The study, led by Monica Feole and Gorazd B. Stokin and published in Acta Neuropathologica, used human neurons derived from the H9 embryonic stem cell line, cultured for forty days until they developed mature axons. The researchers tagged TDP-43 with green fluorescent protein and filmed it under a high-resolution confocal microscope at four frames per second, capturing sixty-second movies of the protein’s movements inside living axons. To interpret what they saw, they compared TDP-43 against three well-characterized axonal cargoes with known transport behaviors: Rab5, an endosomal protein that mostly travels backward toward the cell body; synaptophysin, a synaptic vesicle protein that races forward to nerve terminals; and amyloid precursor protein, or APP, which moves in both directions with a slight forward bias.

The first surprise came from the directionality data. Using semi-automated particle tracking software, the team found that TDP-43 exhibited balanced bidirectional transport, with roughly equal proportions of particles heading toward the synaptic terminal and back toward the soma. This contrasts sharply with the reference cargoes, which showed their canonical patterns: Rab5 was predominantly retrograde, consistent with its role in endosomal recycling, while synaptophysin displayed a strong anterograde bias reflecting its kinesin-driven delivery to synapses. The balanced profile of TDP-43 suggests that its granules deliver messenger RNA to multiple axonal regions rather than a single destination, consistent with the protein’s role in maintaining RNA homeostasis throughout the neuron’s longest compartment.

Direction alone, however, tells only part of the story. The researchers employed a segmental analysis approach that divides each trajectory into vectorial segments, allowing frame-by-frame examination of velocity, pausing behavior, and directional reversals. This revealed that TDP-43 is a fast cargo. It spent significantly more time in anterograde motion than Rab5, paused less often, and reversed direction more frequently, all characteristics that align it with the fast-moving vesicular proteins synaptophysin and APP rather than the slower, pause-prone Rab5. Its total track lengths were comparable to synaptophysin and Rab5 but shorter than APP, which traveled the widest range of distances, hinting that TDP-43 granules may be heterogeneous in composition or in the motors they recruit.

Velocity distributions provided an even sharper clue about the underlying machinery. When the team plotted the speeds of anterograde segments, TDP-43’s velocities almost completely overlapped with those of APP, suggesting that both cargoes may share kinesin-dependent, plus-end-directed transport mechanisms. In the retrograde direction, by contrast, TDP-43’s velocities differed significantly from all three reference cargoes, implying that although the dynein-dynactin complex broadly mediates backward transport, its processivity is tuned in a cargo-specific manner, perhaps through unique adaptor proteins or post-translational modifications on membraneless ribonucleoprotein granules. Correlation analysis further showed that for TDP-43, longer runs correlated with higher speeds in both directions, a hallmark of processive motor engagement, with no significant difference in coupling strength between anterograde and retrograde movement.

To identify the actual molecular motors, the team turned to biochemistry. Co-immunoprecipitation experiments in the mature human neurons revealed that TDP-43 physically associates with kinesin light chain 1, or KLC1, the adaptor that links cargoes to the kinesin-1 family of anterograde motors, and with dynactin 1, a core component of the retrograde dynein complex. Notably, TDP-43’s binding to KLC1 was more robust than its binding to dynactin 1, a preference consistent with the strong anterograde characteristics observed in the live imaging. Reciprocal pulldowns confirmed the interactions: pulling down KLC1 brought along TDP-43 and the expected kinesin-5B partner, while pulling down dynactin 1 co-purified TDP-43 together with dynein intermediate chain 1.

The most striking result came from proximity ligation assays, a technique that detects proteins lying within forty nanometers of each other in their native environment. When the researchers knocked down KLC1 using short hairpin RNA delivered by lentiviral vectors, the TDP-43–KLC1 proximity signal in axons dropped dramatically, validating the specificity of the interaction. Then, using isoform-specific antibodies, they discovered that TDP-43 associates with all three mammalian kinesin-1 heavy chain isoforms: KIF5A, KIF5B, and KIF5C. This is significant because KIF5B is ubiquitously expressed while KIF5A and KIF5C are neuron-specific, and KIF5A is a recognized genetic cause of ALS. The engagement with all three isoforms suggests a flexible, redundant transport system in which distinct motor combinations could deliver different mRNA payloads to different axonal subdomains.

There was one more motor in the fleet. Because TDP-43’s anterograde dynamics resembled those of synaptophysin, which is transported by the kinesin-3 family member KIF1A, the team tested whether TDP-43 also engages this rapid synaptic vesicle motor. Co-immunoprecipitation in both directions confirmed the interaction, and proximity ligation assays placed TDP-43 and KIF1A within nanometers of each other along neuronal projections. The picture that emerges is of a multi-motor system: KIF1A may shuttle selected TDP-43 granules rapidly over long distances to distal synaptic terminals, while the kinesin-1 isoforms support more regulated delivery to intermediate axonal regions. Alternatively, distinct populations of TDP-43 granules, defined by their mRNA cargo or phase-separation properties, may preferentially recruit different motors.

Why does this matter for disease? In ALS, where TDP-43 accumulates abnormally in the cytoplasm, this flexible transport system may become compromised in multiple ways simultaneously. Pathological aggregates could sequester motor proteins or adaptor molecules, disrupting the delivery of mRNAs needed for local translation at synapses, including transcripts for cytoskeletal proteins, synaptic components, and nuclear-encoded mitochondrial proteins. Mutations in TARDBP and C9orf72 repeat expansions have already been linked to impaired mRNA trafficking, and mutations affecting the dynein-dynactin complex are associated with motor neuron disorders. The multi-motor redundancy documented in this study might normally provide resilience, allowing partial compensation when one pathway fails, but combined insults to cytoskeletal integrity, mitochondrial function, and RNA granule homeostasis could eventually overwhelm the system, driving the distal axonal degeneration that characterizes the dying-back model of ALS.

The findings also raise fundamental questions about how transport works without membranes. Classical models of axonal transport were built largely from studies of membrane-bound vesicles, where transmembrane proteins serve as docking sites for motor-adaptor complexes. TDP-43 travels in membraneless organelles, condensates of RNA and protein with no lipid bilayer, yet it clearly engages the same motor machinery. How motors recognize and bind these condensates, whether specific RNA sequences or RNA-binding protein motifs serve as adaptor recruitment sites, and whether the biophysical state of the condensates regulates motor engagement are all open questions. Previous work showed that some RNA granules hitchhike on lysosomes or mitochondria; this study demonstrates that TDP-43 can also associate directly with motors, suggesting both mechanisms may operate in parallel. By establishing a physiological baseline in human neurons, the work provides the framework against which ALS-linked mutations and patient-derived models can now be measured, and it points to axonal TDP-43 transport pathways as promising therapeutic targets for a disease that urgently needs them.

Subject of Research: Axonal transport mechanisms of the RNA-binding protein TDP-43 in human neurons and their relevance to ALS

Article Title: Multiple motor proteins regulate TDP-43 anterograde axonal transport

Article References: Feole, M., Devoto, V. M. P., Dragišić, N., Čarna, M., Klosterman, K., Limbaek-Stokin, C., Forte, G., Moya, K. L., Smith, R. A., Svendsen, C. N., & Stokin, G. B. (2026). Multiple motor proteins regulate TDP-43 anterograde axonal transport. Acta Neuropathologica, 152(1), Article 47. https://doi.org/10.1007/s00401-026-03071-w

Image Credits: AI Generated

DOI: 10.1007/s00401-026-03071-w

Keywords: TDP-43, axonal transport, amyotrophic lateral sclerosis, kinesin, KIF5A, KIF1A, KLC1, dynein, RNA-binding proteins, motor proteins, human neurons, neurodegeneration

The logistics of a motor neuron are staggering. These cells extend axons that can stretch up to a meter in the human body, and their distant terminals need a constant supply of fresh mitochondria, the organelles that generate the chemical energy ATP required for synaptic transmission and cellular survival. Moving mitochondria along the axon is an energy-intensive process in itself, and when that transport falters, the far reaches of the neuron are starved of power while damaged organelles pile up. Disrupted axonal transport has been implicated in ALS for years, and previous work had established that C9ORF72 motor neurons show cell-autonomous defects in mitochondrial motility and function. What remained unknown was whether the astrocytes surrounding those neurons—cells already known to contribute to non-cell-autonomous neurodegeneration through mechanisms such as reduced glutamate uptake and impaired lactate shuttling—also influence this transport machinery.

To find out, the team built a rigorous human stem-cell platform. They used induced pluripotent stem cell lines derived from three patients carrying the C9ORF72 repeat expansion, together with isogenic gene-corrected controls created through CRISPR–Cas9 editing, in which the mutation was removed. From these lines they generated highly enriched spinal cord-patterned astrocytes, more than ninety percent of which expressed the canonical astrocyte markers glial fibrillary acidic protein and S100B, as well as motor neuron cultures of which roughly half to sixty percent were ISL1/2-positive motor neurons. The mutant astrocytes carried hallmark C9ORF72 pathologies, including RNA foci and dipeptide repeat proteins, yet showed no differences in glutamate uptake or calcium wave propagation, meaning any effects on transport could not be attributed to those well-known astrocyte dysfunctions.

The central experiment was elegantly simple. The researchers selectively labeled motor neurons with a fluorescent mitochondrial marker, mito-DsRed2, and then imaged mitochondrial movement along a hundred-micrometer stretch of the proximal axon using live-cell time-lapse microscopy, generating kymographs from which they extracted two established metrics: the percentage of mitochondria that were motile and their average velocity. When genetically normal motor neurons were grown in physical contact with C9ORF72-mutant astrocytes, both measures dropped significantly compared with motor neurons cultured alone. Strikingly, when the same control neurons were paired with gene-corrected astrocytes, the deficit vanished entirely. Even more remarkable, mutant motor neurons cocultured with corrected astrocytes recovered both transport parameters to wild-type levels. In these mixed cultures, the phenotype of the neuron’s mitochondrial traffic was dictated not by the neuron’s own genome but by the genotype of its astrocyte neighbors.

Contact, it turned out, was essential. When the team treated control motor neurons with conditioned medium harvested from mutant astrocytes, mitochondrial motility was unaffected, indicating that secreted factors alone could not reproduce the damage; direct physical interaction between the two cell types was required. The specificity of the defect was equally revealing. Tracking HcT-labeled signaling endosomes, another vital axonal cargo, showed no transport changes in any coculture combination. The astrocyte effect was therefore cargo-specific, targeting mitochondria rather than causing a global collapse of axonal transport. The authors suggest this selectivity reflects distinct energetic dependencies: signaling endosomes fuel their movement with locally generated glycolytic ATP, whereas mitochondrial motility depends on membrane potential and calcium-regulated adaptors such as Miro, directly coupling transport to the bioenergetic state of the organelle itself.

That coupling became the thread the researchers followed next. Using Seahorse extracellular flux analysis, they measured oxygen consumption rates as a proxy for mitochondrial respiration. Because motor neuron cultures respire at nearly eightfold the rate of astrocytes alone, changes in coculture readings could be confidently attributed to the neurons. The results were unambiguous: coculture with astrocytes of either genotype boosted basal and maximal respiration relative to neuron monocultures, but pairing neurons with mutant astrocytes significantly reduced both parameters compared with pairing them with corrected astrocytes. A complementary assay using the potentiometric dye MitoTracker Red CMXRos confirmed that the steady-state mitochondrial membrane potential of motor neurons—mutant or normal—fell in the presence of mutant astrocytes and recovered with corrected astrocytes. The astrocyte genotype was directly modulating the bioenergetic health of neurons it touched.

The root of the problem lay within the astrocytes themselves. Isolated C9ORF72-mutant astrocytes showed significantly reduced basal, ATP-linked and maximal respiration compared with their gene-corrected counterparts, along with diminished glycolytic capacity, lactate production and glucose utilization. RNA sequencing, however, revealed no genotype-driven astrocyte reactivity, making a gain-of-toxic-function inflammatory mechanism unlikely. Intriguingly, when the team measured intracellular lactate in motor neurons using a genetically encoded FRET biosensor called Laconic, they found no significant differences across coculture conditions, suggesting that disrupted oxidative metabolism within astrocytes, rather than altered lactate transfer to neurons, mediates the transport deficit. The astrocytes were not failing to deliver fuel; they were failing in a way that somehow drains the neurons’ own power plants.

The therapeutic implication emerged from a rescue experiment targeting the master regulator of mitochondrial biogenesis. The team overexpressed three key metabolic genes in mutant astrocytes via lentiviral delivery: PPARGC1A (encoding PGC-1α), PPARGC1B (PGC-1β) and TFAM. PGC-1β proved toxic to the astrocytes, and TFAM, consistent with prior reports that it does not drive mitochondrial biogenesis, conferred no benefit. PGC-1α, by contrast, upregulated transcripts of the mitochondrial electron transport chain, including MT-CO2, MT-ND2, MT-CO3, MT-ND4 and MT-ATP6, and produced a trend toward increased respiration. When mutant astrocytes overexpressing PGC-1α were cocultured with motor neurons—mutant or corrected—mitochondrial motility and velocity were restored. Boosting the metabolic engine of the astrocyte alone was sufficient to repair the transport machinery inside genetically distinct neurons.

The findings reframe how scientists think about non-cell-autonomous degeneration in ALS. Rather than astrocytes poisoning neurons through inflammatory or excitotoxic signals, this work describes a bioenergetically coupled mechanism in which the metabolic competence of glia sets the ceiling for mitochondrial dynamics in neurons. The result also resonates with earlier animal work in SOD1 models of ALS, where PGC-1α activation preserved mitochondrial function and delayed disease progression, hinting that metabolic rescue of glia could be a broadly applicable strategy. The authors caution that much remains to be defined, including whether direct mitochondrial exchange, metabolite or lipid transfer, vesicular signaling or other modes of metabolic coupling connect astrocytic energy status to neuronal mitochondrial behavior, and whether similar mechanisms operate across other ALS genotypes and in sporadic disease, which accounts for roughly ninety percent of cases.

For a disease with no cure and only modestly effective therapies, the identification of an actionable, astrocyte-centered node is significant. If pharmacological activation of the PGC-1α pathway in glia can correct non-cell-autonomous mitochondrial dysfunction in C9ORF72-ALS, it opens a therapeutic window that does not require correcting the mutation inside every neuron. The study, funded in part by the Medical Research Council, the UK Dementia Research Institute and the My Name’5 Doddie Foundation among others, demonstrates with unusual clarity that in neurodegeneration, the health of a neuron may depend as much on the metabolic company it keeps as on its own genes—a lesson that could reshape drug discovery for ALS well beyond the C9ORF72 mutation that first revealed it.

Subject of Research: Non-cell-autonomous regulation of axonal mitochondrial transport by C9ORF72-mutant astrocytes in amyotrophic lateral sclerosis

Article Title: Astrocytes regulate axonal mitochondrial transport deficits in C9ORF72 amyotrophic lateral sclerosis motor neurons

Article References: Stavrou, M., Heffernan, Á. B., Carter, R. N., Dando, O., Jiwaji, Z., Burr, K., Nanda, J., Villarroel-Campos, D., Masoud Abdelhafid, A., Cholewa-Waclaw, J., Soong, D., Story, D., Schiavo, G., Hardingham, G. E., Chandran, S., & Selvaraj, B. T. (2026). Astrocytes regulate axonal mitochondrial transport deficits in C9ORF72 amyotrophic lateral sclerosis motor neurons. Nature Neuroscience. https://doi.org/10.1038/s41593-026-02464-0

Image Credits: AI Generated

DOI: 10.1038/s41593-026-02464-0

Keywords: amyotrophic lateral sclerosis, C9ORF72, astrocytes, motor neurons, mitochondrial transport, axonal transport, mitochondrial bioenergetics, PGC-1α, induced pluripotent stem cells, non-cell-autonomous neurodegeneration, CRISPR gene correction, live-cell imaging

The article, led by Carsten Janke of Institut Curie and Université Paris-Saclay together with Anna Akhmanova of Utrecht University and an international consortium of sixteen colleagues from institutions spanning Europe and the United States, lays out a coordinated agenda for the microtubule field. Its authors span structural biology, biophysics, cell biology, neuroscience, parasitology and computational modelling, reflecting their central conviction that progress will come not from any single discipline but from a deliberate fusion of them. The paper is punctuated by the admission that some of the most basic questions remain unexplored, including how the properties and functions of microtubules are affected by the chemical marks known as tubulin post-translational modifications, by disease-related mutations in tubulin genes, or by variation in the microtubule lattice itself.

Microtubules are hollow tubes assembled from αβ-tubulin dimers that stack into protofilaments, typically thirteen of which align laterally to form the wall of the polymer. What makes these structures extraordinary is their behaviour: individual microtubules stochastically switch between phases of growth and rapid shrinkage, a phenomenon called dynamic instability, which was first described by Tim Mitchison and Marc Kirschner in 1984. A stabilizing cap of GTP-bound tubulin at the growing plus end controls this behaviour, and its loss triggers catastrophic depolymerization. Beyond pure polymerization, microtubules can self-repair by incorporating new tubulin along damaged stretches of lattice, a property revealed in studies showing that lattice defects actually induce self-renewal and protect the polymers from destruction by molecular motors. These discoveries have transformed the view of microtubules from static beams into dynamic, self-healing machines.

The Roadmap emphasizes that microtubule function is not determined by the polymer alone but by a dense layer of regulation encoded on the tubulin subunit itself. Enzymes of the tubulin tyrosine ligase-like family decorate the unstructured carboxy-terminal tails of tubulin with modifications such as tyrosination, detyrosination, acetylation, glutamylation and glycylation, creating what has become known as the tubulin code. Recent structural work has shown, for example, that enzymes involved in polyglutamylation recognize microtubules through a quadrivalent mechanism that links microtubule geometry to the generation of localized modification patterns, and that protofilament-specific nanopatterns of these marks tune the mechanics of beating cilia. The Roadmap argues that understanding how this chemical language is written, read and erased across cellular contexts is one of the field’s most pressing challenges, particularly because disruptions in the code are now firmly linked to neurodegeneration, ciliopathies and other disorders.

Nowhere is the clinical relevance more evident than in the nervous system. Neurons depend on microtubule tracks for the long-range transport of organelles, messenger RNAs and signalling endosomes along axons and dendrites, a process powered by kinesin and dynein motor proteins. Mutations in tubulin genes such as TUBA1A cause lissencephaly, a severe developmental brain malformation, by perturbing neuronal migration, and variants in a growing list of tubulin genes underlie a spectrum of neurodevelopmental disorders collectively known as tubulinopathies. Defective axonal transport is a shared hallmark of motor neuron diseases, and recent work has shown that excessive polyglutamylation of tubulin in the brain is sufficient to drive neurodegeneration by disrupting transport, while restoring the balance of these modifications can rescue affected neurons. The Roadmap positions a systems-level understanding of these processes as essential for translating molecular insight into therapies.

The heart and the immune system tell equally compelling stories. In beating cardiomyocytes, detyrosinated microtubules act as load-bearing elements that buckle under contraction and stiffen the cell, and suppressing this modification improves cardiac function in models of heart failure. In immune cells, microtubule networks orchestrate cell shape, migration and the directed release of lytic granules by natural killer cells, while platelets, whose tubulin repertoire is dominated by the β1-tubulin isotype, rely on precisely regulated microtubule assembly during their biogenesis; mutations in the TUBB1 gene cause congenital macrothrombocytopenia and thyroid dysgenesis. The Roadmap argues that these tissue-specific roles cannot be understood piecemeal and demand approaches that bridge the molecular properties of tubulin to the physiology of whole organs.

Technological innovation sits at the centre of the proposed strategy. Cryo-electron microscopy and cryo-electron tomography now reveal microtubules and their associated proteins at near-atomic and in situ resolution, while expansion microscopy and ultrastructure expansion microscopy allow researchers to visualize centrioles, cilia and mitotic spindles in whole cells and tissues with nanometre precision. In vitro reconstitution, a tradition stretching back to the pioneering work of Tim Mitchison and Marc Kirschner and continued in landmark experiments showing that purified microtubules and motors can self-organize into asters and vortices, remains indispensable for dissecting minimal systems. These experimental approaches are increasingly paired with computational and physical models that simulate spindle assembly, microtubule network organization and chromosome movement, and the Roadmap calls for tighter coupling between modelling and experiment, including the development of virtual cells powered by artificial intelligence.

Recombinant tubulin technology is highlighted as a particularly transformative advance. For decades, biochemists were limited to native tubulin purified from brain tissue, a mixture of isotypes and modifications that obscured cause and effect. The ability to produce functional human tubulin dimers in defined isotypes, with controlled post-translational modifications, has now enabled researchers to show directly that different isotypes confer distinct dynamic properties, that detyrosination tunes microtubule stability through selective recruitment of associated factors, and that systematic mutagenesis can map the functional landscape of disease-linked variants. Combined with deep mutational scanning and artificial-intelligence-driven phenotyping, the authors argue, these tools will allow the effects of every clinically observed tubulin mutation to be predicted and tested, a goal that seemed out of reach only a few years ago.

The Roadmap also looks beyond animal cells, drawing attention to the diversity of microtubule arrays across eukaryotes, from the cortical arrays that guide plant cell morphogenesis to the subpellicular arrays of trypanosomes and the specialized mitotic machinery of parasites. Comparative studies of parasites such as Plasmodium have revealed that adaptations in tubulin sequence generate distinct microtubule architectures, mechanics and drug susceptibilities, opening avenues for species-selective therapeutics. The authors contend that this evolutionary breadth is not a curiosity but a resource: organisms that build microtubules with unusual lattices, modifications or assembly mechanisms offer natural experiments that can illuminate principles hidden in familiar model systems.

Ultimately, the Roadmap is an invitation to think bigger. Its authors conclude that the microtubule cytoskeleton will continue to inspire scientists for decades precisely because so many fundamental questions remain open: how microtubule arrays are organized and diversified in different cell types, how the tubulin code is orchestrated across space and time, how mechanical forces reshape and stabilize microtubule lattices in living cells, and how all of this integrates into the physiology of tissues and organisms in health, ageing and disease. By bridging the atomic structure of the tubulin dimer with the behaviour of mitotic spindles, migrating neurons and beating hearts, the field aims to transform its fragmented molecular knowledge into a coherent systems-level picture, one that could reshape how disorders ranging from neurodegeneration and cancer to ciliopathies and heart failure are understood and treated.

The historical depth of the field is worth appreciating. Microtubules were first visualized in the late nineteenth and early twentieth centuries, but their protein building block was only identified in the late 1960s, when colchicine-binding assays led to the isolation of what was soon named tubulin, and amino-acid analysis of sperm flagella confirmed it as the universal subunit of the polymer. The discovery of dynamic instability two decades later established that these polymers are fundamentally nonequilibrium structures, a insight that continues to shape how spindle assembly and chromosome movement are understood today.

Equally important is the cast of regulatory proteins that act on microtubules. Microtubule-associated proteins such as tau can condense on the lattice in regulated phases, altering how motors and severing enzymes engage the polymer, while enzymes that cut microtubules generate new seeds and reshape networks. Proteins like doublecortin, which is mutated in human brain malformations, illustrate how even lattice geometry itself can be read selectively, recognizing only specific protofilament numbers. These layered interactions, spanning motors, maps, severing enzymes and modifying enzymes, form the mechanistic vocabulary that any systems-level account of the cytoskeleton will ultimately need to integrate.

Subject of Research: Systems-level integration of microtubule cytoskeleton structure, regulation and function in physiology and disease

Article Title: Towards a systems-level view of the microtubule cytoskeleton and its functions in physiology and disease

Article References: Janke, C., Akhmanova, A., Bartolini, F., Bodakuntla, S., Del Bene, F., Hamel, V., Mitchison, T. J., Müller-Reichert, T., Nédélec, F., Nogales, E., Pigino, G., Roll-Mecak, A., Schiavo, G., Surrey, T., & Lansky, Z. (2026). Towards a systems-level view of the microtubule cytoskeleton and its functions in physiology and disease. Nature Reviews Molecular Cell Biology. https://doi.org/10.1038/s41580-026-01011-w

Image Credits: AI Generated

DOI: 10.1038/s41580-026-01011-w

Keywords: microtubules, tubulin code, post-translational modifications, dynamic instability, axonal transport, tubulinopathies, ciliopathies, neurodegeneration, mitotic spindle, cryo-electron tomography, in vitro reconstitution, systems biology