Miro GTPase domains regulate the assembly of the mitochondrial motor-adaptor complex.

Mitochondrial transport relies on a motor-adaptor complex containing Miro1, a mitochondrial outer membrane protein with two GTPase domains, and TRAK1/2, kinesin-1, and dynein. Using a peroxisome-directed Miro1, we quantified the ability of GTPase mutations to influence the peroxisomal recruitment of complex components. Miro1 whose N-GTPase is locked in the GDP state does not recruit TRAK1/2, kinesin, or P135 to peroxisomes, whereas the GTP state does. Similarly, the expression of the MiroGAP VopE dislodges TRAK1 from mitochondria. Miro1 C-GTPase mutations have little influence on complex recruitment. Although Miro2 is thought to support mitochondrial motility, peroxisome-directed Miro2 did not recruit the other complex components regardless of the state of its GTPase domains. Neurons expressing peroxisomal Miro1 with the GTP-state form of the N-GTPase had markedly increased peroxisomal transport to growth cones, whereas the GDP-state caused their retention in the soma. Thus, the N-GTPase domain of Miro1 is critical for regulating Miro1's interaction with the other components of the motor-adaptor complex and thereby for regulating mitochondrial motility.

Virtual screening for small-molecule pathway regulators by image-profile matching.

Identifying the chemical regulators of biological pathways is a time-consuming bottleneck in developing therapeutics and research compounds. Typically, thousands to millions of candidate small molecules are tested in target-based biochemical screens or phenotypic cell-based screens, both expensive experiments customized to each disease. Here, our uncustomized, virtual, profile-based screening approach instead identifies compounds that match to pathways based on the phenotypic information in public cell image data, created using the Cell Painting assay. Our straightforward correlation-based computational strategy retrospectively uncovered the expected, known small-molecule regulators for 32% of positive-control gene queries. In prospective, discovery mode, we efficiently identified new compounds related to three query genes and validated them in subsequent gene-relevant assays, including compounds that phenocopy or pheno-oppose YAP1 overexpression and kill a Yap1-dependent sarcoma cell line. This image-profile-based approach could replace many customized labor- and resource-intensive screens and accelerate the discovery of biologically and therapeutically useful compounds.

Serine/Threonine Protein Phosphatase 2A Regulates the Transport of Axonal Mitochondria.

Microtubule-based transport provides mitochondria to distant regions of neurons and is essential for neuronal health. To identify compounds that increase mitochondrial motility, we screened 1,641 small-molecules in a high-throughput screening platform. Indirubin and cantharidin increased mitochondrial motility in rat cortical neurons. Cantharidin is known to inhibit protein phosphatase 2A (PP2A). We therefore tested two other inhibitors of PP2A: LB-100 and okadaic acid. LB-100 increased mitochondrial motility, but okadaic acid did not. To resolve this discrepancy, we knocked down expression of the catalytic subunit of PP2A (PP2CA). This long-term inhibition of PP2A more than doubled retrograde transport of axonal mitochondria, confirming the importance of PP2A as a regulator of mitochondrial motility and as the likely mediator of cantharidin's effect.

Mitochondrial hitch-hiking of Pink1 mRNA supports axonal mitophagy.

Mitostasis, the process of mitochondrial maintenance by biogenesis and degradative mechanisms, is challenged by the extreme length of axons. PINK1 (PTEN induced putative kinase 1) is a mitochondrial protein that targets damaged mitochondria for mitophagy. In reconciling the short half-life of PINK1 with the need for mitophagy of damaged axonal mitochondria, we found that axonal mitophagy depends on local translation of the Pink1 mRNA. Using live-cell imaging, we detected co-transport of the Pink1 mRNA on mitochondria in neurons, which is crucial for mitophagy in distal parts of the cell. Here we discuss how the coupling of the transcript of a short-lived mitochondrial protein to the movement of its target organelles contributes to our understanding of mitostasis in neurons.

APP and DYRK1A regulate axonal and synaptic vesicle protein networks and mediate Alzheimer's pathology in trisomy 21 neurons.

Trisomy 21 (T21) causes Down syndrome and an early-onset form of Alzheimer's disease (AD). Here, we used human induced pluripotent stem cells (hiPSCs) along with CRISPR-Cas9 gene editing to investigate the contribution of chromosome 21 candidate genes to AD-relevant neuronal phenotypes. We utilized a direct neuronal differentiation protocol to bypass neurodevelopmental cell fate phenotypes caused by T21 followed by unbiased proteomics and western blotting to define the proteins dysregulated in T21 postmitotic neurons. We show that normalization of copy number of APP and DYRK1A each rescue elevated tau phosphorylation in T21 neurons, while reductions of RCAN1 and SYNJ1 do not. To determine the T21 alterations relevant to early-onset AD, we identified common pathways altered in familial Alzheimer's disease neurons and determined which of these were rescued by normalization of APP and DYRK1A copy number in T21 neurons. These studies identified disruptions in T21 neurons in both the axonal cytoskeletal network and presynaptic proteins that play critical roles in axonal transport and synaptic vesicle cycling. These alterations in the proteomic profiles have functional consequences: fAD and T21 neurons exhibit dysregulated axonal trafficking and T21 neurons display enhanced synaptic vesicle release. Taken together, our findings provide insights into the initial molecular alterations within neurons that ultimately lead to synaptic loss and axonal degeneration in Down syndrome and early-onset AD.

Neuronal mitochondria transport Pink1 mRNA via synaptojanin 2 to support local mitophagy.

PTEN-induced kinase 1 (PINK1) is a short-lived protein required for the removal of damaged mitochondria through Parkin translocation and mitophagy. Because the short half-life of PINK1 limits its ability to be trafficked into neurites, local translation is required for this mitophagy pathway to be active far from the soma. The Pink1 transcript is associated and cotransported with neuronal mitochondria. In concert with translation, the mitochondrial outer membrane proteins synaptojanin 2 binding protein (SYNJ2BP) and synaptojanin 2 (SYNJ2) are required for tethering Pink1 mRNA to mitochondria via an RNA-binding domain in SYNJ2. This neuron-specific adaptation for the local translation of PINK1 provides distal mitochondria with a continuous supply of PINK1 for the activation of mitophagy.

FHL2 anchors mitochondria to actin and adapts mitochondrial dynamics to glucose supply.

Mitochondrial movement and distribution are fundamental to their function. Here we report a mechanism that regulates mitochondrial movement by anchoring mitochondria to the F-actin cytoskeleton. This mechanism is activated by an increase in glucose influx and the consequent O-GlcNAcylation of TRAK (Milton), a component of the mitochondrial motor-adaptor complex. The protein four and a half LIM domains protein 2 (FHL2) serves as the anchor. FHL2 associates with O-GlcNAcylated TRAK and is both necessary and sufficient to drive the accumulation of F-actin around mitochondria and to arrest mitochondrial movement by anchoring to F-actin. Disruption of F-actin restores mitochondrial movement that had been arrested by either TRAK O-GlcNAcylation or forced direction of FHL2 to mitochondria. This pathway for mitochondrial immobilization is present in both neurons and non-neuronal cells and can thereby adapt mitochondrial dynamics to changes in glucose availability.

zapERtrap: A light-regulated ER release system reveals unexpected neuronal trafficking pathways.

Here we introduce zapalog-mediated endoplasmic reticulum trap (zapERtrap), which allows one to use light to precisely trigger forward trafficking of diverse integral membrane proteins from internal secretory organelles to the cell surface with single cell and subcellular spatial resolution. To demonstrate its utility, we use zapERtrap in neurons to dissect where synaptic proteins emerge at the cell surface when processed through central (cell body) or remote (dendrites) secretory pathways. We reveal rapid and direct long-range trafficking of centrally processed proteins deep into the dendritic arbor to synaptic sites. Select proteins were also trafficked to the plasma membrane of the axon initial segment, revealing a novel surface trafficking hotspot. Proteins locally processed through dendritic secretory networks were widely dispersed before surface insertion, challenging assumptions for precise trafficking at remote sites. These experiments provide new insights into compartmentalized secretory trafficking and showcase the tunability and spatiotemporal control of zapERtrap, which will have broad applications for regulating cell signaling and function.

QuoVadoPro, an Autonomous Tool for Measuring Intracellular Dynamics using Temporal Variance.

Trafficking of intracellular cargo is essential to cellular function and can be defective in pathological states including cancer and neurodegeneration. Tools to quantify intracellular traffic are thus necessary for understanding this fundamental cellular process, studying disease mechanisms, and testing the effects of therapeutic pharmaceuticals. In this article we introduce an algorithm called QuoVadoPro that autonomously quantifies the movement of fluorescently tagged intracellular cargo. QuoVadoPro infers the extent of intracellular motility based on the variance of pixel illumination in a series of time-lapse images. The algorithm is an unconventional approach to the automatic measurement of intracellular traffic and is suitable for quantifying movements of intracellular cargo under diverse experimental paradigms. QuoVadoPro is particularly useful to measure intracellular cargo movement in non-neuronal cells, where cargo trafficking occurs as short movements in mixed directions. The algorithm can be applied to images with low temporal or spatial resolutions and to intracellular cargo with varying shapes or sizes, like mitochondria or endoplasmic reticulum: situations in which conventional methods such as kymography and particle tracking cannot be applied. In this article we present a stepwise protocol for using the QuoVadoPro software, illustrate its methodology with common examples, discuss critical parameters for reliable data analysis, and demonstrate its use with a previously published example. © 2020 Wiley Periodicals LLC. Basic Protocol: QuoVadoPro, an autonomous tool for measuring intracellular dynamics using temporal variance.

Kymolyzer, a Semi-Autonomous Kymography Tool to Analyze Intracellular Motility.

The movement of intracellular cargo, such as transcripts, proteins, and organelles, is fundamental to cellular function. Neurons, due to their long axons and dendrites, are particularly dependent on proper intracellular trafficking and vulnerable to defects in the movement of intracellular cargo that are noted in neurodegenerative and neurodevelopmental disorders. Accurate quantification of intracellular transport is therefore needed for studying the mechanisms of cargo trafficking, the influence of mutations, and the effects of potentially therapeutic pharmaceuticals. In this article, we introduce an algorithm called "Kymolyzer." The algorithm can quantify intracellular trafficking along a defined path, such as that formed by the aligned microtubules of axons and dendrites. Kymolyzer works as a semi-autonomous kymography software application. It constructs and analyzes kymographs to measure the movement and distribution of fluorescently tagged objects along a user-defined path. The algorithm can be used under a wide variety of experimental conditions and can extract a diverse array of motility parameters describing intracellular movement, including time spent in motion, percentage of objects in motion, percentage of objects that are stationary, and velocities of motile objects. This article serves as a user manual describing the design of Kymolyzer, providing a stepwise protocol for its use and illustrating its functions with common examples. © 2020 Wiley Periodicals LLC Basic Protocol: Kymolyzer, a semi-autonomous kymography tool to analyze intracellular motility.

A High-Content Screen Identifies TPP1 and Aurora B as Regulators of Axonal Mitochondrial Transport.

Dysregulated axonal trafficking of mitochondria is linked to neurodegenerative disorders. We report a high-content screen for small-molecule regulators of the axonal transport of mitochondria. Six compounds enhanced mitochondrial transport in the sub-micromolar range, acting via three cellular targets: F-actin, Tripeptidyl peptidase 1 (TPP1), or Aurora Kinase B (AurKB). Pharmacological inhibition or small hairpin RNA (shRNA) knockdown of each target promotes mitochondrial axonal transport in rat hippocampal neurons and induced pluripotent stem cell (iPSC)-derived human cortical neurons and enhances mitochondrial transport in iPSC-derived motor neurons from an amyotrophic lateral sclerosis (ALS) patient bearing one copy of SOD1A4V mutation. Our work identifies druggable regulators of axonal transport of mitochondria, provides broadly applicable methods for similar image-based screens, and suggests that restoration of proper axonal trafficking of mitochondria can be achieved in human ALS neurons.

The light-sensitive dimerizer zapalog reveals distinct modes of immobilization for axonal mitochondria.

Controlling cellular processes with light can help elucidate their underlying mechanisms. Here we present zapalog, a small-molecule dimerizer that undergoes photolysis when exposed to blue light. Zapalog dimerizes any two proteins tagged with the FKBP and DHFR domains until exposure to light causes its photolysis. Dimerization can be repeatedly restored with uncleaved zapalog. We implement this method to investigate mitochondrial motility and positioning in cultured neurons. Using zapalog, we tether mitochondria to constitutively active kinesin motors, forcing them down the axon towards microtubule (+) ends until their instantaneous release via blue light, which results in full restoration of their endogenous motility. We find that one-third of stationary mitochondria cannot be pulled away from their position and that these firmly anchored mitochondria preferentially localize to VGLUT1-positive presynapses. Furthermore, inhibition of actin polymerization with latrunculin A reduces this firmly anchored pool. On release from exogenous motors, mitochondria are preferentially recaptured at presynapses.

Kinetochore Proteins Have a Post-Mitotic Function in Neurodevelopment.

The kinetochore is a complex of proteins, broadly conserved from yeast to man, that resides at the centromere and is essential for chromosome segregation in dividing cells. There are no known functions of the core complex outside of the centromere. We now show that the proteins of the kinetochore have an essential post-mitotic function in neurodevelopment. At the embryonic neuromuscular junction of Drosophila melanogaster, mutation or knockdown of many kinetochore components cause neurites to overgrow and prevent formation of normal synaptic boutons. Kinetochore proteins were detected in synapses and axons in Drosophila. In post-mitotic cultured hippocampal neurons, knockdown of mis12 increased the filopodia-like protrusions in this region. We conclude that the proteins of the kinetochore are repurposed to sculpt developing synapses and dendrites and thereby contribute to the correct development of neuronal circuits in both invertebrates and mammals.

Mitostasis in Neurons: Maintaining Mitochondria in an Extended Cellular Architecture.

Neurons have more extended and complex shapes than other cells and consequently face a greater challenge in distributing and maintaining mitochondria throughout their arbors. Neurons can last a lifetime, but proteins turn over rapidly. Mitochondria, therefore, need constant rejuvenation no matter how far they are from the soma. Axonal transport of mitochondria and mitochondrial fission and fusion contribute to this rejuvenation, but local protein synthesis is also likely. Maintenance of a healthy mitochondrial population also requires the clearance of damaged proteins and organelles. This involves degradation of individual proteins, sequestration in mitochondria-derived vesicles, organelle degradation by mitophagy and macroautophagy, and in some cases transfer to glial cells. Both long-range transport and local processing are thus at work in achieving neuronal mitostasis-the maintenance of an appropriately distributed pool of healthy mitochondria for the duration of a neuron's life. Accordingly, defects in the processes that support mitostasis are significant contributors to neurodegenerative disorders.

A high mitochondrial transport rate characterizes CNS neurons with high axonal regeneration capacity.

Improving axonal transport in the injured and diseased central nervous system has been proposed as a promising strategy to improve neuronal repair. However, the contribution of each cargo to the repair mechanism is unknown. DRG neurons globally increase axonal transport during regeneration. Because the transport of specific cargos after axonal insult has not been examined systematically in a model of enhanced regenerative capacity, it is unknown whether the transport of all cargos would be modulated equally in injured central nervous system neurons. Here, using a microfluidic culture system we compared neurons co-deleted for PTEN and SOCS3, an established model of high axonal regeneration capacity, to control neurons. We measured the axonal transport of three cargos (mitochondria, synaptic vesicles and late endosomes) in regenerating axons and found that the transport of mitochondria, but not the other cargos, was increased in PTEN/SOCS3 co-deleted axons relative to controls. The results reported here suggest a pivotal role for this organelle during axonal regeneration.

O-GlcNAc Transferase Is Essential for Sensory Neuron Survival and Maintenance.

O-GlcNAc transferase (OGT) regulates a wide range of cellular processes through the addition of the O-GlcNAc sugar moiety to thousands of protein substrates. Because nutrient availability affects the activity of OGT, its role has been broadly studied in metabolic tissues. OGT is enriched in the nervous system, but little is known about its importance in basic neuronal processes in vivo Here, we show that OGT is essential for sensory neuron survival and maintenance in mice. Sensory neuron-specific knock-out of OGT results in behavioral hyposensitivity to thermal and mechanical stimuli accompanied by decreased epidermal innervation and cell-body loss in the dorsal root ganglia. These effects are observed early in postnatal development and progress as animals age. Cultured sensory neurons lacking OGT also exhibit decreased axonal outgrowth. The effects on neuronal health in vivo are not solely due to disruption of developmental processes, because inducing OGT knock-out in the sensory neurons of adult mice results in a similar decrease in nerve fiber endings and cell bodies. Significant nerve-ending loss occurs before a decrease in cell bodies; this phenotype is indicative of axonal dieback that progresses to neuronal death. Our findings demonstrate that OGT is important in regulating axonal maintenance in the periphery and the overall health and survival of sensory neurons.SIGNIFICANCE STATEMENT We show the importance of O-GlcNAc transferase (OGT) for sensory neuron health and survival in vivo This study is the first to find that loss of OGT results in neuronal cell death. Moreover, it suggests that aberrant O-GlcNAc signaling can contribute to the development of neuropathy. The sensory neurons lie outside of the blood-brain barrier and therefore, compared to central neurons, may have a greater need for mechanisms of metabolic sensing and compensation. Peripheral sensory neurons in particular are subject to degeneration in diabetes. Our findings provide a foundation for understanding the role of OGT under normal physiological conditions in the peripheral nervous system. This knowledge will be important for gaining greater insight into such disease states as diabetic neuropathy.

Neurotoxic mechanisms of paclitaxel are local to the distal axon and independent of transport defects.

Chemotherapy-induced peripheral neuropathy (CIPN) is a dose-limiting side effect of paclitaxel and other chemotherapeutic agents. Paclitaxel binds and stabilizes microtubules, but the cellular mechanisms that underlie paclitaxel's neurotoxic effects are not well understood. We therefore used primary cultures of adult murine dorsal root ganglion neurons, the cell type affected in patients, to examine leading hypotheses to explain paclitaxel neurotoxicity. We address the role of microtubule hyperstabilization and its downstream effects. Paclitaxel administered at 10-50nM for 1-3days induced retraction bulbs at the tips of axons and arrested axon growth without triggering axon fragmentation or cell death. By correlating the toxic effects and microtubule stabilizing activity of structurally different microtubule stabilizing compounds, we confirmed that microtubule hyperstabilization, rather than an off-target effect, is the likely primary cause of paclitaxel neurotoxicity. We examined potential downstream consequences of microtubule hyperstabilization and found that changes in levels of tubulin posttranslational modifications, although present after paclitaxel exposure, are not implicated in the paclitaxel neurotoxicity we observed in the cultures. Additionally, defects in axonal transport were not implicated as an early, causative mechanism of paclitaxel's toxic effects on dorsal root ganglion neurons. By using microfluidic chambers to selectively treat different parts of the axon with paclitaxel, we found that the distal axon was primarily vulnerable to paclitaxel, indicating that paclitaxel acts directly on the distal axon to induce degenerative effects. Together, our findings point to local effects of microtubule hyperstabilization on the distal-most portion of the axon as an early mediator of paclitaxel neurotoxicity. Because sensory neurons have a unique and ongoing requirement for distal growth in order to reinnervate the epidermis as it turns over, we propose that the ability of paclitaxel to arrest their growth accounts for the selective vulnerability of sensory neurons to paclitaxel neurotoxicity.

The Mammalian-Specific Protein Armcx1 Regulates Mitochondrial Transport during Axon Regeneration.

Mitochondrial transport is crucial for neuronal and axonal physiology. However, whether and how it impacts neuronal injury responses, such as neuronal survival and axon regeneration, remain largely unknown. In an established mouse model with robust axon regeneration, we show that Armcx1, a mammalian-specific gene encoding a mitochondria-localized protein, is upregulated after axotomy in this high regeneration condition. Armcx1 overexpression enhances mitochondrial transport in adult retinal ganglion cells (RGCs). Importantly, Armcx1 also promotes both neuronal survival and axon regeneration after injury, and these effects depend on its mitochondrial localization. Furthermore, Armcx1 knockdown undermines both neuronal survival and axon regeneration in the high regenerative capacity model, further supporting a key role of Armcx1 in regulating neuronal injury responses in the adult central nervous system (CNS). Our findings suggest that Armcx1 controls mitochondrial transport during neuronal repair.