CRMP/UNC-33 organizes microtubule bundles for KIF5-mediated mitochondrial distribution to axon

Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.     

PINK1- and PARK2-mediated local mitophagy in distal neuronal axons

Mutations in the PINK1 and PARK2/PARKIN genes are associated with hereditary early onset Parkinson disease (PD), and in cell lines the corresponding gene products play a critical role in mitophagic clearance of damaged mitochondria. In neurons, however, where the extraordinary cellular shapes pose particular challenges for maintaining healthy mitochondria, the pathways of mitophagy are less well understood. Both the location at which mitophagy occurs and the involvement of PINK1 and PARK2 have been controversial. Here we review our recent study where we found that selective damage to a subset of axonal mitochondria causes them to be engulfed within autophagosomes and cleared locally within the axon without the need for transport back to the soma. We also found this process to be completely dependent on neuronal PINK1 and PARK2.     

FANCJ lets chromatin stay true

Axonal mitochondria are recruited to synaptic terminals in response to neuronal activity, but the mechanisms underlying activity-dependent regulation of mitochondrial transport are largely unknown. In this paper, using genetic mouse model combined with live imaging, we demonstrate that syntaphilin (SNPH) mediates the activity-dependent immobilization of axonal mitochondria through binding to KIF5. In vitro analysis showed that the KIF5–SNPH coupling inhibited the motor adenosine triphosphatase. Neuronal activity further recruited SNPH to axonal mitochondria. This motor-docking interplay was induced by Ca²⁺ and synaptic activity and was necessary to establish an appropriate balance between motile and stationary axonal mitochondria. Deleting snph abolished the activity-dependent immobilization of axonal mitochondria. We propose an “Engine-Switch and Brake” model, in which SNPH acts both as an engine off switch by sensing mitochondrial Rho guanosine triphosphatase-Ca²⁺ and as a brake by anchoring mitochondria to the microtubule track. Altogether, our study provides new mechanistic insight into the molecular interplay between motor and docking proteins, which arrests axonal mitochondrial transport in response to changes in neuronal activity.     

ASSOCIATION OF CALCIUM WITH MEMBRANES OF SQUID GIANT AXON : Ultrastructure and Microprobe Analysis

Giant axons from the squid, Loligo pealei, were fixed in glutaraldehyde and postfixed in osmium tetroxide. Calcium chloride (5 mM/liter) was added to all aqueous solutions used for tissue processing. Electron-opaque deposits were found along the axonal plasma membranes, within mitochondria, and along the basal plasma membranes of Schwann cells. X-ray microprobe analysis (EMMA-4) yielded signals for calcium and phosphorus when deposits were probed, whereas these elements were not detected in the axoplasm.     

Drosophila Growth Cones Advance by Forward Translocation of the Neuronal Cytoskeletal Meshwork In Vivo

In vitro studies conducted in Aplysia and chick sensory neurons indicate that in addition to microtubule assembly, long microtubules in the C-domain of the growth cone move forward as a coherent bundle during axonal elongation. Nonetheless, whether this mode of microtubule translocation contributes to growth cone motility in vivo is unknown. To address this question, we turned to the model system Drosophila. Using docked mitochondria as fiduciary markers for the translocation of long microtubules, we first examined motion along the axon to test if the pattern of axonal elongation is conserved between Drosophila and other species in vitro. When Drosophila neurons were cultured on Drosophila extracellular matrix proteins collected from the Drosophila Kc167 cell line, docked mitochondria moved in a pattern indicative of bulk microtubule translocation, similar to that observed in chick sensory neurons grown on laminin. To investigate whether the C-domain is stationary or advances in vivo, we tracked the movement of mitochondria during elongation of the aCC motor neuron in stage 16 Drosophila embryos. We found docked mitochondria moved forward along the axon shaft and in the growth cone C-domain. This work confirms that the physical mechanism of growth cone advance is similar between Drosophila and vertebrate neurons and suggests forward translocation of the microtubule meshwork in the axon underlies the advance of the growth cone C-domain in vivo. These results highlight the need for incorporating en masse microtubule translocation, in addition to assembly, into models of axonal elongation.     

Misfolded SOD1 associated with motor neuron mitochondria alters mitochondrial shape and distribution prior to clinical onset

Mutations in superoxide dismutase (SOD1) are causative for inherited amyotrophic lateral sclerosis. A proportion of SOD1 mutant protein is misfolded onto the cytoplasmic face of mitochondria in one or more spinal cord cell types. By construction of mice in which mitochondrially targeted enhanced green fluorescent protein is selectively expressed in motor neurons, we demonstrate that axonal mitochondria of motor neurons are primary in vivo targets for misfolded SOD1. Mutant SOD1 alters axonal mitochondrial morphology and distribution, with dismutase active SOD1 causing mitochondrial clustering at the proximal side of Schmidt-Lanterman incisures within motor axons and dismutase inactive SOD1 producing aberrantly elongated axonal mitochondria beginning pre-symptomatically and increasing in severity as disease progresses. Somal mitochondria are altered by mutant SOD1, with loss of the characteristic cylindrical, networked morphology and its replacement by a less elongated, more spherical shape. These data indicate that mutant SOD1 binding to mitochondria disrupts normal mitochondrial distribution and size homeostasis as early pathogenic features of SOD1 mutant-mediated ALS.     

Translating m-AAA protease function in mitochondria to hereditary spastic paraplegia

Hereditary spastic paraplegia (HSP) is a genetically heterogeneous neurodegenerative disorder that is characterized by progressive and cell-specific axonal degeneration. An autosomal recessive form of the disease is caused by mutations in paraplegin, which is a conserved subunit of the ubiquitous and ATP-dependent m-AAA protease in mitochondria. The m-AAA protease carries out protein quality control in the inner membrane of the mitochondria, suggesting a pathogenic role of misfolded proteins in HSP. A recent study demonstrates that the m-AAA protease regulates ribosome assembly and translation within mitochondria by controlling proteolytic maturation of a ribosomal subunit. Here, we will discuss implications of the dual role of the m-AAA protease in protein activation and degradation for mitochondrial dysfunction and axonal degeneration.     

Docking of axonal mitochondria by syntaphilin controls their mobility and affects short-term facilitation

Proper distribution of mitochondria within axons and at synapses is critical for neuronal function. While one-third of axonal mitochondria are mobile, a large proportion remains in a stationary phase. However, the mechanisms controlling mitochondrial docking within axons remain elusive. Here, we report a role for axon-targeted syntaphilin (SNPH) in mitochondrial docking through its interaction with microtubules. Axonal mitochondria that contain exogenously or endogenously expressed SNPH lose mobility. Deletion of the mouse snph gene results in a substantially higher proportion of axonal mitochondria in the mobile state and reduces the density of mitochondria in axons. The snph mutant neurons exhibit enhanced short-term facilitation during prolonged stimulation, probably by affecting calcium signaling at presynaptic boutons. This phenotype is fully rescued by reintroducing the snph gene into the mutant neurons. These findings demonstrate a molecular mechanism for controlling mitochondrial docking in axons that has a physiological impact on synaptic function.     

Axoplasmic transport in the crayfish nerve cord. The role of fibrillar constituents of neurons

(a) Axoplasmic transport of tritium-labeled proteins in crayfish nerve cord was confirmed at a slow rate of 1 mm/day. A second proteinaceous component which moves at a rate of 10 mm/day was also detected. Radioautography and biochemical analysis indicate that proteins migrating at these velocities have a perikaryal origin and move caudad within axons as sharply defined peaks. (b) Evidence is presented for the blockage of the slow and the fast movement of proteins by intraganglionic injection of the anti-mitotic agent vinblastine sulfate (0.1 mM). (c) Electron microscope observations of vinblastine-treated ganglia revealed a reduction in the number of axonal microtubules and the formation of intracellular aggregates presumably composed of microtubular protein. (d) These findings would be compatible with the involvement of microtubules in both slow and fast axoplasmic transport. However, the block induced by vinblastine was detected in regions of the cord (up to 10 mm away from the injection site) where the number and morphology of microtubules appeared unaltered. In addition, axons showing effects of vinblastine occasionally contained mitochondria with remarkably dense and thickened membranes. (e) In association with the surfaces of axonal microtubules are lateral filamentous elements (40–80 A in diameter) which also showed vinblastine-induced alterations. Our observations indicate that such filiform structures, associated with microtubules, may be a necessary component in the transport mechanism(s).     

Direct evidence for axonal transport defects in a novel mouse model of mutant spastin-induced hereditary spastic paraplegia (HSP) and human HSP patients

Mutations in spastin are the most common cause of hereditary spastic paraplegia (HSP) but the mechanisms by which mutant spastin induces disease are not clear. Spastin functions to regulate microtubule organisation, and because of the essential role of microtubules in axonal transport, this has led to the suggestion that defects in axonal transport may underlie at least part of the disease process in HSP. However, as yet there is no direct evidence to support this notion. Here we analysed axonal transport in a novel mouse model of spastin-induced HSP that involves a pathogenic splice site mutation, which leads to a loss of spastin protein. A mutation located within the same splice site has been previously described in HSP. Spastin mice develop gait abnormalities that correlate with phenotypes seen in HSP patients and also axonal swellings containing cytoskeletal proteins, mitochondria and the amyloid precursor protein (APP). Pathological analyses of human HSP cases caused by spastin mutations revealed the presence of similar axonal swellings. To determine whether mutant spastin influenced axonal transport we quantified transport of two cargoes, mitochondria and APP-containing membrane bound organelles, in neurons from mutant spastin and control mice, using time-lapse microscopy. We found that mutant spastin perturbs anterograde transport of both cargoes. In neurons with axonal swellings we found that the mitochondrial axonal transport defects were exacerbated; distal to axonal swellings both anterograde and retrograde transport were severely reduced. These results strongly support a direct role for defective axonal transport in the pathogenesis of HSP because of spastin mutation.     

Kinesin-1 and Dynein are the primary motors for fast transport of mitochondria in Drosophila motor axons

To address questions about mechanisms of filament-based organelle transport, a system was developed to image and track mitochondria in an intact Drosophila nervous system. Mutant analyses suggest that the primary motors for mitochondrial movement in larval motor axons are kinesin-1 (anterograde) and cytoplasmic dynein (retrograde), and interestingly that kinesin-1 is critical for retrograde transport by dynein. During transport, there was little evidence that force production by the two opposing motors was competitive, suggesting a mechanism for alternate coordination. Tests of the possible coordination factor P150(Glued) suggested that it indeed influenced both motors on axonal mitochondria, but there was no evidence that its function was critical for the motor coordination mechanism. Observation of organelle-filled axonal swellings ("organelle jams" or "clogs") caused by kinesin and dynein mutations showed that mitochondria could move vigorously within and pass through them, indicating that they were not the simple steric transport blockades suggested previously. We speculate that axonal swellings may instead reflect sites of autophagocytosis of senescent mitochondria that are stranded in axons by retrograde transport failure; a protective process aimed at suppressing cell death signals and neurodegeneration.     

In vivo imaging reveals mitophagy independence in the maintenance of axonal mitochondria during normal aging

Mitophagy is thought to be a critical mitochondrial quality control mechanism in neurons and has been extensively studied in neurological disorders such as Parkinson's disease. However, little is known about how mitochondria are maintained in the lengthy neuronal axons in the context of physiological aging. Here, we utilized the unique Drosophila wing nerve model and in vivo imaging to rigorously profile changes in axonal mitochondria during aging. We revealed that mitochondria became fragmented and accumulated in aged axons. However, lack of Pink1 or Parkin did not lead to the accumulation of axonal mitochondria or axonal degeneration. Further, unlike in in vitro cultured neurons, we found that mitophagy rarely occurred in intact axons in vivo, even in aged animals. Furthermore, blocking overall mitophagy by knockdown of the core autophagy genes Atg12 or Atg17 had little effect on the turnover of axonal mitochondria or axonal integrity, suggesting that mitophagy is not required for axonal maintenance; this is regardless of whether the mitophagy is PINK1‐Parkin dependent or independent. In contrast, downregulation of mitochondrial fission–fusion genes caused age‐dependent axonal degeneration. Moreover, Opa1 expression in the fly head was significantly decreased with age, which may underlie the accumulation of fragmented mitochondria in aged axons. Finally, we showed that adult‐onset, neuronal downregulation of the fission–fusion, but not mitophagy genes, dramatically accelerated features of aging. We propose that axonal mitochondria are maintained independently of mitophagy and that mitophagy‐independent mechanisms such as fission–fusion may be central to the maintenance of axonal mitochondria and neural integrity during normal aging.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Ultrastructural studies of severed medial giant and other CNS axons in crayfish

Summary The distal stumps of severed medial giant axons (MGAs) and of non-giant axons (NGAs) in the CNS of the crayfish Procambarus clarkii show long-term (5–9 months) survival associated with disorientation of mitochondria and thickening of the glial sheath. However, the morphological responses of the two axonal types differ in that neither the proximal nor the distal stump of severed MGAs ever fills with mitochondria as is observed in some severed NGAs. Furthermore, the adaxonal glial layer never completely encircles portions of MGA axoplasm as occurs in many severed NGAs; in fact, ultrastructural changes in the adaxonal layer around severed MGAs are often difficult to detect. No multiple axonal profiles are ever seen within the glial sheath of the proximal or distal stumps of severed MGAs whereas these structures are easily located within severed NGAs.This work was supported by NIH research grant #NS-14412 and an RCDA#00070 to GDB     

CSPGs inhibit axon branching by impairing mitochondria‐dependent regulation of actin dynamics and axonal translation

Chondroitin sulfate proteoglycans (CSPGs) inhibit the formation of axon collateral branches. The regulation of the axonal cytoskeleton and mitochondria are important components of the mechanism of branching. Actin‐dependent axonal plasticity, reflected in the dynamics of axonal actin patches and filopodia, is greatest along segments of the axon populated by mitochondria. It is reported that CSPGs partially depolarize the membrane potential of axonal mitochondria, which impairs the dynamics of the axonal actin cytoskeleton and decreases the formation and duration of axonal filopodia, the first steps in the mechanism of branching. The effects of CSPGs on actin cytoskeletal dynamics are specific to axon segments populated by mitochondria. In contrast, CSPGs do not affect the microtubule content of axons, or the localization of microtubules into axonal filopodia, a required step in the mechanism of branch formation. It is also reported that CSPGs decrease the mitochondria‐dependent axonal translation of cortactin, an actin associated protein involved in branching. Finally, the inhibitory effects of CSPGs on axon branching, actin cytoskeletal dynamics and the axonal translation of cortactin are reversed by culturing neurons with acetyl‐l ‐carnitine, which promotes mitochondrial respiration. Collectively these data indicate that CSPGs impair mitochondrial function in axons, an effect which contributes to the inhibition of axon branching. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

A new method for quantifying mitochondrial axonal transport

Axonal transport of mitochondria is critical for neuronal survival and function. Automatically quantifying and analyzing mitochondrial movement in a large quantity remain challenging. Here, we report an efficient method for imaging and quantifying axonal mitochondrial transport using microfluidic-chamber-cultured neurons together with a newly developed analysis package named “MitoQuant”. This tool-kit consists of an automated program for tracking mitochondrial movement inside live neuronal axons and a transient-velocity analysis program for analyzing dynamic movement patterns of mitochondria. Using this method, we examined axonal mitochondrial movement both in cultured mammalian neurons and in motor neuron axons of Drosophila in vivo. In 3 different paradigms (temperature changes, drug treatment and genetic manipulation) that affect mitochondria, we have shown that this new method is highly efficient and sensitive for detecting changes in mitochondrial movement. The method significantly enhanced our ability to quantitatively analyze axonal mitochondrial movement and allowed us to detect dynamic changes in axonal mitochondrial transport that were not detected by traditional kymographic analyses.     

Amyloid beta impairs mitochondrial anterograde transport and degenerates synapses in Alzheimer's disease neurons

Loss of synapses and synaptic damage are the best correlates of cognitive decline identified in patients with Alzheimer's disease (AD), and mitochondrial oxidative damage and synaptic pathology have been identified as early events in the progression of AD. The progressive accumulation of amyloid beta (Aβ) in synapses and synaptic mitochondria are hypothesized to cause synaptic degeneration and cognitive decline in patients with AD. However, the precise mechanistic link between Aβ and mitochondria is not well understood. The purpose of this study was to better understand the effects of Aβ on mitochondrial axonal transport and synaptic alterations in AD. Using mouse hippocampal neurons and Aβ(25-35) peptide, we studied axonal transport of mitochondria, including mitochondrial motility, mitochondrial length and size, mitochondrial index per neurite, and synaptic alterations of the hippocampal neurons. In the PBS-treated neurons, 36.4±4.7% of the observed mitochondria were motile, with 21.0±1.3% moving anterograde and 15.4±3.4% moving retrograde and the average speed of movement was 12.1±1.8μm/min. In contrast, in the Aβ-treated neurons, the number of motile mitochondria were significantly less, at 20.4±2.6% (P<0.032), as were those moving anterograde (10.1±2.6%, P<0.016) relative to PBS-treated neurons, suggesting that the Aβ(25-35) peptide impairs axonal transport of mitochondria in AD neurons. In the Aβ-treated neurons, the average speed of motile mitochondria was also less, at 10.9±1.9μm/min, and mitochondrial length was significantly decreased. Further, synaptic immunoreactivity was also significantly less in the Aβ-treated neurons relative to the PBS-treated neurons, indicating that Aβ affects synaptic viability. These findings suggest that, in neurons affected by AD, Aβ is toxic, impairs mitochondrial movements, reduces mitochondrial length, and causes synaptic degeneration.     

HDAC6 Inhibitor Blocks Amyloid Beta-Induced Impairment of Mitochondrial Transport in Hippocampal Neurons

Even though the disruption of axonal transport is an important pathophysiological factor in neurodegenerative diseases including Alzheimer's disease (AD), the relationship between disruption of axonal transport and pathogenesis of AD is poorly understood. Considering that α-tubulin acetylation is an important factor in axonal transport and that Aβ impairs mitochondrial axonal transport, we manipulated the level of α-tubulin acetylation in hippocampal neurons with Aβ cultured in a microfluidic system and examined its effect on mitochondrial axonal transport. We found that inhibiting histone deacetylase 6 (HDAC6), which deacetylates α-tubulin, significantly restored the velocity and motility of the mitochondria in both anterograde and retrograde axonal transports, which would be otherwise compromised by Aβ. The inhibition of HDAC6 also recovered the length of the mitochondria that had been shortened by Aβ to a normal level. These results suggest that the inhibition of HDAC6 significantly rescues hippocampal neurons from Aβ-induced impairment of mitochondrial axonal transport as well as mitochondrial length. The results presented in this paper identify HDAC6 as an important regulator of mitochondrial transport as well as elongation and, thus, a potential target whose pharmacological inhibition contributes to improving mitochondrial dynamics in Aβ treated neurons.     

Loss of Axonal Mitochondria Promotes Tau-Mediated Neurodegeneration and Alzheimer's Disease-Related Tau Phosphorylation Via PAR-1

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.     

Increased axonal mitochondrial mobility does not slow amyotrophic lateral sclerosis (ALS)-like disease in mutant SOD1 mice

Reduced axonal mitochondrial transport has been observed in major neurodegenerative diseases, including fALS patients and SOD1(G93A) mice. However, it is unclear whether this defect plays a critical role in axonal degeneration or simply reflects sequelae of general transport alteration. Using genetic mouse models combined with time-lapse imaging of live neurons, we previously discovered that axon-targeted syntaphilin (SNPH) acts as a docking receptor specific for axonal mitochondria. Deletion of the snph gene in mice results in a substantially higher proportion of axonal mitochondria in the mobile state without any effect on the transport of other axonal organelles. Here we address whether increased (rescued) axonal mitochondrial mobility changes the disease course by crossing fALS-linked transgenic SOD1(G93A) and snph(-/-) knock-out mice. We found that a 2-fold increase in axonal mitochondrial mobility in SOD1(G93A)/snph(-/-) mice did not affect the onset of ALS-like symptoms. Both SOD1(G93A) and SOD1(G93A)/snph(-/-) mice exhibit similar weight loss, deterioration in motor function and motor neuron loss, significant gliosis, and a lifespan of 152-154 days. Thus, for the first time, our study provides genetic and pathological evidence that the impairment of mitochondrial transport seen in SOD1(G93A) mice plays a minimal role in the rapid-onset of fALS-linked pathology.