Specific Aim 1. Mobile axonal mitochondria contribute to the variability of synaptic strength (Sun et al., Cell Reports 2013). One of the most notable characteristics of synaptic physiology in the CNS is the wide pulse-to-pulse variability of synaptic strength in response to identical stimulation. A long-standing question for decades is how this variability arises. This raises a fundamental question: Can those motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? We demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Selectively enhancing axonal mitochondrial motility increases the pulse-to-pulse variability, while immobilizing axonal mitochondria reduces the variability. We further showed that mitochondrial movement, either into or out of presynaptic terminals significantly influences SV release due to dynamic fluctuation of synaptic ATP levels. A motile mitochondrion passing through synapses changes synaptic energy levels and influence synaptic activities. Our study revealed, for the first time, that axonal mitochondrial transport is one of the primary mechanisms underlying the presynaptic variation.
Specific Aim 2. Parkin-independent mechanism removing damaged mitochondria from axonal terminals (Cai et al., Current Biology 2012; Lin et al., Neuron 2017) Mitochondrial dysfunction and impaired transport emerge as central problems associate with major neurodegenerative disorders. Early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. Mature neurons often show delayed Parkin-mediated mitophagy in response to mitochondrial depolarization, raising a question as to whether mitophagy is an early mechanism of neuronal mitochondrial quality control. Acute damage to mitochondria unlikely occurs in vivo. We recently investigat axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in amytrophic lateral sclerosis (ALS)- and Alzheimers disease (AD)-linked neurons. We show that stressed mitochondria are removed from axons triggered by the bulk release of mitochondrial anchoring protein SNPH via a new class of mitochondria-derived cargos independent of mitophagy. We also show the budding of SNPH cargos, which then share a ride on late endosomes for transport toward the soma. Releasing SNPH is also activated in the early pathological stages of ALS- and AD-linked mutant neurons. Our study provides new mechanistic insights into the maintenance of axonal mitochondrial quality through SNPH-mediated coordination of mitochondrial stress and motility before activation of mitophagy.
Specific Aim 3. Enhancing mitochondrial transport facilitates axon regeneration by rescuing energy deficits (Zhou et al., JCB 2016). While young neurons possess robust axon growth during early development, mature CNS axons typically fail to regrow after injury. While regeneration is a highly energy-demanding process, axonal mitochondrial transport progressively declines with maturation, thus raising a fundamental question as to whether mitochondrial transport is necessary to support regeneration. By applying microfluidic culture system, which allows physical separation of axons from cell bodies and dendrites, we reveal that reduced mitochondrial motility and energy deficits in injured axons are intrinsic mechanisms controlling regrowth in mature neurons. Axotomy induces acute mitochondrial damage and ATP depletion in injured axons. Thus, mature neuron-associated increase in SNPH and decrease in mitochondrial transport cause local energy deficits. Strikingly, enhancing mitochondrial transport facilitates regenerative capacity by replenishing healthy mitochondria in injured axons, thereby rescuing energy deficits. An in vivo sciatic nerve crush study further shows that enhanced mitochondrial transport in snph KO mice accelerates axon regeneration. Therefore, our study suggests a new target for stimulating axon regeneration and functional recovery after nerve injury and disease.
Specific Aim 4. Endo-lysosomal deficits augment mitochondria pathology in asymptomatic fALS mice (Xie et al., Neuron 2015). One pathological hallmark in ALS motor neurons (MNs) is axonal accumulation of damaged mitochondria. Proper clearance of those mitochondria via mitophagy may serve as an early protective mechanism. A fundamental question remains: does reduced degradation of those mitochondria due to impaired autophagy-lysosomal system contribute to mitochondrial pathology? We reveal MN-targeted progressive lysosome defects in the hSOD1G93A mice starting as early as postnatal day 40, accompanied by accumulation of damaged mitochondria engulfed by autophagosomes in axons. In vitro and in vivo studies demonstrate that endo-lysosomal transport is crucial to remove damaged mitochondria from axons. Such deficits are attributable to impaired retrograde transport of late endosomes by mutant hSOD1G93A, thus reducing the recruitment of dynein motors to the organelles for transport. AAV9-snapin injection in hSOD1G93A mice reverses mitochondria pathology, reduces MN loss, and ameliorates the fALS-linked disease phenotype. Elucidation of this early pathological mechanism is broadly relevant, because defective transport, lysosomal deficits, and mitochondrial pathology are associated with major neurodegenerative diseases.
Specific Aim 5. Organismal aging affects neuronal mitochondrial maintenance (Morsci et al., Journal of Neuroscience 2016). Aging is associated with cognitive decline and increasing risk of neurodegeneration. The critical understanding of how organismal aging affects lifetime neuronal mitochondrial maintenance remains unknown-particularly in physiologically relevant context. To address this issue, we performed a comprehensive in vivo analysis of age-associated changes in mitochondrial morphology, density, trafficking, and stress resistance in individual Caenorhabditis elegans neurons throughout adult life. Adult neurons display three distinct stages of increase, maintenance and decrease in mitochondrial size and density during adulthood. Mitochondrial trafficking in the distal neuronal processes declines progressively with age starting from early adulthood. In contrast, genetic long-lived daf-2 mutants exhibit delayed age-associated changes in mitochondrial morphology, constant mitochondrial density, and maintained trafficking rates during adulthood. Reduced mitochondrial load at late adulthood correlates with decreased mitochondrial resistance to oxidative stress. Revealing aging-associated changes in neuronal mitochondrial maintenance in vivo is an essential precedent that will allow future elucidation of the mechanistic causes of mitochondrial aging profile. Publications: Yuxiang Xie, Bing Zhou, Mei-Yao Lin, Shiwei Wang, Kevin D. Foust, and Zu-Hang Sheng. (2015) Endolysosome deficits augment mitochondria pathology in spinal motor neurons of asymptomatic fALS-linked mice. Neuron 87, 355-370. N. S. Morsci, D. H. Hall, M. Driscoll, and Z.-H. Sheng (2016). Age-related phasic patterns of mitochondrial maintenance in adult C. elegans neurons. Journal of Neuroscience 36, 1373-1385. Zhou, B., P. Yu, MY. Lin, T. Sun, Y. Chen, and Z.-H. Sheng (2016). Facilitation of axon regeneration by enhancing mitochondrial transport and rescuing energy deficits. Journal of Cell Biology 214, 203-119. Lin*, M-Y., X-T. Cheng* (equal contributions), P Tammineni, Y. Xie, B. Zhou, Q. Cai, and Z-H. Sheng (2017). Releasing syntaphilin removes stressed mitochondria from axons independent of mitophagy under pathophysiological conditions. Neuron 94, 595-610. Sheng, Z.-H. (2017) The interplay of axonal energy homeostasis and mitochondrial trafficking and anchoring. (Invited review) Trends in Cell Biology 27, 403-416.
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