While lysosomes have been traditionally viewed as degradative compartments, evidence suggests that they are signaling organelles that are highly mobile within or across cells to maintain local and global homeostasis 1,2 . Spatial distribution of lysosomes has been shown to influence their acidification and functions 3 , which are essential to support normal cellular activities in the brain 4- 6 . Although lysosome transport is important in all neural cells, this is particularly critical in neurons, given their highly polarized and extended morphology. Lysosomal activity can vary across cellular compartments, such as the soma and axon, highlighting the need for organelle transport to meet compartment-specific functional demands 7 . To sustain this transport, neurons rely on regulated trafficking of lysosomes along microtubules and other cytoskeletal elements within the cells 8 . Beyond intracellular trafficking, intercellular transfer of organelles, such as through tunneling nanotubes (TNTs), is a unique mechanism where neurons, astrocytes, and microglia appear to be capable of transferring organelles including lysosomes to one another under stress conditions 9,10 . Defective lysosomal acidification, function, and trafficking and associated accumulation of toxic intrinsically disordered proteins (IDPs) have emerged as a disease mechanism across neurodegenerative disorders, including Alzheimer's disease (AD) and related tauopathies and Parkinson's disease (PD) 111213 . Lysosomal trafficking defects indicate that neurodegeneration arises not only from organelle deacidification and impaired degradative function, but also from the failure to properly deliver lysosomes to regions of need within cells and from disrupted exchange of functional and damaged lysosomes between cells in the brain.In neurons, lysosome formation and positioning depend on long-range transport mediated by microtubule motors. Anterograde transport, the process of trafficking toward the axon termini, is driven by kinesins, specifically kinesin-1 and kinesin-3, which interact with lysosomal membranes through the BORC-ARL8-SKIP complex 14 . Retrograde transport, moving in the direction toward the soma, is mediated by the GTPase Rab7, Rab-interacting lysosomal protein (RILP), and the dynein-dynactin complex as the motor 15 . These opposing motors continuously reposition lysosomes to maintain degradative homeostasis 16,17 . Spatial compartmentalization of lysosomes based on acidity adds another layer to the transport of these organelles within neurons. Axonal lysosomes are often in the process of maturation as they move closer to the soma, representing late endosomal intermediates 3,18 . This maturation gradient works through pH modulation, where lysosomes moving from the distal axon start around a pH of 6 and increasingly acidify to around pH 4.5 as they move towards the soma 3,19 . Lysosomal acidification state can influence spatial distribution, as acidified lysosomes undergo retrograde transport toward the soma, with fully acidified lysosomes localizing near the nucleus. Less acidic lysosomes are localized in distal neuronal compartments and towards the periphery of the cell 20 . Acidity of lysosomes follows a retrograde pathway, and the final destination of the lysosome depends on acidity and function. A recent study supports that spatial regulation of lysosomes is influenced by complete assembly of the lysosomal vacuolar (H + )-ATPase (V-ATPase) complex, with fully assembled V-ATPase-containing vesicles exhibiting mostly retrograde transport from the axon toward the soma 21 . Perturbation of this transport process, such as disrupting microtubule stability, causes lysosomes to cluster near aggregated materials, potentially preventing distal degradation and increasing accumulation of autophagic vesicles 22 .Tau, a microtubule-associated protein abundant along axon microtubules, plays a vital role in maintaining cytoskeletal stability 23 . When hyperphosphorylated, tau detaches from microtubules 24,25 , leading to cytoskeletal disorganization and impaired cargo transport 26 . Similarly, genetic mutations of tau, such as tauP301L or tauP301S, have been shown to disrupt microtubule stability in vitro, which can impair vesicle transport 27 . This microtubule instability has significant effects on lysosome mobility and therefore affects overall degradation capacity within the cell. In post-mortem brain samples from tauopathy patients, cathepsin D-spilling lysosomes accumulate around the nucleus with autophagic vesicles accumulating at distant locations from the soma 28 . This relationship indicates a breakdown in lysosomal maturity, potentially due to an inability to transport these lysosomes from distal areas of the neuron, exacerbating the degradation issue of these toxic tau proteins.Pathological tau not only obstructs microtubule tracks but also perturbs motor interactions, disrupting kinesin function 29 . Interestingly, under normal conditions, patches of non-pathological tau regulate dynein and kinesin proteins, reversing the direction of dynein while preferentially influencing kinesin detachment 30 . This mechanism ensures balanced anterograde and retrograde transport. However, this suggests that when tau becomes hyperphosphorylated and detaches from microtubules, this regulatory pattern becomes dysregulated and leads to inefficient transport. Loss of tau from axons increases kinesin access to microtubules which may allow for more anterograde transport, but the overall effect is the disruption of cargo delivery and neuronal homeostasis.Alpha-synuclein (αSyn), an IDP implicated in PD, interacts directly with lysosomal membranes. αSyn has been shown to bind LAMP2A to be taken into the lysosome for degradation 31 . In dopaminergic neurons, αSyn overexpression leads to accumulation of acidic lysosomes in the soma and a slowing of retrograde transport 32 , preventing degradation near the distal axon and potentially contributing to synaptic dysfunction 33 . Interestingly, stimulation of the trafficking-related, small GTPase Rab7 in PD models has been shown to reduce αSyn toxicity 34 . In PD, it has been shown that lysosome, hydrolase, and substrate trafficking are disrupted by αSyn aggregates, worsening the disease pathology 35 . This suggests that lysosomal positioning and motility are tightly coupled to autophagosome-lysosome fusion and the efficient degradation of αSyn aggregates.Recently, it was observed in a mouse model of AD that knocking down ATP citrate lyase (ACLY), an enzyme involved in the conversion of citrate into acetyl-CoA and oxaloacetate that is normally decreased in AD patients, destabilized microtubules, disrupted autophagic-lysosomal flux, and accelerated β-amyloid (Aβ) deposition 36 . While this study highlights the relationship of Aβ, lysosomes, and microtubules, much less is known about how Aβ affects intracellular transport of lysosomes as compared to tau and αSyn. While Aβ impairs both the endolysosomal pathway and kinesins 37,38 , more research on the mechanisms of dysfunctional transport needs to be conducted to show intracellular lysosomal transport defects. In addition, the intracellular transport of lysosomes within astrocytes and microglia, under both basal and pathological conditions, remains poorly understood. As glial cells play essential roles in maintaining neuronal health throughout life, it is crucial to investigate their lysosomal trafficking mechanisms for understanding neurodegenerative disease progression.Among various mechanisms that enable intercellular movement and exchange of biological molecules 39,40 , the formation of TNTs appears to be the sole pathway that allows dynamic intercellular trafficking of organelles such as lysosomes. TNTs are F-actin structures that form cytoplasmic continuity between cells, promoting the exchange of organelles, proteins, and macromolecules 41,42 . These cytoplasmic protrusions can span distances of approximately 10-250 m and exhibit a wide range of diameters, normally between 50-700 nm wide 434445 . While most TNTs formed by following the characteristics of F-actin-based structures allowing for cytoplasmic continuity, TNT variations have been observed in vitro 43 . Some TNTs have been reported to contain microtubules 46 , while some projections lack cytoplasmic continuity 43 . TNT structures have also been seen to terminate near neighboring cell membranes, or invaginate into the neighboring membrane without fusing 47 . In a recent study, TNT-like nonsynaptic filopodia were observed in ex vivo mouse somatosensory cortex slices, being the first examples of TNT-like structures observed in mature mouse brains 48 .Multiple drivers and pathways have been implicated in TNT regulation. Small GTPases, like CDC42, and the cytosolic protein tumor necrosis factor (TNF) alpha-induced protein 2 (TNFAIP2, also known as m-Sec) are key drivers that promote actin polymerization and membrane protrusion, aiding in the initiation of TNT formation 49 . Their activity can be triggered by the inflammatory signal TNF, which activates NF-κB 50 , increasing TNFAIP2 expression and activating CDC42 51,52 . TNFAIP2 is typically expressed in both the initiating and receiving cells to begin actin remodeling and membrane extension, with kinesins being essential to facilitating cargo movement along these cytoplasmic extensions 53 . The kinesins function in both intercellular and intracellular transport, although tauopathies and synucleinopathies have been shown to inhibit or disrupt kinesin activity 54,55 .Other players have been implicated within this process such as myosin-X (Myo10), specifically in neuronal TNT formation 56 . Myo10 may play a role in inducing TNTs by transporting cargo to ends of filopodia that aid in actin polymerization, contributing to elongation of filopodia and conversion into TNTs 56,57 . A mechanical theory of TNT formation has also been proposed, where cells that exhibit physical contact create cytoplasmic bridges as they begin to relocate. Membranes stay physically connected as the cells distant themselves, with TNTs being the remnants of that physical connectivity 58 . Insulin receptor substrate (IRSp53) has also been linked to TNT initiation through recruitment of VASP, an actin polymerase, which promotes localized actin remodeling essential for nanotube formation 59 . Another form of induction is oxidative stress, such as H₂O₂ treatment, which induces TNT formation through activation of the PI3K/AKT/mTOR pathway, with inhibitors of these pathways showing reduced TNT formation 60 . Collectively, TNT formation is driven by cytoskeletal remodeling through small GTPases, TNFAIP2/m-Sec, and stress or inflammation-activated signaling pathways that initiate protrusion and nanotube extension.Due to their fragile and dynamic nature, examining TNTs and their associated intercellular trafficking can be challenging. Fluorescent microscopy is employed for live imaging of these TNTs, mainly using cell cultures and more recently in isolated brain tissue sections from transgenic AD mice 48 . Cell cultures can be treated with stressors, such as IDP aggregates or H2O2, to induce TNT formation 61,62 . Immunohistochemistry of F-actin or plasmids that fluorescently tag F-actin can be used in live-cell or tissue imaging. Fixed cells or tissues can also be imaged using a probe that binds to F-actin, phalloidin, coupled with a fluorophore 62 . Similar approaches can be used to track lysosomes, with use of lysosome trackers to label live lysosomes, or expression of lysosome membrane proteins, such as LAMP1/2A, with fluorescent protein tags. While these approaches may be successful in tagging and monitoring of lysosomal movement across TNTs, the challenges encountered are related to the preservation and identification of TNTs for subsequent characterizations 62 . Additionally, presence of cytonemes, similar F-actin-containing cytoplasmic protrusions, complicate the identification of TNTs, as they also influence organelle positioning and there is currently no known marker to specifically stain TNTs. However, cytonemes are unable to transfer organelles and can contain signaling protein receptors, being involved more in cell signaling, unlike TNTs 63,64 .In neurons, imaging shows transfer of lysosomes with toxic species through TNTs. Neurons under stress with toxic IDP aggregates can export defective, less acidic lysosomes to other cells for support in degradation 65 . This exchange may represent a support network, where neurons offload toxic species to glia and glia support neurons by donating functional lysosomes. Communication is bidirectional in neuron-glia interactions, with neuron-microglia and neuron-astrocyte relationships exhibiting bidirectional exchange of organelles and proteins 66,67 . This allows for both neurons and glia to donate and receive lysosomes, enhancing communication through reciprocal movement of organelles in neural systems. There was also evidence of transfer of toxic protein aggregates such as αSyn within individual astrocytes and microglia 68,69 , although glia-glia interaction between cell types via TNTs remains to be investigated. Although TNT-mediated lysosome transfer may initially dampen stress, the transfer can also facilitate the spread of disease through prion-like pathologic proteins and accumulation of diseased lysosomes 65 . In tauopathy models, tau fibrils have been shown to be transferred intercellularly through TNTs. As observed in embryonic rat neurons, when exchanged to recipient cells, tau cargo propagates disease through prion-like abilities that cause endogenous tau misfolding and seeding 70 . αSyn shows similar capabilities, as αSyn fibrils can propagate through TNTs contained within the lysosome 65 . Aβ is also transported across TNTs, from neurons to astrocytes, causing propagation of toxic proteins, although it remains unclear whether they can be transported via lysosomes 71 . These observations support the idea that TNTs exhibit both beneficial and detrimental effects with the trafficking of lysosomes. Selective export of lysosomes may serve as a short-term coping mechanism, but loss of control transforms it into an amplifier of disease spreading.Lysosomes are not all equally mobile or transferable, as their fates appear to lie in the molecular compositions of their membranes. Variations in the ratios of LAMP1 and LAMP2A proteins that make up half of all lysosome membrane proteins, may signal distinct functions of the lysosome 72 . Gene ontology network analysis has shown that LAMP1 interacts more with transport proteins, while LAMP2A interacts more with synaptic proteins 73 . Composition of LAMP1/2A in the lysosomal membrane may influence intracellular (potentially LAMP1) versus intercellular transport (potentially LAMP2A). Other membrane proteins, like VAMP7 or other SNARE proteins, may influence the fusion properties and destination of lysosomes. Lack of SNARE proteins provide a cluster of lysosomes that are unable to fuse with autophagosomes, potentially causing the lysosome to be exocytosed or transferred through TNTs 74 . Small GTPases, such as Rab7, Rab9, and ARL8b, are also involved in the selectivity of this pathway and influence whether lysosomes engage in anterograde, retrograde, or intercellular movement 75 . The presence of these various players in the autophagy and lysosome transport pathways may determine the final destination and function of lysosomes.The lipid composition of the lysosomal membrane provides an additional layer of selectivity. Phosphoinositides, particularly phosphatidylinositol 3,5-bisphosphate (PI(3,5)P₂), regulate organelle trafficking by controlling Ca² + -dependent fusion events through activation of TRPML1 76 . Disturbances in PIKfyve, the kinase responsible for the phosphorylation to produce PI(3,5)P₂, disrupts lipid composition balance, leading to swollen, immobile lysosomes commonly observed in lysosomal storage and neurodegenerative disorders 77 . These highlight how lipids and membrane composition have the ability to influence lysosomal identity and trafficking. Furthermore, lysosomal acidity could serve as both a functional purpose and a transport signal. As lysosomes travel from the axon to the soma, they become fully acidified (pH 4.5-5), implying normal interactions with motor proteins 3 . Therefore, mechanisms of deacidification, such as inhibition of V-ATPase, may act as a regulator that determines lysosomal fate. This pH-dependent transport and localization could be the cause of selective lysosome export observed in stressed neurons.We propose a two-step model where intracellular transport acts as a primary response and intercellular lysosome transport as a secondary attempt to prevent neuronal death, with both representing coordinated responses to facilitate cellular transport and mitigate cellular stress. Pathological stressors, such as IDPs, pro-inflammatory cytokines like TNF, and oxidative stress, disrupt lysosome function by interfering with acidification and V-ATPase assembly and activity 78- 81 . Reduced degradative capacity allows for accumulation of aggregates which can interfere with motor proteins, enhancing aggregation in the cell periphery 82,83 . Furthermore, accumulation of toxic protein aggregates stresses the lysosome by damaging the membrane, weakening lysosomal integrity, and increasing proton leakage and deacidification 84 . However, reacidification of impaired lysosomes has been shown to restore organelle function and improve the clearance of aggregates, easing lysosomal stress on cells 85 .As a normal stress response, the transcription factor EB (TFEB) signaling pathway is activated to increase lysosome biogenesis and autophagic flux is upregulated to restore degradative capacity 86 . When cells experience lysosomal stress from accumulation of dysfunctional lysosomes or disrupted trafficking, TFEB upregulates coordinated lysosomal expression and regulation (CLEAR) genes to increase lysosome biogenesis and autophagy 87 . This increased autophagy allows for the clearance of aggregates and may aid in lysosome transport restoration with a decrease in pathological stressors, like IDPs, that inhibit motor proteins, such as kinesins 29 . Furthermore, lysosomal acidification influences transport as fully acidified lysosomes exhibit enhanced retrograde trafficking toward perinuclear regions, while less acidic vesicles are more prevalent in distal regions 88 . These links between stressors, acidification, and transport provide a for understanding how lysosomal stress responses influence both intracellular positioning and intercellular stress conditions, neurons transport lysosomes within the cell to prevent a of aggregates . from interactions for anterograde transport of lysosomes for degradation in distal regions . lysosome delivery near the and soma When intracellular due to cytoskeletal acidification or IDP cells begin to form TNTs allowing for the exchange of and lysosomes 65 . accumulation causes neural cells to become where signaling and the NF-κB pathway are activated to induce TNT formation through cytoskeletal in neurons and glial cells . of stress signaling, such as may the of TNT formation in cells . TNT formation astrocytes for of functional lysosomes to stressed neurons, while the neurons . Neurons offload vesicles to astrocytes or other neurons to stress for their . Selective lysosome movement across TNTs as an cellular response that neuronal by facilitating the bidirectional exchange of and impaired lysosomes between neurons and astrocytes to neuronal and glia for clearance As pathology and both and this process and the response becomes a for disease propagation . The transport that serve as for disease moving lysosomes that like to propagate tau, Aβ, and αSyn pathology throughout neural whether promoting lysosome exchange or lysosomal acidity can mitigate cellular stress. promoting lysosome transfer through TFEB increased lysosome and TNFAIP2 TNT could determine whether acidic lysosomes neuronal function and IDP accumulation . While there are currently no known molecules that to directly protein interactions, trafficking can be through inhibitors and motor protein such as inhibitors have been shown to microtubules, while is a that activates kinesins, where may allow for trafficking of lysosomes . Small molecules and acidic that lysosomal acidification may represent to lysosome positioning and reduce the intercellular spread of these approaches how selective lysosome trafficking across intracellular and intercellular a dynamic network for maintaining in the and diseased brain lysosome is not only the of degradation but a dynamic organelle movement determines the or of neural In neurons, selective lysosome transport within and between cells degradation with signaling and When properly this allows spatial control of lysosomes and under stressed When it becomes an for disease tau microtubule and disrupts lysosomal transport. Aβ homeostasis and lysosomal while αSyn trafficking and autophagic function. These produce an accumulation of lysosomes that are and for TNTs, these lysosomes can to neighboring cells, pathology and seeding disease across neural This suggests that neurodegeneration not only from the accumulation of proteins, but from a breakdown in the spatial regulation of lysosomal transport. trafficking, whether through impaired loss of or intercellular exchange of toxic species via TNTs, the transport, and function of degradative TNTs also of organelle promoting lysosome and transfer from to neurons or glial These organelles may be to promote through increased ATP by and decreased through increased lysosome presence and activity . that restore transport, interactions, and regulate intercellular lysosome exchange may degradative capacity and the propagation of pathology across neural transport of towards the soma allows for lysosomal fusion and on the lysosome from aggregates causes TFEB to have effects that on genes related to lysosome biogenesis and activating kinesins to increase anterograde transport of lysosomes to distal lysosome formation and transport allow for efficient degradation across the nanotube formation by cellular stresses including protein and lysosomal stress causes leading to activation of NF-κB and of an actin TNT formation the exchange of lysosomes and aggregates between neural cells such as between neurons and Selective lysosome transport between and diseased neural cells a role in maintaining cellular cells toxic protein aggregates and lysosomes near the membrane for export and transport to cells, where the protein can be properly Similarly, cells transport functional lysosomes across TNTs to diseased cells which could in their cellular
Murphy et al. (Mon,) studied this question.