Ligand-free mitochondria-localized mutant AR-induced cytotoxicity in spinal bulbar muscular atrophy.

Spinal bulbar muscular atrophy (SBMA), the first identified CAG-repeat expansion disorder, is an X-linked neuromuscular disorder involving CAG-repeat-expansion mutations in the androgen receptor (AR) gene. We utilized CRISPR-Cas9 gene editing to engineer novel isogenic human induced pluripotent stem cell (hiPSC) models, consisting of isogenic AR knockout, control and disease lines expressing mutant AR with distinct repeat lengths, as well as control and disease lines expressing FLAG-tagged wild-type and mutant AR, respectively. Adapting a small-molecule cocktail-directed approach, we differentiate the isogenic hiPSC models into motor neuron-like cells with a highly enriched population to uncover cell-type-specific mechanisms underlying SBMA and to distinguish gain- from loss-of-function properties of mutant AR in disease motor neurons. We demonstrate that ligand-free mutant AR causes drastic mitochondrial dysfunction in neurites of differentiated disease motor neurons due to gain-of-function mechanisms and such cytotoxicity can be amplified upon ligand (androgens) treatment. We further show that aberrant interaction between ligand-free, mitochondria-localized mutant AR and F-ATP synthase is associated with compromised mitochondrial respiration and multiple other mitochondrial impairments. These findings counter the established notion that androgens are requisite for mutant AR-induced cytotoxicity in SBMA, reveal a compelling mechanistic link between ligand-free mutant AR, F-ATP synthase and mitochondrial dysfunction, and provide innovative insights into motor neuron-specific therapeutic interventions for SBMA.

Programming axonal mitochondrial maintenance and bioenergetics in neurodegeneration and regeneration.

Mitochondria generate ATP essential for neuronal growth, function, and regeneration. Due to their polarized structures, neurons face exceptional challenges to deliver mitochondria to and maintain energy homeostasis throughout long axons and terminal branches where energy is in high demand. Chronic mitochondrial dysfunction accompanied by bioenergetic failure is a pathological hallmark of major neurodegenerative diseases. Brain injury triggers acute mitochondrial damage and a local energy crisis that accelerates neuron death. Thus, mitochondrial maintenance defects and axonal energy deficits emerge as central problems in neurodegenerative disorders and brain injury. Recent studies have started to uncover the intrinsic mechanisms that neurons adopt to maintain (or reprogram) axonal mitochondrial density and integrity, and their bioenergetic capacity, upon sensing energy stress. In this review, we discuss recent advances in how neurons maintain a healthy pool of axonal mitochondria, as well as potential therapeutic strategies that target bioenergetic restoration to power neuronal survival, function, and regeneration.

Neuronal endolysosomal transport and lysosomal functionality in maintaining axonostasis.

Lysosomes serve as degradation hubs for the turnover of endocytic and autophagic cargos, which is essential for neuron function and survival. Deficits in lysosome function result in progressive neurodegeneration in most lysosomal storage disorders and contribute to the pathogenesis of aging-related neurodegenerative diseases. Given their size and highly polarized morphology, neurons face exceptional challenges in maintaining cellular homeostasis in regions far removed from the cell body where mature lysosomes are enriched. Neurons therefore require coordinated bidirectional intracellular transport to sustain efficient clearance capacity in distal axonal regions. Emerging lines of evidence have started to uncover mechanisms and signaling pathways regulating endolysosome transport and maturation to maintain axonal homeostasis, or "axonostasis," that is relevant to a range of neurologic disorders. In this review, we discuss recent advances in how axonal endolysosomal trafficking, distribution, and lysosomal functionality support neuronal health and become disrupted in several neurodegenerative diseases.

Lipid-mediated impairment of axonal lysosome transport contributing to autophagic stress.

Efficient degradation of autophagic vacuoles (AVs) generated at axon terminals by mature lysosomes enriched in the cell body represents an exceptional challenge that neurons face in maintaining cellular homeostasis. Here, we discuss our recent findings revealing a lipid-mediated impairment of lysosome transport to distal axons contributing to axonal AV accumulation in the neurodegenerative lysosomal storage disorder Niemann-Pick disease type C (NPC). Using transmission electron microscopy, we observed a striking buildup of endocytic and autophagic organelles in NPC dystrophic axons, indicating defects in the clearance of organelles destined for lysosomal degradation. We further revealed that elevated cholesterol on NPC lysosome membranes abnormally sequesters motor-adaptors of axonal lysosome delivery, resulting in impaired anterograde lysosome transport into distal axons that disrupts maturation of axonal AVs during their retrograde transport route. Together, our study demonstrates a mechanism by which altered membrane lipid composition compromises axonal lysosome trafficking and positioning and shows that lowering lysosomal lipid levels rescues lysosome transport into NPC axons, thus reducing axonal autophagic stress at early stages of NPC disease.

Neurobiology: A pathogenic tug of war.

Pathogenic mutations in the kinase LRRK2 have been implicated in Parkinson's disease. A new study shows that hyperactivation of this kinase reduces the processivity of autophagosomal retrograde transport in axons through an unproductive 'tug-of-war' between anterograde and retrograde motors, thus contributing to autophagy dysfunction and axonal degeneration.

Lipid-mediated motor-adaptor sequestration impairs axonal lysosome delivery leading to autophagic stress and dystrophy in Niemann-Pick type C.

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-ß-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.

Defects in syntabulin-mediated synaptic cargo transport associate with autism-like synaptic dysfunction and social behavioral traits.

The formation and maintenance of synapses require long-distance delivery of newly synthesized synaptic proteins from the soma to distal synapses, raising the fundamental question of whether impaired transport is associated with neurodevelopmental disorders such as autism. We previously revealed that syntabulin acts as a motor adapter linking kinesin-1 motor and presynaptic cargos. Here, we report that defects in syntabulin-mediated transport and thus reduced formation and maturation of synapses are one of core synaptic mechanisms underlying autism-like synaptic dysfunction and social behavioral abnormalities. Syntabulin expression in the mouse brain peaks during the first 2 weeks of postnatal development and progressively declines during brain maturation. Neurons from conditional syntabulin-/- mice (stb cKO) display impaired transport of presynaptic cargos, reduced synapse density and active zones, and altered synaptic transmission and long-term plasticity. Intriguingly, stb cKO mice exhibit core autism-like traits, including defective social recognition and communication, increased stereotypic behavior, and impaired spatial learning and memory. These phenotypes establish a new mechanistic link between reduced transport of synaptic cargos and impaired maintenance of synaptic transmission and plasticity, contributing to autism-associated behavioral abnormalities. This notion is further confirmed by the human missense variant STB-R178Q, which is found in an autism patient and loses its adapter capacity for binding kinesin-1 motors. Expressing STB-R178Q fails to rescue reduced synapse formation and impaired synaptic transmission and plasticity in stb cKO neurons. Altogether, our study suggests that defects in syntabulin-mediated transport mechanisms underlie the synaptic dysfunction and behavioral abnormalities that bear similarities to autism.

Developmental regulation of microtubule-based trafficking and anchoring of axonal mitochondria in health and diseases.

Mitochondria are cellular power plants that supply most of the ATP required in the brain to power neuronal growth, function, and regeneration. Given their extremely polarized structures and extended long axons, neurons face an exceptional challenge to maintain energy homeostasis in distal axons, synapses, and growth cones. Anchored mitochondria serve as local energy sources; therefore, the regulation of mitochondrial trafficking and anchoring ensures that these metabolically active areas are adequately supplied with ATP. Chronic mitochondrial dysfunction is a hallmark feature of major aging-related neurodegenerative diseases, and thus, anchored mitochondria in aging neurons need to be removed when they become dysfunctional. Investigations into the regulation of microtubule (MT)-based trafficking and anchoring of axonal mitochondria under physiological and pathological circumstances represent an important emerging area. In this short review article, we provide an updated overview of recent in vitro and in vivo studies showing (1) how mitochondria are transported and positioned in axons and synapses during neuronal developmental and maturation stages, and (2) how altered mitochondrial motility and axonal energy deficits in aging nervous systems link to neurodegeneration and regeneration in a disease or injury setting. We also highlight a major role of syntaphilin as a key MT-based regulator of axonal mitochondrial trafficking and anchoring in mature neurons.

The secret life of degradative lysosomes in axons: delivery from the soma, enzymatic activity, and local autophagic clearance.

Lysosomal degradation of protein aggregates and damaged organelles is essential for maintaining cellular homeostasis. This process in neurons is challenging due to their highly polarized architecture. While enzymatically active degradative lysosomes are enriched in the cell body, their trafficking and degradation capacity in axons remain elusive. We recently characterized the axonal delivery of degradative lysosomes by applying a set of fluorescent probes that selectively label active forms of lysosomal hydrolases on cortical neurons in microfluidic devices. We revealed that soma-derived degradative lysosomes rapidly influx into distal axons and target to autophagosomes and Parkinson disease-related SNCA/α-synuclein cargos for local degradation. Disrupting axon-targeted delivery of degradative lysosomes induces axonal autophagic stress. We demonstrate that the axon is an active compartment for local degradation, establishing a foundation for future investigations into axonal lysosome trafficking and functionality in neurodegenerative diseases and lysosomal storage disorders associated with axonal pathology and macroautophagy/autophagy stress.

Defending stressed mitochondria: uncovering the role of MUL1 in suppressing neuronal mitophagy.

Chronic mitochondrial stress is associated with major neurodegenerative diseases; and thus, the recovery of those mitochondria constitutes a critical step of energy maintenance in early stages of neurodegeneration. Our recent study provides the first lines of evidence showing that the MUL1-MFN2 pathway acts as an early checkpoint to maintain mitochondrial integrity by regulating mitochondrial morphology and interplay with the endoplasmic reticulum (ER). This mechanism ensures that degradation through mitophagy is restrained in neurons under early stress conditions. MUL1 deficiency increases MFN2 activity, triggering the first phase of mitochondrial hyperfusion and acting as an antagonist of ER-mitochondria (ER-Mito) tethering. Reduced ER-Mito interplay enhances the cytoplasmic Ca2+ load that induces the DNM1L/Drp1-dependent second phase of mitochondrial fragmentation and mitophagy. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions. Identifying this pathway is particularly relevant because chronic mitochondrial dysfunction and altered ER-Mito contacts have been reported in major neurodegenerative diseases.

Mul1 restrains Parkin-mediated mitophagy in mature neurons by maintaining ER-mitochondrial contacts.

Chronic mitochondrial stress associates with major neurodegenerative diseases. Recovering stressed mitochondria constitutes a critical step of mitochondrial quality control and thus energy maintenance in early stages of neurodegeneration. Here, we reveal Mul1-Mfn2 pathway that maintains neuronal mitochondrial integrity under stress conditions. Mul1 deficiency increases Mfn2 activity that triggers the first phasic mitochondrial hyperfusion and also acts as an ER-Mito tethering antagonist. Reduced ER-Mito coupling leads to increased cytoplasmic Ca2+ load that activates calcineurin and induces the second phasic Drp1-dependent mitochondrial fragmentation and mitophagy. Overexpressing Mfn2, but not Mfn1, mimics Mul1-deficient phenotypes, while expressing PTPIP51, an ER-Mito anchoring protein, suppresses Parkin-mediated mitophagy. Thus, by regulating mitochondrial morphology and ER-Mito contacts, Mul1-Mfn2 pathway plays an early checkpoint role in maintaining mitochondrial integrity. Our study provides new mechanistic insights into neuronal mitochondrial maintenance under stress conditions, which is relevant to several major neurodegenerative diseases associated with mitochondrial dysfunction and altered ER-Mito interplay.

Neuronal Soma-Derived Degradative Lysosomes Are Continuously Delivered to Distal Axons to Maintain Local Degradation Capacity.

Neurons face the challenge of maintaining cellular homeostasis through lysosomal degradation. While enzymatically active degradative lysosomes are enriched in the soma, their axonal trafficking and positioning and impact on axonal physiology remain elusive. Here, we characterized axon-targeted delivery of degradative lysosomes by applying fluorescent probes that selectively label active forms of lysosomal cathepsins D, B, L, and GCase. By time-lapse imaging of cortical neurons in microfluidic devices and standard dishes, we reveal that soma-derived degradative lysosomes rapidly influx into distal axons and target to autophagosomes and Parkinson disease-related α-synuclein cargos for local degradation. Impairing lysosome axonal delivery induces an aberrant accumulation of autophagosomes and α-synuclein cargos in distal axons. Our study demonstrates that the axon is an active compartment for local degradation and reveals fundamental aspects of axonal lysosomal delivery and maintenance. Our work establishes a foundation for investigations into axonal lysosome trafficking and functionality in neurodegenerative diseases.

Revisiting LAMP1 as a marker for degradative autophagy-lysosomal organelles in the nervous system.

Lysosomes serve as the degradation hubs for macroautophagic/autophagic and endocytic components, thus maintaining cellular homeostasis essential for neuronal survival and function. LAMP1 (lysosomal associated membrane protein 1) and LAMP2 are distributed among autophagic and endolysosomal organelles. Despite widespread distribution, LAMP1 is routinely used as a lysosome marker and LAMP1-positive organelles are often referred to as lysosomal compartments. By applying immuno-electron microscopy (iTEM) and confocal imaging combined with Airyscan microscopy, we expand on the limited literature to provide a comprehensive and quantitative analysis of LAMP1 distribution in various autophagic and endolysosomal organelles in neurons. Our study demonstrates that a significant portion of LAMP1-labeled organelles lack major lysosomal hydrolases. BSA-gold pulse-chase assay further shows heterogeneous degradative capacities of LAMP1-labled organelles. In addition, LAMP1 intensity is not a sensitive readout to assess lysosomal deficits in familial amyotrophic lateral sclerosis-linked motor neurons in vivo. Our study thus calls for caution when interpreting LAMP1-labeled organelles in the nervous system where LAMP1 intensity, trafficking, and distribution do not necessarily represent degradative lysosomes or autolysosomes under physiological and pathological conditions. ABBREVIATIONS: ALS: amyotrophic lateral sclerosis; BSA: bovine serum albumin; DRG: dorsal root ganglion; IGF2R/CI-M6PR: insulin like growth factor 2 receptor; iTEM: immuno-transmission electron microscopy; LAMP1/2: lysosomal associated membrane protein 1/2; P80: postnatal day 80; sMNs: spinal motor neurons.

Characterization of LAMP1-labeled nondegradative lysosomal and endocytic compartments in neurons.

Despite widespread distribution of LAMP1 and the heterogeneous nature of LAMP1-labeled compartments, LAMP1 is routinely used as a lysosomal marker, and LAMP1-positive organelles are often referred to as lysosomes. In this study, we use immunoelectron microscopy and confocal imaging to provide quantitative analysis of LAMP1 distribution in various autophagic and endolysosomal organelles in neurons. Our study demonstrates that a significant portion of LAMP1-labeled organelles do not contain detectable lysosomal hydrolases including cathepsins D and B and glucocerebrosidase. A bovine serum albumin-gold pulse-chase assay followed by ultrastructural analysis suggests a heterogeneity of degradative capacity in LAMP1-labeled endolysosomal organelles. Gradient fractionation displays differential distribution patterns of LAMP1/2 and cathepsins D/B in neurons. We further reveal that LAMP1 intensity in familial amyotrophic lateral sclerosis-linked motor neurons does not necessarily reflect lysosomal deficits in vivo. Our study suggests that labeling a set of lysosomal hydrolases combined with various endolysosomal markers would be more accurate than simply relying on LAMP1/2 staining to assess neuronal lysosome distribution, trafficking, and functionality under physiological and pathological conditions.

Removing dysfunctional mitochondria from axons independent of mitophagy under pathophysiological conditions.

Chronic mitochondrial dysfunction has been implicated in major neurodegenerative diseases. Long-term cumulative pathological stress leads to axonal accumulation of damaged mitochondria. Therefore, the early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. We recently investigated the axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in amyotrophic lateral sclerosis (ALS)- and Alzheimer disease (AD)-linked neurons. We demonstrated that remobilizing stressed mitochondria is critical for maintaining axonal mitochondrial integrity. The selective release of the mitochondrial anchoring protein SNPH (syntaphilin) from stressed mitochondria enhances their retrograde transport toward the soma before PARK2/Parkin-mediated mitophagy is activated. This SNPH-mediated response is robustly activated during the early disease stages of ALS-linked motor neurons and AD-related cortical neurons. Our study thus reveals a new mechanism for the maintenance of axonal mitochondrial integrity through SNPH-mediated coordination of mitochondrial stress and motility that is independent of mitophagy.

Releasing Syntaphilin Removes Stressed Mitochondria from Axons Independent of Mitophagy under Pathophysiological Conditions.

Chronic mitochondrial stress is a central problem associated with neurodegenerative diseases. Early removal of defective mitochondria from axons constitutes a critical step of mitochondrial quality control. Here we investigate axonal mitochondrial response to mild stress in wild-type neurons and chronic mitochondrial defects in Amytrophic Lateral Sclerosis (ALS)- and Alzheimer's disease (AD)-linked neurons. We show that stressed mitochondria are removed from axons triggered by the bulk release of mitochondrial anchoring protein syntaphilin via a new class of mitochondria-derived cargos independent of Parkin, Drp1, and autophagy. Immuno-electron microscopy and super-resolution imaging show the budding of syntaphilin cargos, which then share a ride on late endosomes for transport toward the soma. Releasing syntaphilin 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 Parkin-mediated mitophagy. VIDEO ABSTRACT.

Axonal autophagosomes use the ride-on service for retrograde transport toward the soma.

Degradation of autophagic vacuoles (AVs) via lysosomes is an important homeostatic process in cells. Neurons are highly polarized cells with long axons, thus facing special challenges to transport AVs generated at distal processes toward the soma where mature acidic lysosomes are relatively enriched. We recently revealed a new motor-adaptor sharing mechanism driving autophagosome transport to the soma. Late endosome (LE)-loaded dynein-SNAPIN motor-adaptor complexes mediate the retrograde transport of autophagosomes upon their fusion with LEs in distal axons. This motor-adaptor sharing mechanism enables neurons to maintain effective autophagic clearance in the soma, thus reducing autophagic stress in axons. Therefore, our study reveals a new cellular mechanism underlying the removal of distal AVs engulfing aggregated misfolded proteins and dysfunctional organelles associated with several major neurodegenerative diseases.

Axonal autophagosomes recruit dynein for retrograde transport through fusion with late endosomes.

Efficient degradation of autophagic vacuoles (AVs) via lysosomes is an important cellular homeostatic process. This is particularly challenging for neurons because mature acidic lysosomes are relatively enriched in the soma. Although dynein-driven retrograde transport of AVs was suggested, a fundamental question remains how autophagosomes generated at distal axons acquire dynein motors for retrograde transport toward the soma. In this paper, we demonstrate that late endosome (LE)-loaded dynein-snapin complexes drive AV retrograde transport in axons upon fusion of autophagosomes with LEs into amphisomes. Blocking the fusion with syntaxin17 knockdown reduced recruitment of dynein motors to AVs, thus immobilizing them in axons. Deficiency in dynein-snapin coupling impaired AV transport ,: resulting in AV accumulation in neurites and synaptic terminals. Altogether, our study provides the first evidence that autophagosomes recruit dynein through fusion with LEs and reveals a new motor-adaptor sharing mechanism by which neurons may remove distal AVs engulfing aggregated proteins and dysfunctional organelles for efficient degradation in the soma.