Mitofusin activation enhances mitochondrial motility and promotes neuroregeneration in CMT2A

Human brains represent only 2% of body mass, but their high relative metabolic activity accounts for ~20% total adenosine triphosphate (ATP) consumption. ATP generated by neuronal mitochondria fuels nerve signaling and homeostatic repair. In the peripheral nervous system, which has greater capacity regeneration after physical, toxic or genetic injury than central also powers actin polymerization/depolymerization growth cone formation axon extension. Mitochondrial generation is therefore a component functioning in systems. The contextual roles are largely determined two factors, fitness individual organelles localization within host neurons. role mitochondrial widely acknowledged: healthy perform ATP-producing function without compromising neurons, whereas damaged respiratory chain partially uncoupled from production elaborate cytotoxic reactive oxygen species (ROS) that both damage neuron can activate programmed cell death pathways. By comparison, transport recently become appreciated: almost all ~1000 proteins (i.e. except 13 encoded genome) nuclear genes synthesized ribosomes. Therefore, biogenesis takes place primarily soma newly formed actively directed to distal areas activity, such as synapses cones. unique architecture especially long motor neurons innervating upper lower extremities, requires traverse approximately 1 meter length spine neuromuscular junctions. Damaged senescent buds likewise require export mitophagic elimination, necessary prevent mitochondrial-mediated ROS cytotoxicity at nerves. Thus, an interruption might be consequential health as, contribute to, impaired fitness. Recently, Franco et al. (2020) demonstrated directly enhancing trafficking axons helps regenerate mouse model rare sensorimotor neuropathy, Charcot-Marie-Tooth disease type 2A (CMT2A). These studies provide critical vitro vivo support mechanistically linking regeneration. Two neuropathies serve prototypical neurodegenerative diseases having dysfunction: Familial amyotrophic lateral sclerosis caused mutations superoxide dismutase (SOD1) example loss (Wong al., 1995), this case precipitated increased levels. CMT2A mitofusin 2 (MFN2) interrupted (Baloh 2007), although precise mechanism dysfunction fusion protein impairs remains unclear. MFN2 closely related MFN1 were originally described mediating mammalian outer membrane (Dorn, 2019). plays important mitophagy (the selective elimination via autophagy pathways) (Chen Dorn, 2013) axonal (Rocha 2018). observations motility CMT2A-associated dysmotility reversed small molecule activators 2018), suggested pharmacological activation endogenous normal overcome mutant suppression autosomal dominant CMT2A. While conceptually simple, putative mechanistic link between progression least 4 degrees separation: (1) MFN1/MFN2-mediated suppressed mutants; (2) away from, junctions diminished; (3) consequently impaired; (4) nerves die back muscles; (5) muscle mass decreases due neurogenic atrophy. This pathophysiological complexity confounded expectations whether could reverse vivo. testing idea activating mitofusins developed three major objectives. first was assess relevance human replicating patient previous (Franco 2016; Rocha 2018; Dang 2020). Because iPSC-derived lose characteristic phenotypes (e.g. fragmentation stasis motility), used microRNA-mediated conversion reprogram dermal fibroblasts four different into HB9/MNX1-positive Reprogramming efficiency > 90% (β-III tubulin positive) 85% (HB9 cells. Compared reprogrammed multiple subjects, lines exhibited hallmark (loss fusion), depolarization (impaired function), (measured proportion velocity motile mitochondria). Notwithstanding mechanisms (two GTPase domain coiled-coiled hydrophobic core), responded addition activator normalizing morphology, polarization status motility. like respond reversing phenotypes. Moreover, sample benefit on appeared agnostic causal mutation. second goal establish efficacy similar condition. there no published models reproduce time course progression, pattern dysfunction, neuroelectrophysiological features, histological/ultrastructural characteristics absence confounding developmental defects 2020), they new combining specific Mnx1-Cre flox-stop MFN2-T105M transgenes resulting mice express (h) T105M Cre evokes expression extinguished week life, not continuing variable. Paralleling childhood onset with young adulthood, hMFN2 up 10 weeks age then progressive decline RotaRod latency) 20 30 weeks, stabilized. clinically distinguished more common CMT1 neuroelectrophysiologic showing decreased amplitude compound action potentials (reflecting neurons) conduction (because myelin); recapitulated mice. Finally, histological ultrastructural features reproduced Remarkably, daily intramuscular injection trans-MiM111 full-fledged phenotype these abnormalities 8 final elucidate observed reversal activation. As currently understood, pathophysiology forearm foreleg degeneration loss-of-function consists following: leads dearth junctions; compromises die-back; 3. end result muscular atrophy (Figure 1). If correct, restoration activator-treated implies had previously died regenerated, thus reestablishing integrity. Alternately, alone have engendered neuroprotective effects. However, connection regeneration, genetically ablating anchoring syntaphilin shown promote regrowth physical (Zhou 2016).Figure 1: Schematic depiction relationship transport, die-back, CMT2A.Normal (top) bidirectional delivers removes termini. (middle) repair, prompting dying Restoration activated (bottom) promotes reverses dysfunction. CMT2A: 2A.Franco performed series experiments fully understand activation, transport/localization, Reasoning die-back would occupancy junctional reduced number synapses, respectively, immunohistology quantify (anti-acetylcholine receptor) resident (anti-cytochrome oxidase IV) muscles. Consistent regrowth, tibialis muscles vehicle-treated fewer half per myocyte, ~half density those synapses; abnormal metrics above supported thesis CMT2A, data entirely correlative. enhanced inferential. isolated dorsal root ganglion (DRG) carrying allele, transgene using adenoviral Cre, measured DRG outgrowth presence activator. (Note, adeno-Cre experiment because DRGs contain sensory MNX1/HB9 promoter drives predominantly expression.) platform permitted live aspect ratio (an indirect measure residency termini (linking localization), growth/branching (a metric isolation plating), possible fixed tissue. Mitofusin strikingly improved each endpoints, ratio, residency, markedly accelerating outgrowth. Importantly, courses responses, maximized hours accelerated 24 hours, corrected 48 de novo distinct neonatal cortical cultured wells connected empty linear microchannels. Cortical resulted long, non-branching grew through microchannels wells, wherein underwent terminal branching. arrangement mimics soma, branched axon. grow very poorly (as studies), induce grown complex arborizations (i.e., days vitro). way, mature created subsequently aspiration axotomy, amputating while leaving proximal intact. Amputated maintained culture early readouts (aspect ratio) (% velocity) 3 days. affected (already diminished compared treated identically) further restored doubled regions neuron, novel employed integrated demonstrate underlying principle achieved facilitating delivery areas. Indeed, paradigm may apply other diseases, including sclerosis, Huntington’s disease, Parkinson’s structure, and/or neuropathology (Knott 2008). conditions we envision vicious pathophysiologic cycle primary encoding diverse indirectly evoke oxidative stress mitotoxicity, additional feedback loop culminates insufficiency drop-out pathway apoptosis. According scenario, interrupting any point prove therapeutic. interventions general resistance array noxious insults broad therapeutic utility. present work NIH R35HL135736, R41NS113642, R41NS115184, Research Grant 628906 Muscular Dystrophy Association (to GWD). GWD Philip Sima K. Needleman-endowed Professor Scholar-Innovator awardee Harrington Discovery Institute. inventor patent applications PCT/US18/028514 submitted Washington University PCT/US19/46356 Mitochondria Emotion, Inc. cover use agonists treat chronic founder Motion, Inc., Saint Louis based biotech R&D company focused diseases.