Specific Aims: Syntaphilin (SNPH) is a neuron-specific and axon-mitochondria targeted protein (Kang et al., Cell 2008). 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-based cytoskeleton. Deletion of the snph gene in mice resulted in a substantially higher proportion of axonal mitochondria in the mobile state. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation by affecting calcium signaling at presynaptic boutons. Thus, SNPH acts as a static anchor for docking/retaining mitochondria in axons and at synapses.
Aim 1. To investigate mechanisms of recruiting motile mitochondria to stationary pool. Mitochondria in axons display complex mobility patterns with frequent stops and changes in direction suggesting their coupling with two opposing molecular motors, kinesins and dynein, along with docking/anchoring machinery. Recent advances in identifying SNPH as a mitochondrial docking receptor and KIF5 motor adaptor Miro as a calcium sensor in arresting mitochondrial movement provide molecular targets for such regulation. Using snph KO neurons combined with time-lapse imaging, we are addressing whether SNPH-mediated docking is required for Ca-dependent arrest of axonal mitochondrial movement. Our ongoing study highlights a critical role of SNPH in controlling axonal mitochondrial movement in response to synaptic activity. These results allow us to propose that KIF5-Miro and SNPH share a single system of regulation that could involve the physical displacement of the motor-adaptor complex by docking interactions. Our study will advance knowledge how axonal mitochondria are recruited between the motile and stationary pools in response to neuronal activity.
Aim 2. To examine contribution of mitochondrial mobility to the variation of synaptic strength. Synaptic variability is increasingly recognized as a characteristic feature of cellular, synaptic and network properties. Some degree of variability may be essential for the faithful processing of the signal in flexible or adaptive systems. Synaptic mitochondria play important roles in the ATP-dependent SV mobilization from the reserve pool to RRP and in calcium homeostasis by buffering extra intracellular Ca2+ during tetanic stimulation, thus changing synaptic strength and plasticity. In addition, in mature neurons, approximate one-third of mitochondria dynamically move along axons;they either pass by or enter and exit presynaptic boutons dynamically, which raises a fundamental question whether those mobile mitochondria influence synaptic homeostasis, thus contributing to the synaptic variability. Our snph mouse is a unique model to address this question. Our ongoing study using snph (-/-) hippocampal neurons examines whether axonal mitochondrial mobility has a significant influence on the variation of EPSC amplitudes. in particular, we will address whether mobile mitochondria passing presynaptic boutons contribute to synaptic variability.
Aim 3. To determine the impact of mitochondrial mobility on mitochondria quality control and axonal degeneration. Proper transport and distribution of axonal mitochondria are critical for neuronal function. Mitochondrial dysfunction and fragmentation and their impaired transport in axons have been implicated in the pathogenesis of major neurodegenerative diseases such as Alzheimers, Huntingtons and amyotrophic lateral sclerosis (ALS). However, whether altered mitochondrial transport plays a critical role in axonal degeneration or manifests as a side effect of general defective axonal transport has been a subject for debate. Our snph mouse provides a valuable model to assess the impact of mitochondrial mobility on axonal degeneration. ALS is a late onset neurodegenerative disease specifically affecting motor neurons. We selected ALS-linked SOD1G93A mutant mice as our fiirst disease model to study because several labs independently reported altered axonal mitochondrial transport in this mouse line or following expression of the SOD1G93A mutant in neurons. By crossing SOD1G93A and snph-/- mice we address whether increasing (rescued) mitochondrial mobility has any impact on the pathogenesis of the fALS-linked SOD1G93A mouse model by comparing clinical and histological observations of SOD1G93A mice to the crossed SOD1G93A/snph-/- mice. To our surprise, although the crossed SOD1G93A/snph-/- mice exhibit a 2-fold increase in axonal mitochondrial mobility at late disease stages, there is no observable improvement in the deterioration of motor function or in disease progression and lifespan. Furthermore, immunostaining of spinal cord sections from both SOD1G93A and SOD1G93A/snph-/- mice show similar ALS-like disease histopathology that includes motor neuron loss and gliosis. Thus, our study provides genetic, cellular, and pathological evidence that challenges the prevailing hypothesis that defective transport of axonal mitochondria contributes to rapid-onset motor neuron degeneration in this mouse model. Thus, mitochondria dysfunction, rather than impaired mobility, may be more important in rapid-onset motor neuron degeneration (Zhu and Sheng JBC, 2011). Recent emerging evidence suggests that selective mitochondrial degradation through autophagy (mitophagy) plays an important role in the quality control of mitochondria, thus providing a direct relation between mitophagy and neurodegenerative diseases. However, significance of mitophagy in neuronal mitochondria quality control is not well characterized. In particular, whether and how mitochondrial dysfunction and altered mobility impact mitophagy within axons is not clear. We are addressing this question by determining whether axonal mitochondria can be eliminated by mitophagy when they are dysfunctional, for example following treatment with mitochondria decoupler CCCP. We are also determining whether the mobility of axonal mitochondria has any impact on mitophagy using snph (-/-) neurons. Given the established role of Snapin in regulating autophagy-lysosomal function, we will elevate Snapin expression in neurons and determine whether Snapin enhances mitophagy, thereby facilitating the turnover of dysfunctional mitochondria. These studies will provide cellular and genetic clues as to whether manipulating mitochondrial transport and turnover may leads to new therapeutic approaches. Pursuing these investigations will advance our knowledge of fundamental processes that may affect human neurological disorders and is thus the very essence of the mission of the National Institute of Neurological Disorders and Stroke. Selected publications related the projects: Qian Cai, Claudia Gerwin, and Zu-Hang Sheng. (2005). Syntabulin-mediated anterograde transport of mitochondria along the neuronal processes. Journal of Cell Biology 170, 959-969. Jian-Sheng Kang,Jin-Hua Tian, Philip Zald, Ping-Yue Pan, Cuiling Li, Chuxia Deng, and Zu-Hang Sheng. (2008). Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation. Cell 132, 137-148. Yan-Min Chen, Claudia Gerwin, and Zu-Hang Sheng. (2009). Dynein light chain LC8 regulates syntaphilin-mediated mitochondrial docking in axons. Journal of Neuroscience 29, 9428-9437. Huan Ma, Qian Cai, Wenbo Lu, Zu-Hang Sheng (co-corresponding author), and Sumiko Mochida. (2009). KIF5 motor adaptor syntabulin maintains synaptic transmission in sympathetic neurons. Journal of Neuroscience 29, 13019-13029.
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