: 1. Guifang Lao, Volker Scheuss, Claudia M. Gerwin, Qingning Su, Sumiko Mochida, Jens Rettig, and Zu-Hang Sheng (2000). Neuron 25, 191-201. 2. Jian-Sheng Kang,Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng. (2008). Cell 132, 137-148. 3. Yan-Min Chen, Claudia Gerwin, and Zu-Hang Sheng. (2009). Journal of Neuroscience 29, 9428-9437.
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