Using genetic mouse models combined with time-lapse imaging in live neurons, we identified syntaphilin (SNPH) as a static anchor for axonal mitochondria required for maintaining a large number of axonal mitochondria in a stationary state through an interaction with the microtubule-based cytoskeleton. Deleting snph results in a robust increase of axonal mitochondria in motile pools, while over-expressing SNPH abolishes axonal mitochondrial transport. The snph knockout mouse is an ideal genetic model to examine the impact of altered motility of axonal mitochondria on presynaptic function, mitochondrial quality control, axonal degeneration and regeneration.
Specific Aim 1. Mobile Axonal Mitochondria Contribute to the Variability of Synaptic Strength 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. In hippocampal neurons, approximately one-third of axonal mitochondria are highly motile and some dynamically pass through presynaptic boutons. This raises a fundamental question: Can those motile mitochondria contribute to the pulse-to-pulse variability of presynaptic strength? We are solving this puzzle by combining live neuron imaging and electrophysiological analysis of snph genetic mouse model in which axonal mitochondrial motility was selectively manipulated. Using hippocampal neurons and slices of snph knockout mice, we demonstrate that the motility of axonal mitochondria correlates with presynaptic variability. Enhancing mitochondrial motility increases the pulse-to-pulse variability, while immobilizing mitochondria reduces the variability. Using dual-channel imaging of axonal mitochondria motility and SV release at single-bouton levels, we further showed that mitochondrial movement, either into or out of presynaptic boutons, significantly influences SV release due to fluctuation of synaptic ATP levels. An anchored mitochondrion provides stable and continuous ATP supply. A motile mitochondrion passing through synapses temporally supplies ATP, thus changing synaptic energy levels and influencing ATP-dependent synaptic activities. Therefore, the fluctuation of presynaptic ATP levels resulting from axonal mitochondrial transport contributes to the wide variability of presynaptic strength. Our study revealed, for the first time, that the fast movement of axonal mitochondria is one of the primary mechanisms underlying the presynaptic variation. Thus, our study brings new insight into the fundamental properties of the CNS to ensure the plasticity and reliability of synaptic transmission.
Specific Aim 2. Mechanism Removing Damaged Mitochondria from Axonal Terminals for Mitophagy Mitochondrial dysfunction, altered dynamics, and impaired transport emerge as central problems associate with major neurodegenerative disorders. Sequestration of damaged mitochondria into autophagosomes and subsequent degradation within mature lysosomes constitute a key cellular pathway in mitochondrial quality control. We provide multiple lines of in vitro and in vivo evidence showing mature acidic lysosomes are mainly localized in soma and proximal regions. Thus, an important mechanistic question, critical toward understanding axonal degeneration emerges: How are damaged mitochondria at distal synapses efficiently eliminated? By chronically dissipating mitochondrial membrane potential (Δψm) in mature cortical neurons, we demonstrate that Parkin-mediated mitophagy mainly occurs in the soma and proximal regions of processes. Under these chronic stress conditions, the majority of neurons survive and a significant portion of mitochondria remains motile, thus reflecting chronic mitochondrial stress in vivo under pathophysiological conditions. We further detect a significant decrease of axonal mittochondrial docking protein syntaphilin (SNPH) following chronic Δψm dissipation. Mitochondrial retrograde transport is increased after mitochondrial depolarization. This phenotype is correlated with reduced levels of SNPH. Live neuron imaging analysis revealed that SNPH-containing vesicles are derived from depolarized mitochondria. Our study demonstrates that SNPH is highly regulated by sensing Δψm. Degradation of SNPH is required for enhanced retrograde transport, thus preferring damaged mitochondria trafficking from distal axons to the soma where major lysosome are mainly localized. Altogether, our study provides new mechanistic insight into the fine coordination of mitochondrial motility and the quality control in distal axons of neurons.
Specific Aim 3. Axonal Mitochondrial Transport Impacts Axon Regeneration Mitochondria supply ATP to power various activities essential for axonal growth and regeneration, thus raising a fundamental question as whether mitochondrial transport toward growth cones controls axonal regeneration capacity following axon injury. By applying microfluidic neuron culture system, which allows physical and fluidic separation of axons from cell bodies and dendrites, we found that snph deficiency, which selectively increases axonal mitochondrial motility, facilitates axonal regeneration capacity of mature cortical neurons after axotomy. Loss of axon regeneration capacity in mature cortical neurons correlates with reduced mitochondria motility and increased SNPH expression. Interestingly, mature and adult neurons regain axon regeneration capacity by enhancing mitochondrial transport, while blocking mitochondrial ATP generation by oligomycin abolishes such regrowth capacity. Furthermore, altered axonal mitochondrial motility impacts distal energy homeostasis. Our study provides mechanistic insights into how axonal regeneration capacity in mature or adult neurons is controlled by mitochondrial motility and energy homeostasis at axonal tips.
Specific Aim 4. Endolysosomal Deficits Augment Mitochondria Pathology in Spinal Motor Neurons of Asymptomatic fALS Mice 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 by 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 (P40), accompanied by aberrant accumulation of damaged mitochondria engulfed by autophagosomes in axons. Our in vitro and in vivo studies demonstrate that endo-lysosomal transport is crucial to maintain mitochondrial integrity and MN survival. Such deficits are attributable to impaired retrograde transport of late endosomes by mutant hSOD1G93A, which interferes with dynein-snapin (motor-adaptor) coupling, thus reducing the recruitment of dynein motors to the organelles for transport. These deficits can be rescued by elevated snapin expression, which competes with hSOD1G93A to restore dynein-driven transport. AAV9-snapin injection in hSOD1G93A mice reverses mitochondria pathology, reduces MN loss, and ameliorates the fALS-linked disease phenotype. Our study reveals a new cellular target for development of early therapeutic intervention. Elucidation of this early pathological mechanism is broadly relevant, because defective retrograde transport, lysosomal deficits, and mitochondrial pathology are associated with major neurodegenerative diseases including ALS, Huntingtons, Parkinsons and Alzheimers diseases. Therefore, enhancing clearance of damaged mitochondria by regulating endolysosomal trafficking may be a potential therapeutic strategy for ALS and perhaps other neurodegenerative diseases.
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