Specific Aim 1. Mechanisms recruiting motile mitochondria to stationary pool in response to synaptic activity The mechanism by which mitochondrial mobility is regulated in response to neuronal activity and synaptic modification is unknown. Recent advances in identifying SNPH as a mitochondrial docking protein 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 and distribution in response to synaptic activity. These results allow us to propose the working hypothesis 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 (or vice versa). Our study advances knowledge how axonal mitochondria are recruited between the motile and stationary pools in response to neuronal activity and how the motility coordinates with the clearance of dysfunctional mitochondria from distal axons and synapses in healthy conditions and in axonal degeneration (Chen et al., submitted).
Specific Aim 2. Mobile axonal mitochondria contribute to the variability of synaptic strength One of the most notable characteristics of synaptic transmission is the pulse-to-pulse variation in synaptic strength. A fundamental question is how this variation of synaptic strength arises. Numerous studies focused on the structural and stochastic properties of molecular events underlying synaptic variability. Most of these factors are likely the basis for marked heterogeneity in synaptic transmission from neuron-to-neuron or from synapse-to-synapse. It is not known, however, which dynamic process contributes to pulse-to-pulse variability at single-bouton levels in response to identical stimulation. Mitochondria maintain synaptic transmission by buffering Ca2+ and producing ATP. Approximately one-third of axonal mitochondria are mobile, some of which dynamically pass through presynaptic boutons. This raises a question: Can these mobile mitochondria influence SV release in a dynamic manner, thereby contributing to the pulse-to-pulse variability of synaptic strength? The snph mouse provides us with a unique genetic tool to address whether selective changes of axonal mitochondrial mobility could compromise the variability of synaptic strength. Using electrophysiological analysis in hippocampal neurons and acute hippocampal slices, we recently reveal that enhancing mobility of axonal mitochondria increases the pulse-to-pulse variability of synaptic strength, while immobilizing axonal mitochondria effectively reduces the variability. By dual-imaging mitochondria and SVs with DsRed-mito and synapto-pHluorin, we further show that mitochondrial movement, either into or out of the boutons, influences presynaptic variability during repeated trains of stimulation. Altered ATP homeostasis resulting from mitochondrial movement is the primary source for the variability of SV release. Thus, our study reveals, for the first time, that the dynamic movement of axonal mitochondria is one of the primary mechanisms underlying the pulse-to-pulse variability of synaptic strength and the efficiency of SV release in the CNS (Sun et al., submitted).
Specific Aim 3. Impact of mitochondrial mobility on mitochondrial quality control. PINK1/Parkin-mediated pathways ensure mitochondrial integrity and function. Translocation of Parkin to damaged mitochondria induces mitophagy in many non-neuronal cell types. However, evidence showing Parkin translocation in primary neurons is controversial. We recently revealed several unique features of dissipating mitochondrial Δψm-induced and Parkin-mediated mitophagy in mature cortical neurons: (1) Parkin translocation onto depolarized mitochondria is slower than in non-neuronal cells and occurs in a small percentage of neurons;lysosomal degradation is critical for the efficacy of Parkin-mediated mitophagy;(2) Parkin translocation is restricted to the somatodendritic regions, where mature lysosomes are predominantly localized;(3) During Parkin translocation, anterograde transport was reduced while retrograde transport was relatively increased. Parkin-mediated process prevents dysfunctional mitochondria from traveling peripherally. Altered mitochondrial mobility may be protective for neurons under stressful conditions, where healthy mitochondria remain distally while damaged mitochondria return to the soma for degradation. Therefore, our study demonstrates selective and dynamic Parkin translocation to depolarized mitochondria and subsequent degradation via the autophagy-lysosomal pathway in live neurons (Cai et al., Current Biology 2012, Cai et al., Autophagy 2012).
Specific Aim 4. Impact of mitochondrial mobility on axonal degeneration. Mitochondrial dysfunction and their impaired transport in axons have been implicated in the pathogenesis of major neurodegenerative diseases. However, whether altered mitochondrial transport plays a critical role in axonal degeneration or manifests as a side effect of general transport defects has been a subject for debate. Our snph mouse provides a valuable model to assess the impact of mitochondrial mobility on axonal degeneration. Amyotrophic lateral sclerosis (ALS) is a late onset neurodegenerative disease specifically affecting motor neurons. We selected ALS-linked SOD1G93A mutant mice for our study. By crossing SOD1G93A and snph-/- mice we addressed whether increasing (rescued) mitochondrial mobility has any impact on the pathogenesis of the fALS-linked SOD1G93A mouse model. To our surprise, although the crossed 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. The crossed mice show similar ALS-like disease histopathology that includes motor neuron loss and gliosis. Thus, our study suggests that mitochondria dysfunction, rather than impaired mobility, may play a more important role in rapid-onset motor neuron degeneration (Zhu and Sheng JBC, 2011). In summery, 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. Yi-Bing Zhu and Zu-Hang Sheng (2011). Increased Axonal Mitochondrial Mobility Does Not Slow ALS-like Disease in Mutant SOD1 Mice. The Journal of Biological Chemistry 286, 23432-23440. Qian Cai, Hesham Mostafa Zakaria, Anthony Simone, and Zu-Hang Sheng (2012) Spatial Parkin Translocation and Degradation of Depolarized Mitochondria via Mitophagy in Live Cortical Neurons. Current Biology 22, 545-552. Zu-Hang Sheng &Qian Cai (2012) Mitochondrial Transport in Neurons: Impact on Synaptic Homeostasis and Neurodegeneration. Nature Reviews Neuroscience 13, 77-93.
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