Lysosomes In Axons Research Articles

Axonal Transport Defect in Gigaxonin Deficiency Rescued by Tubastatin A

SummaryGiant axonal neuropathy (GAN) is a disease caused by a deficiency of gigaxonin, a mediator of the degradation of intermediate filament (IF) proteins. A lack of gigaxonin alters the turnover of IF proteins, provoking accumulation and disorganization of neurofilaments (NFs) in neurons, a hallmark of the disease. However, the effects of IF disorganization on neuronal function remain unknown. Here, we report that cultured embryonic dorsal root ganglia (DRG) neurons derived from Gan−/− mice exhibit accumulations of IF proteins and defects in fast axonal transport of organelles. Kymographs generated by time-lapse microscopy revealed substantial reduction of anterograde movements of mitochondria and lysosomes in axons of Gan−/− DRG neurons. Treatment of Gan−/− DRG neurons with Tubastatin A (TubA) increased the levels of acetylated tubulin and it restored the normal axonal transport of these organelles. Furthermore, we tested the effects of TubA in a new mouse model of GAN consisting of Gan−/− mice with overexpression of peripherin (Prph) transgene. Treatment of 12-month-old Gan−/−;TgPer mice with TubA led to a slight amelioration of motor function, especially a significant improvement of gait performance as measured by footprint analyses. Moreover, TubA treatment reduced the abnormal accumulations of Prph and NF proteins in spinal neurons and it boosted the levels of Prph transported into peripheral nerve axons. These results suggest that drug inhibitors of histone deacetylase aiming to enhance axonal transport should be considered as a potential treatment for GAN disease.

Defective axonal transport of endo-lysosomes and dense core vesicles in a Drosophila model of C9-ALS/FTD.

A GGGGCC (G4 C2 ) repeat expansion in the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although disruptions in axonal transport are implicated in the pathogenesis of multiple neurodegenerative diseases, the underlying mechanisms causing these defects remain unclear. Here, we performed live imaging of Drosophila motor neurons expressing expanded G4 C2 repeats in third-instar larvae and investigated the axonal transport of multiple organelles in vivo. Expression of expanded G4 C2 repeats causes an increase in static axonal lysosomes, while it impairs trafficking of late endosomes (LEs) and dense core vesicles (DCVs). Surprisingly, however, axonal transport of mitochondria is unaffected in motor axons expressing expanded G4 C2 repeats. Thus, our data indicate that expanded G4 C2 repeat expression differentially impacts axonal transport of vesicular organelles and mitochondria in Drosophila models of C9orf72-associated ALS/FTD.

Loss of MAPK8IP3 Affects Endocytosis in Neurons.

Perturbations in endo-lysosomal trafficking pathways are linked to many neurodevelopmental and neurodegenerative diseases. Of relevance to our current study, MAPK8IP3/JIP3, a brain enriched putative adaptor between lysosomes and motors has been previously implicated as a key regulator of axonal lysosome transport. Since de novo variants in MAPK8IP3 have recently been linked to a neurodevelopmental disorder with intellectual disability, there is a need to better understand the functioning of this protein in human neurons. To this end, using induced neurons (i3Neurons) derived from human iPSCs lacking MAPK8IP3, we demonstrate that loss of hMAPK8IP3 affects endocytic uptake in neurons but does not affect the proteolytic activity of lysosomes in neuronal cell bodies. Our findings indicate that MAPK8IP3 may be a regulator of bulk endocytosis in neurons and that altered endocytic uptake may play a role in MAPK8IP3-linked neurodevelopmental disorders.

JIP3 links lysosome transport to regulation of multiple components of the axonal cytoskeleton

Lysosome axonal transport is important for the clearance of cargoes sequestered by the endocytic and autophagic pathways. Building on observations that mutations in the JIP3 (MAPK8IP3) gene result in lysosome-filled axonal swellings, we analyzed the impact of JIP3 depletion on the cytoskeleton of human neurons. Dynamic focal lysosome accumulations were accompanied by disruption of the axonal periodic scaffold (spectrin, F-actin and myosin II) throughout each affected axon. Additionally, axonal microtubule organization was locally disrupted at each lysosome-filled swelling. This local axonal microtubule disorganization was accompanied by accumulations of both F-actin and myosin II. These results indicate that transport of axonal lysosomes is functionally interconnected with mechanisms that control the organization and maintenance of the axonal cytoskeleton. They have potential relevance to human neurological disease arising from JIP3 mutations as well as for neurodegenerative diseases associated with the focal accumulations of lysosomes within axonal swellings such as Alzheimer’s disease.

ER\xa0\u2013\xa0lysosome contacts at a pre-axonal region regulate axonal lysosome availability

Neuronal function relies on careful coordination of organelle organization and transport. Kinesin-1 mediates transport of the endoplasmic reticulum (ER) and lysosomes into the axon and it is increasingly recognized that contacts between the ER and lysosomes influence organelle organization. However, it is unclear how organelle organization, inter-organelle communication and transport are linked and how this contributes to local organelle availability in neurons. Here, we show that somatic ER tubules are required for proper lysosome transport into the axon. Somatic ER tubule disruption causes accumulation of enlarged and less motile lysosomes at the soma. ER tubules regulate lysosome size and axonal translocation by promoting lysosome homo-fission. ER tubule – lysosome contacts often occur at a somatic pre-axonal region, where the kinesin-1-binding ER-protein P180 binds microtubules to promote kinesin-1-powered lysosome fission and subsequent axonal translocation. We propose that ER tubule – lysosome contacts at a pre-axonal region finely orchestrate axonal lysosome availability for proper neuronal function.

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

ABSTRACT 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.

Disease-causing mutated ATLASTIN 3 is excluded from distal axons and reduces axonal autophagy

Mutations in the ER-network forming GTPase atlastin3 (ATL3) can cause axon degeneration of sensory neurons by not fully understood mechanisms. We here show that the hereditary sensory and autonomous neuropathy (HSAN)-causing ATL3 Y192C or P338R are excluded from distal axons by a barrier at the axon initial segment (AIS). This barrier is selective for mutated ATL3, but not wildtype ATL3 or unrelated ER-membrane proteins. Actin-depolymerization partially restores the transport of ATL3 Y192C into distal axons. The results point to the existence of a selective diffusion barrier in the ER membrane at the AIS, analogous to the AIS-based barriers for plasma membrane and cytosolic proteins. Functionally, the absence of ATL3 at the distal axon reduces axonal autophagy and the ER network deformation in the soma causes a reduction in axonal lysosomes. Both could contribute to axonal degeneration and eventually to HSAN.

Lysosomes to the rescue: Anterograde axonal lysosome transport and neuronal proteostasis.

Niemann-Pick is a lysosomal storage disease caused by loss of the lysosomal cholesterol exporter NPC1 and leads to axon degeneration. Roney et al. report that immature autophagosomes accumulate in axons because cholesterol-laden lysosomes in the soma are not transported to the axon for autophagosome fusion and maturation because they aberrantly sequester non-functioning kinesin-1.

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-β-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.

Overlapping roles of JIP3 and JIP4 in promoting axonal transport of lysosomes in human iPSC-derived neurons.

The dependence of neurons on microtubule-based motors for the movement of lysosomes over long distances raises questions about adaptations that allow neurons to meet these demands. Recently, JIP3/MAPK8IP3, a neuronally enriched putative adaptor between lysosomes and motors, was identified as a critical regulator of axonal lysosome abundance. In this study, we establish a human induced pluripotent stem cell (iPSC)-derived neuron model for the investigation of axonal lysosome transport and maturation and show that loss of JIP3 results in the accumulation of axonal lysosomes and the Alzheimer’s disease–related amyloid precursor protein (APP)-derived Aβ42 peptide. We furthermore reveal an overlapping role of the homologous JIP4 gene in lysosome axonal transport. These results establish a cellular model for investigating the relationship between lysosome axonal transport and amyloidogenic APP processing and more broadly demonstrate the utility of human iPSC–derived neurons for the investigation of neuronal cell biology and pathology.

Lysosome Function and Dysfunction in Hereditary Spastic Paraplegias.

Hereditary Spastic Paraplegias (HSPs) are a genetically diverse group of inherited neurological diseases with over 80 associated gene loci. Over the last decade, research into mechanisms underlying HSPs has led to an emerging interest in lysosome dysfunction. In this review, we highlight the different classes of HSPs that have been linked to lysosome defects: (1) a subset of complex HSPs where mutations in lysosomal genes are causally linked to the diseases, (2) other complex HSPs where mutation in genes encoding membrane trafficking adaptors lead to lysosomal defects, and (3) a subset of HSPs where mutations affect genes encoding proteins whose function is primarily linked to a different cellular component or organelle such as microtubule severing and Endoplasmic Reticulum-shaping, while also altering to lysosomes. Interestingly, aberrant axonal lysosomes, associated with the latter two subsets of HSPs, are a key feature observed in other neurodegenerative diseases such as Alzheimer’s disease. We discuss how altered lysosome function and trafficking may be a critical contributor to HSP pathology and highlight the need for examining these features in the cortico-spinal motor neurons of HSP mutant models.

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

SUMMARYNeurons 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.

Activity-Dependent Secretion of Synaptic Organizer Cbln1 from Lysosomes in Granule Cell Axons

Synapse formation is achieved by various synaptic organizers. Although this process is highly regulated by neuronal activity, the underlying molecular mechanisms remain largely unclear. Here we show that Cbln1, a synaptic organizer of the C1q family, is released from lysosomes in axons but not dendrites of cerebellar granule cells in an activity- and Ca2+-dependent manner. Exocytosed Cbln1 was retained on axonal surfaces by binding to its presynaptic receptor neurexin. Cbln1 further diffused laterally along the axonal surface and accumulated at boutons by binding postsynaptic δ2 glutamate receptors. Cbln1 exocytosis was insensitive to tetanus neurotoxin, accompanied by cathepsin B release, and decreased by disrupting lysosomes. Furthermore, overexpression of lysosomal sialidase Neu1 not only inhibited Cbln1 and cathepsin B exocytosis invitro but also reduced axonal bouton formation invivo. Our findings imply that co-release of Cbln1 and cathepsin B from lysosomes serves as a new mechanism of activity-dependent coordinated synapse modification.

De Novo Variants in MAPK8IP3 Cause Intellectual Disability with Variable Brain Anomalies

Using exome sequencing, we have identified de novo variants in MAPK8IP3 in 13 unrelated individuals presenting with an overlapping phenotype of mild to severe intellectual disability. The de novo variants comprise six missense variants, three of which are recurrent, and three truncating variants. Brain anomalies such as perisylvian polymicrogyria, cerebral or cerebellar atrophy, and hypoplasia of the corpus callosum were consistent among individuals harboring recurrent de novo missense variants. MAPK8IP3 has been shown to be involved in the retrograde axonal-transport machinery, but many of its specific functions are yet to be elucidated. Using the CRISPR-Cas9 system to target six conserved amino acid positions in Caenorhabditis elegans, we found that two of the six investigated human alterations led to a significantly elevated density of axonal lysosomes, and five variants were associated with adverse locomotion. Reverse-engineering normalized the observed adverse effects back to wild-type levels. Combining genetic, phenotypic, and functional findings, as well as the significant enrichment of de novo variants in MAPK8IP3 within our total cohort of 27,232 individuals who underwent exome sequencing, we implicate de novo variants in MAPK8IP3 as a cause of a neurodevelopmental disorder with intellectual disability and variable brain anomalies.

Impaired JIP3-dependent axonal lysosome transport promotes amyloid plaque pathology.

Lysosomes robustly accumulate within axonal swellings at Alzheimer's disease (AD) amyloid plaques. However, the underlying mechanisms and disease relevance of such lysosome accumulations are not well understood. Motivated by these problems, we identified JNK-interacting protein 3 (JIP3) as an important regulator of axonal lysosome transport and maturation. JIP3 knockout mouse neuron primary cultures accumulate lysosomes within focal axonal swellings that resemble the dystrophic axons at amyloid plaques. These swellings contain high levels of amyloid precursor protein processing enzymes (BACE1 and presenilin 2) and are accompanied by elevated Aβ peptide levels. The in vivo importance of the JIP3-dependent regulation of axonal lysosomes was revealed by the worsening of the amyloid plaque pathology arising from JIP3 haploinsufficiency in a mouse model of AD. These results establish the critical role of JIP3-dependent axonal lysosome transport in regulating amyloidogenic amyloid precursor protein processing and support a model wherein Aβ production is amplified by plaque-induced axonal lysosome transport defects.

UNC-16 (JIP3) Acts Through Synapse-Assembly Proteins to Inhibit the Active Transport of Cell Soma Organelles to Caenorhabditis elegans Motor Neuron Axons.

The conserved protein UNC-16 (JIP3) inhibits the active transport of some cell soma organelles, such as lysosomes, early endosomes, and Golgi, to the synaptic region of axons. However, little is known about UNC-16's organelle transport regulatory function, which is distinct from its Kinesin-1 adaptor function. We used an unc-16 suppressor screen in Caenorhabditis elegans to discover that UNC-16 acts through CDK-5 (Cdk5) and two conserved synapse assembly proteins: SAD-1 (SAD-A Kinase), and SYD-2 (Liprin-α). Genetic analysis of all combinations of double and triple mutants in unc-16(+) and unc-16(-) backgrounds showed that the three proteins (CDK-5, SAD-1, and SYD-2) are all part of the same organelle transport regulatory system, which we named the CSS system based on its founder proteins. Further genetic analysis revealed roles for SYD-1 (another synapse assembly protein) and STRADα (a SAD-1-interacting protein) in the CSS system. In an unc-16(-) background, loss of the CSS system improved the sluggish locomotion of unc-16 mutants, inhibited axonal lysosome accumulation, and led to the dynein-dependent accumulation of lysosomes in dendrites. Time-lapse imaging of lysosomes in CSS system mutants in unc-16(+) and unc-16(-) backgrounds revealed active transport defects consistent with the steady-state distributions of lysosomes. UNC-16 also uses the CSS system to regulate the distribution of early endosomes in neurons and, to a lesser extent, Golgi. The data reveal a new and unprecedented role for synapse assembly proteins, acting as part of the newly defined CSS system, in mediating UNC-16's organelle transport regulatory function.

Long-distance autophagy

Long-distance autophagy

Expression of axonal protein degradation machinery in sympathetic neurons is regulated by nerve growth factor

Deficiencies in protein degradation and proteolytic function within neurons are linked to a number of neurodegenerative diseases and developmental disorders. Compartmentalized cultures of peripheral neurons were used to investigate the properties and relative abundance of the proteolytic machinery in the axons and cell bodies of sympathetic and sensory neurons. Immunoblotting of axonal proteins demonstrated that LAMP2, LC3, and PSMA2 were abundant in axons, suggesting that lysosomes, autophagosomes and proteasomes were located in axons. Interestingly, the expression of proteins associated with lysosomes and proteasomes were upregulated selectively in axons by NGF stimulation of the distal axons of sympathetic neurons, suggesting that axonal growth and maintenance requires local protein turnover. The regulation of the abundance of both proteasomes and lysosomes in axons by NGF provides a link between protein degradation and the trophic status of peripheral neurons. Inhibition of proteasomes located in axons resulted in an accumulation of ubiquitinated proteins in these axons. In contrast, lysosome inhibition in axons did not result in an accumulation of ubiquitinated proteins or the transferrin receptor, a transmembrane protein degraded by lysosomes. Interestingly, lysosomes were transported both retrogradely and anterogradely, so it is likely that ubiquitinated proteins that are normally destined for degradation by lysosomes in axons can be transported to the cell bodies for degradation. In summary, proteasomal degradation occurs locally, whereas proteins degraded by lysosomes can most likely either be degraded locally in axons or be transported to cell bodies for degradation.

Nudel Promotes Axonal Lysosome Clearance and Endo‐lysosome Formation via Dynein‐Mediated Transport

Axonal transport is critical for neuronal function and survival. Cytoplasmic dynein and its accessory complex dynactin form a microtubule minus end-directed motor in charge of retrograde transport. In this study, we show that Nudel, a dynein regulator, was highly expressed in dorsal root ganglion (DRG) neurons. Microinjection of anti-Nudel antibody into cultured DRG neurons abolished retrograde transport of membranous organelles in the axon and led to dispersions of Golgi cisternae in the soma. As a result, lysosomes, which are normally enriched in the soma, moved persistently into and thus accumulated in axons. Endo-lysosome formation was also markedly delayed. As anterograde motility of mitochondria was not inhibited, the antibody apparently did not abolish retrograde transport by destructing axonal microtubule tracks. Similar results were obtained by microinjecting N-terminal Nudel, anti-dynein antibody or a p150(Glued) mutant capable of abrogating the dynein-dynactin association. These results indicate a critical role of Nudel in dynein-mediated axonal transport. Moreover, the effects of dynein on endolysosome formation and regional sequestration of lysosomes may contribute to defects in the endocytic pathway seen in neurons of patients or animals with malfunction of dynein.

Two motifs target Batten disease protein CLN3 to lysosomes in transfected nonneuronal and neuronal cells.

Batten disease is a neurodegenerative disorder resulting from mutations in CLN3, a polytopic membrane protein, whose predominant intracellular destination in nonneuronal cells is the lysosome. The topology of CLN3 protein, its lysosomal targeting mechanism, and the development of Batten disease are poorly understood. We provide experimental evidence that both the N and C termini and one large loop domain of CLN3 face the cytoplasm. We have identified two lysosomal targeting motifs that mediate the sorting of CLN3 in transfected nonneuronal and neuronal cells: an unconventional motif in the long C-terminal cytosolic tail consisting of a methionine and a glycine separated by nine amino acids [M(X)9G], and a more conventional dileucine motif, located in the large cytosolic loop domain and preceded by an acidic patch. Each motif on its own was sufficient to mediate lysosomal targeting, but optimal efficiency required both. Interestingly, in primary neurons, CLN3 was prominently seen both in lysosomes in the cell body and in endosomes, containing early endosomal antigen-1 along neuronal processes. Because there are few lysosomes in axons and peripheral parts of dendrites, the presence of CLN3 in endosomes of neurons may be functionally important. Endosomal association of the protein was independent of the two lysosomal targeting motifs.