Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model

Asakawa, Kazuhide; Tomita, Takuya; Shioya, Shinobu; Handa, Hiroshi; Saeki, Yasushi; Kawakami, Koichi

doi:10.1038/s41467-025-65097-0

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Published: 27 October 2025

Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model

Nature Communications volume 16, Article number: 9213 (2025) Cite this article

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Amyotrophic lateral sclerosis

Abstract

Selective neuronal vulnerability is a defining feature of neurodegenerative disorders, exemplified by motor neuron degeneration in amyotrophic lateral sclerosis (ALS). The nature of motor neurons underlying this selectivity remains unresolved. Here, by monitoring autophagy at single-cell resolution across the translucent zebrafish spinal cord, we identify motor neurons as the cell population with the highest autophagic flux. Large spinal motor neurons (SMNs), most susceptible to ALS, exhibit higher flux compared to smaller SMNs and ALS-resistant ocular motor neurons. Notably, large SMNs accelerates both autophagy and proteasome-mediated degradation, which are further augmented by TDP-43 loss. Additionally, acceleration of multiple unfolded protein response pathways indicates their innate tendency to accumulate misfolded proteins. Enhanced cellular degradation in large SMNs is neuroprotective as its inhibition halts axon outgrowth. These findings propose that cell size-associated degradation load underlies selective neuronal vulnerability in ALS, highlighting the alleviation of catabolic stress as a target of therapy and prevention.

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Introduction

Cells constituting the central nervous system (CNS) exhibit varying degrees of susceptibility to various diseases. Amyotrophic lateral sclerosis (ALS), a currently incurable neurodegenerative disorder, exemplifies disease-specific neuronal susceptibility in which motor neurons responsible for muscle contraction undergo selective degeneration. Remarkably, selective vulnerability exists, even within this functionally similar neuronal population. For instance, the motor neurons innervating the extraocular muscles are resistant to ALS, though the underlying mechanisms remain poorly understood^1,2,3. Consequently, eye movements tend to be relatively preserved, even in the end-stage of the disease in ALS, whereas most bodily movements become progressively impaired. By contrast, large motor neurons are particularly vulnerable to ALS and normal aging^4,5,6. Large motor neurons, or α-motor neurons, are characterized by large cell bodies with highly branched dendritic arbors that receive a multitude of synaptic inputs. These neurons have a high conduction velocity due to their long axons with a large diameter and thick myelination and control large numbers of muscle fibers to generate strong voluntary body movements. However, the physiological and molecular mechanisms by which these cellular traits contribute to the selective vulnerability to ALS and aging remain unclear.

The accumulation of misfolded proteins or protein aggregates is a characteristic of most neurodegenerative diseases. Deposition of ubiquitin-positive inclusion bodies containing aggregates of the trans-activation response element (TAR) DNA-binding protein 43 (TDP-43) in motor neurons is a major hallmark of ALS^7,8, suggesting that a decline in cellular degradation could be intrinsically linked to the as-yet-unidentified root causes of ALS. Macroautophagy (hereafter referred to as autophagy) is a critical cellular degradation mechanism for eliminating and recycling damaged proteins and organelles. Dysfunction of this process may impair neural function and result in neurodegeneration^9,10. Human genetics have identified multiple autophagy-related genes as causative factors of neurodegenerative diseases, including ALS^{11,12,13,14,15,16,17}. The accumulation of autophagosomes, a double-membraned vesicles that engulf degradation substrates and deliver them to lysosomes, in spinal motor neurons (SMNs) has been reported in sporadic cases with ALS and animal models of the disease^18,19,20. These observations substantiate that autophagy-related cellular degradation plays a protective role in physiology and pathophysiology of motor neurons^21,22, although autophagy can have adverse effects during certain stages of the disease course²¹. In neuronal cells, autophagy operates constitutively at certain levels, and the physiological role of such basal autophagic flux may differ from that of the canonical response to starvation²³. The involvement of autophagy in the regulation of motor neuron sub-compartments, including axon formation and synaptogenesis, has been reported in multiple models^{21,24,25,26,27}. In addition to autophagy, the ubiquitin-proteasome system (UPS) is a key cellular degradation system involved in ALS pathogenesis. In mice, motor neuron-specific knockout of a proteasome subunit results in ALS phenotypes, whereas knockout of the autophagy-related gene ATG7 does not, suggesting that the UPS and autophagy may play distinct roles in ALS pathogenesis^28,29. The UPS exhibits varying levels of activity in mouse embryonic stem cell-derived cranial motor neurons and SMNs, which correspond to ALS-resistant and ALS-vulnerable neurons, respectively³, indicating a potential role for UPS activity in determining neuronal susceptibility to ALS. Currently, it remains unclear whether the activity of major cellular degradation pathways, such as autophagy and the UPS, differs across various CNS cell types and how such variations contribute to the differing neuronal vulnerability observed in ALS. To address these questions, comparing the dynamic aspects of cellular catabolism in vivo between ALS-vulnerable and ALS-resistant neurons is imperative. However, such real-time in vivo comparisons are not readily feasible, even in model animals, let alone in humans.

In the present study, we performed a cellular-resolution scanning of the autophagic flux across the translucent zebrafish spinal cord and identified motor neurons as the cell population with the highest level of autophagic flux. A comparison among the motor neuron subtypes revealed that large SMNs displayed higher autophagic flux than smaller SMNs as well as brainstem ocular motor neurons, both of which are relatively resistant to ALS. Notably, large SMNs not only exhibited enhanced autophagy but also demonstrated proteasome-mediated degradation. These processes were further intensified by the loss of the ALS-related protein TDP-43. The naturally activated multiple unfolded protein response pathways in large SMNs suggest an inherent tendency to accumulate misfolded proteins. These results posit that the cellular degradation load linked to cell size underlies motor neuron vulnerability and is a crucial determinant of selective susceptibility to ALS.

Results

Diversity of autophagy activity among neuronal cells in the spinal cord

Differential basal autophagic activity across various tissues has been revealed in zebrafish using the ratiometric autophagic flux probe GFP-LC3-RFP-LC3∆G³⁰. The probe assesses the stability of the key autophagosome marker, microtubule-associated protein light chain 3 (LC3), by releasing degradable GFP-LC3 and a non-degradable internal control RFP-LC3∆G through cleavage by the ATG4 protease upon autophagy activation and reflects autophagy flux acceleration by a decrease in the GFP-LC3/RFP-LC3∆G ratio. Using this probe, the basal autophagic flux was demonstrated to be relatively lower in the spinal cord than in the skeletal muscle³⁰. To investigate the diversity of autophagic flux within the spinal cord at single-cell resolution, we generated transgenic zebrafish (Tg[UAS:GFP-LC3-RFP-LC3∆G]) in which GFP-LC3-RFP-LC3∆G can be expressed ubiquitously or in a defined cell type in a Gal4-dependent manner (Fig. 1a). First, we broadly expressed GFP-LC3-RFP-LC3∆G in the spinal cord using the ubiquitous Gal4 driver Tg[SAGFF73A]³¹. Expression levels of GFP-LC3 and RFP-LC3∆G varied among the spinal cord cells in Tg[SAGFF73A] Tg[UAS:GFP-LC3-RFP-LC3∆G] double transgenic fish (Fig. 1b, Supplementary Movie. 1). We noticed a cell population with oval-shaped cell bodies and a markedly low GFP-LC3/RFP-LC3∆G ratio, indicating high autophagic flux, in the ventrolateral side of the spinal cord at 30 h post-fertilization (hpf) (Fig. 1b). Neuronal populations in the spinal cord commonly exhibit spatially characteristic clustering according to cell type³². Neurons responsible for sensory functions are primarily positioned dorsally, whereas those controlling motor execution are predominantly located ventrally. Thus, we hypothesized that the ventral cells with a low GFP-LC3/RFP-LC3∆G ratio include SMNs, which cluster to form a motor column on the ventral side of the spinal cord in zebrafish^33,34. To address this possibility, we restricted GFP-LC3-RFP-LC3∆G expression to the caudal primary motor neurons (CaPs) in the ventral side and tactile sensing Rohon-Beard (RB) cells in the dorsal side of the spinal cord, using the Gal4 driver Tg[SAIG213A]³⁵ (Fig. 1c). We found that GFP-LC3/RFP-LC3∆G values in the soma regions were significantly lower in the CaPs than in RB cells at 30 hpf (Fig. 1d, Supplementary Fig. 1a–c), showing that CaPs have a higher level of basal autophagic flux than the RB cells. The Tg[SAIG213A] driver also labeled some types of interneurons with diverse cell morphologies. The GFP-LC3/RFP-LC3∆G ratio in these interneurons was intermediate between those of observed in CaPs and RB cells (Fig. 1d). Taken together, these observations suggest that the cell populations with high autophagic flux in the ventrolateral spinal cord includes SMNs. Zebrafish assembles a neural circuit for touch-evoked swimming movements as early as by 21 hpf, where tactile afferent signals elicited from RB cells are transmitted to SMNs, including CaPs^36,37. Therefore, these results demonstrate that the functional neural circuit for sensory-evoked locomotion consists of cells with different levels of autophagic flux.

**Fig. 1: Autophagic flux scanning across spinal cord neurons.**

Modest inverse correlation between basal autophagic flux and soma size of SMNs

Having discovered that SMNs exhibit the highest level of autophagic flux in the spinal cord, we further explored whether the autophagic flux varies among motor neuron subtypes. The SMNs represent a heterogeneous population with diverse cell sizes corresponding to their distinct physiological roles and connectivity patterns³⁸. The fact that CaPs represent one of the largest SMN types in zebrafish³⁹ prompted us to examine the relationship between cell size and autophagic flux intensity in SMNs. Using the pan-motor neuronal Gal4 driver Tg[mnr2b-hs:Gal4]⁴⁰, we expressed GFP-LC3-RFP-LC3∆G in most of the SMNs and measured the GFP-LC3/RFP-LC3∆G ratio in their soma regions at 48–50 hpf (Fig. 1e). Intriguingly, by plotting the GFP-LC3/RFP-LC3∆G values against soma size, we observed a modest, but significant, trend that the GFP-LC3/RFP-LC3∆G ratio decreased as soma size increased (Fig. 1f). This indicates that large SMNs tend to have more enhanced autophagic flux than small SMNs.

Motor neurons are the primary neuronal type that degenerates in the motor neuron disease ALS, with large SMNs being more vulnerable than smaller SMNs^4,5. Enhanced autophagic flux in the large SMNs of zebrafish suggests a correlation between the intensity of autophagic flux and neuronal vulnerability to ALS. To further explore this idea, we compared the autophagic flux between SMNs and ocular motor neurons in the brainstem, which are known to be resistant to ALS. Purposing this, we took advantage of Gal4 expression in the abducens motor neurons (ABNs) of the brainstem in the Tg[mnr2b-hs:Gal4] line (Fig. 1g)⁴⁰. The GFP-LC3/RFP-LC3∆G values of the ABNs and SMNs were compared in the same confocal sections including the junction between the brainstem and spinal cord at 72 hpf, when most ABNs had already connected to the lateral rectus muscle⁴⁰. We noted that the GFP-LC3/RFP-LC3∆G values in the somas were highly variable in ABNs compared with those in SMNs (Fig. 1h, Supplementary Fig. 2a–c). Nonetheless, ABNs displayed higher GFP-LC3/RFP-LC3∆G values than SMNs, indicating that autophagic flux was generally lower in the ABNs than in SMNs. These observations further highlighted the correlation between enhanced autophagic flux and neuronal vulnerability in ALS.

Loss of TDP-43 function accelerates basal autophagic flux in the SMNs

To explore the significance of accelerated autophagy in large SMNs in ALS, we examined how an ALS-related genetic mutations affect autophagic flux in these neurons. We focused on the tardbp/TARDBP gene which encodes TDP-43 protein, mutations and protein aggregation of which are associated with most cases of ALS^7,8,41. Zebrafish have two genes that encode TDP-43, tardbp and tardbpl. Using stable mutant alleles with frame-shift mutations introducing premature termination codons in both genes (tardbp-n115 and tardbpl-n94)⁴², we expressed the GFP-LC3-RFP-LC3∆G reporter in the CaPs, RB cells, and interneurons of the TDP-43 double knockout (DKO) fish. Intriguingly, we found that the GFP-LC3/RFP-LC3∆G values were significantly reduced in each of these neuronal types in the TDP-43 DKO fish at 48 hpf (Fig. 2a), showing that the loss of TDP-43 function accelerates autophagic flux regardless of neuronal type. Therefore, under normal conditions, TDP-43 function contributes to a reduction in autophagic flux across different neuronal types. This observation is consistent with the notion that TDP-43 prevents the production of aberrant proteins by suppressing cryptic exons and thereby reducing mis-splicing⁴³, and that loss of TDP-43 function imposes cellular degradation stress and accelerates autophagic flux in SMNs.

**Fig. 2: TDP-43 loss accelerates autophagic flux in the SMNs.**

Autophagic acceleration is independent of MiT/TFE transcription factors

Next, we investigated the potential upstream mechanisms driving the sustained, cell-type-specific activation of autophagy in large SMNs. In cultured human cells (HEK 293 and HeLa cells), the knockdown of TDP-43 results in the nuclear accumulation of the MiT/TFE transcription factor TFEB⁴⁴, which enhances global gene expression in the autophagy-lysosome pathway (ALP)^45,46. To determine the contribution of MiT/TFE transcription factors to autophagic flux acceleration in SMNs in vivo, we first examined the subcellular localization of Tfeb, as well as the other MiT/TFE transcription factors, Tfe3a and Tfe3b, in CaPs. Each of these proteins was tagged with an enhanced green fluorescent protein (EGFP) and expressed along with the nuclear marker mCherry-tagged histone H2A (H2az2a-mCherry) using the Tg[SAIG213A] driver. We found that Tfeb-EGFP was enriched in the cytoplasm at 48 hpf (Fig. 2b). Unexpectedly, Tfeb-EGFP remained in the cytoplasm under the TDP-43 DKO condition, unlike in cultured cells⁴⁴. We also found that Tfe3a-EGFP was predominantly enriched in the nucleus, whereas Tfeb-EGFP and Tfe3b-EGFP were mainly localized in the cytoplasm at 48 hpf, and these localization patterns remained unchanged in TDP-43 DKO fish. To determine the causal relationships between the subcellular localization patterns of the MiT/TFE transcription factors and accelerated autophagy in SMNs, we introduced a frameshift mutation that resulted in premature termination in the MiT/TFE domain at the amino terminus of the tfeb, tfe3a, and tfe3b genes using the CRISPR-Cas9 system (tfeb-n115, tfe3a-n141 and tfe3b-n134, respectively). However, contrary to our expectations, the homozygous tfe3a-n141 mutation did not affect autophagic flux in CaPs or other neuronal cell types (Fig. 2c, d), indicating that nuclear enrichment of Tfe3a did not contribute to autophagy enhancement in CaPs. Consistent with the predominant cytoplasmic localization of Tfe3b-EGFP and Tfeb-EGFP, the homozygous tfe3b-n134 mutation or tfeb-n115 mutation did not cause a significant change in autophagic flux in CaPs (Fig. 2e–h). These observations collectively suggest that MiT/TFE transcription factors and the canonical ALP that they regulate play little to no role, if any, in accelerating autophagic flux in SMNs. We noted that heterozygous carriers of the tfe3b-n134 or tfeb-n115 mutations displayed a slight but significant acceleration of autophagic flux in CaPs, implying a dominant effect on the wild-type allele in this process. We also noticed that the homozygous and heterozygous tfeb-n115 mutations caused a significant acceleration of autophagic flux in RB cells and interneurons, respectively, suggesting neuronal type-specific roles of tfeb/TFEB. The precise mechanisms underlying these cell-type specific roles of MiT/TFE transcription factors remain to be determined.

The UPS is accelerated in large SMNs

To further explore why autophagic flux is enhanced in large SMNs, we investigated whether autophagy is particularly activated relative to other cellular degradation pathways. We specifically investigated the status of the ubiquitin-proteasome system (UPS), another major cellular degradation system. To this end, we developed a fluorescence-based reporter with a moderate half-life, EGFP-d410m, in which EGFP was tagged with a degron sequence (amino acids 410–461) from mouse ornithine decarboxylase carrying the T436A mutation, extending its half-life by twofold⁴⁷ (Fig. 3a, Supplementary Fig. 3a). EGFP-d410m-T436A was stoichiometrically expressed alongside the internal control mCherry through self-cleaving P2A peptide-dependent translation. When expressed using the Tg[SAIG213A] driver, the EGFP-d410m-T436A/mCherry ratio significantly increased following bath treatment with the proteasome inhibitor MG132 (Supplementary Fig. 3b), confirming that EGFP-d410m-T436A was indeed degraded via the UPS. We found that the EGFP-d410m-T436A/mCherry ratio was significantly lower in CaPs than in RB cells or interneurons (Fig. 3b, c), indicating that the UPS is intrinsically more active in SMNs than in other types of neuronal cells. Furthermore, among the SMNs, the EGFP-d410m-T436A/mCherry ratio significantly decreased as soma size increased (Fig. 3d, e), demonstrating that the UPS was significantly active in larger SMNs. Notably, under TDP-43 DKO conditions, the EGFP-d410m-T436A/mCherry ratio was significantly reduced in CaPs, as well as in RB cells and interneurons, indicating an acceleration of the UPS (Fig. 3c). We noted that TDP-43 disruption led to a reduction in the EGFP-d410m-T436A signal in most CaP cells. However, a subset of cells exhibited an increase, suggesting that the loss of TDP-43 function may differentially affect UPS activity, enhancing it in some cells and impairing it in others. Together, these results indicate that in SMNs, both autophagy and UPS are accelerated under normal and loss-of-TDP-43 function conditions, suggesting a general activation of intracellular degradation systems in large SMNs.

**Fig. 3: UPS and UPR activities in SMNs.**

Intrinsic propensity of large SMNs to accumulate misfolded proteins

A possible cause for the inherently and generally elevated cellular degradation activity in large SMNs could be an increased accumulation of degradation substrates, such as misfolded proteins. To test this possibility, we investigated the status of the unfolded protein response (UPR), a cellular stress response mechanism that is activated when misfolded or unfolded proteins accumulate in the endoplasmic reticulum (ER). The activity of the three UPR sensors located in the ER membrane (IRE1α, PERK, and ATF6) is modulated by different specific factors⁴⁸. We first investigated the activity of the IRE1α-Xbp1 pathway by constructing a UAS reporter line (Tg[UAS:Gxbp1uS]). In this reporter, EGFP and mScarlet were fused via an ER stress-dependent xbp1 intron of 26 base pairs (Fig. 3f). An EGFP-Xbp1-mScarlet fusion protein would be expressed when the stress-dependent xbp1 intron was removed by IRE1α-dependent unconventional splicing, leading to an increase of mScarlet/EGFP ratio^49,50. When driven by the pan-motor neuronal driver, Tg[mnr2b-hs:Gal4], the mScarlet/EGFP ratio was higher in the large somas (Fig. 3g, h), suggesting that larger SMNs experienced higher levels of ER stress than small SMNs under normal conditions. To determine whether the UPR activation is specific to the IRE1α-Xbp1 pathway, we also examined the status of the PERK-ATF4 pathway in the SMNs. Based on the principle of the SPOTlight reporter⁵¹, we established a UAS reporter line (Tg[UAS:zSPOTlight]) to monitor the PERK-ATF4 pathway activity in zebrafish. Briefly, Gal4-dependent transcripts encoding the green (EGFP) and red (mScarlet) fluorophores were translated in a mutually exclusive manner depending on the activation of the PERK-atf4a pathway (Fig. 3i). This regulation is mediated by the differential usage of upstream open reading frames (uORFs) in the 5’ untranslated region of Atf4 mRNA⁵², with mScarlet translation being enhanced under ER stress. When driven by the pan-motor neuronal driver, Tg[mnr2b-hs:Gal4], the mScarlet/EGFP ratio was significantly increased in the large SMNs (Fig. 3j, k). This indicates that the PERK-atf4a pathway was significantly active in large SMNs. Taken together, these results suggest that large SMNs inherently accumulate high levels of misfolded proteins in the ER, which may contribute to the global and inherent activation of cellular degradation systems.

Accelerated cellular degradation is neuroprotective in large SMNs

To investigate whether enhanced cellular degradation is beneficial or detrimental to SMNs, we examined the consequences of autophagy perturbation in SMNs. To selectively manipulate autophagy in CaP, we decided to perturb an ATG5-dependent pathway, as the conjugation of ATG12 to ATG5 promotes targeting of LC3 to autophagosome, thereby autophagy flux^53,54. We found that simultaneous expression of wild type ATG5 and GFP-LC3–RFP-LC3ΔG via bi-directional Gal4/UAS-mediated expression was sufficient to stabilize GFP-LC3 in RB cell and CaPs at 48 hpf, suggesting that wild type ATG5 overexpression exerts a dominant-negative effect on autophagy flux in these neuronal types (Fig. 4a–c). Furthermore, the K132R substitution, which disrupts ATG12-ATG5 conjugation, significantly enhanced the inhibitory effect on autophagy flux, particularly in interneurons and SMNs (Fig. 4c). Accordingly, we found that the total length of motor axons expressing wild-type ATG5 was reduced by an average of 7% and that the inhibitory effect was further exaggerated by the K132R mutation, resulting in a 24% reduction (Fig. 4d, e). Furthermore, CaP axons overexpressing ATG5^K132R were less ramified, although they still innervated their inherent target muscles in the ventral myotome. Based on these observations, we concluded that enhanced cellular degradation promotes motor axon outgrowth and is thus neuroprotective in large SMNs.

**Fig. 4: Autophagic acceleration is neuroprotective in large SMNs.**

Discussion

Through single-cell resolution scanning of autophagy, UPS, and UPR activities in the zebrafish spinal cord, we found that large SMNs inherently experience high catabolic stress and sustain the highest levels of cellular degradation activity in the spinal cord. These findings suggest that the junctions between the CNS and peripheral muscles are under significant catabolic stress even in healthy animals. This intrinsic vulnerability of the CNS-muscle connection may contribute to susceptibility to ALS in humans.

The intrinsically accelerated cellular degradation of large SMNs is a neuroprotective response to catabolic stress, as the disruption of ATG5-dependent autophagy leads to deficits in motor axon outgrowth. This finding aligns with a previous observation that motor neuron-specific ATG7 deletion in mice results in some denervated endplates in the tibialis anterior muscle, which are primarily innervated by fast-fatigable motor neurons²¹. One potential reason autophagy positively influences motor axon outgrowth is its role in active metabolic turnover. Large SMNs, including fast-fatigable motor neurons, exhibit an active cellular metabolism to sustain their connections with muscles, regulate contractions, and provide trophic support^38,55. These motor circuit functions are underpinned by the formation, maintenance, and elimination of synapses^{21,24,27,42,56}, which are high energy-demanding subcellular compartments⁵⁷. Thus, the impairment of autophagy-dependent metabolic turnover could disrupt synaptic development and function, ultimately halting axonal growth in motor neurons. Beyond the role of this process in metabolic control, our findings are also open to the possibility that autophagy has a specialized function in degrading unidentified key proteins that inhibit motor axon outgrowth.

The further amplification of the already elevated autophagy and UPS activity following the loss of TDP-43 function in large SMNs supports the idea that degradation substrates increase abnormally as TDP-43 function declines from the early phase through the progression of ALS, compelling SMNs to upregulate their degradation machinery. TDP-43 is an RNA-binding protein whose primary functions include RNA splicing. Recent studies have demonstrated that loss of TDP-43 function leads to the incorporation of cryptic exons into mature mRNA^{43,58,59,60,61}, resulting in the production of proteins with low folding fidelity⁴⁸. UPR imaging further indicated that misfolded protein accumulation is already evident under normal physiological conditions in large SMNs. Elevated ER stress has also been previously described in an SOD1-ALS model (SOD1-G93A mice)^62,63 and in patients with sporadic ALS⁶². Moreover, not only do misfolded proteins accumulate but also damaged organelles including mitochondria, owing to reduced TDP-43 function⁶⁴. Taken together, the increased degradation load from accumulated misfolded proteins and damaged organelles may be a common feature of both normal physiology and the pathophysiology of ALS-vulnerable neurons. In neurons that innately experience high degradation stress, risk factors affecting the cellular degradation systems, such as genetic mutations in autophagy and UPS-related components, may readily amplify proteostatic stress and reduce the threshold for degeneration⁶⁵. This type of vulnerability may share underlying mechanisms with the motor neurons that are lost during normal aging⁶. Notably, the accumulation of autophagosomes in SMNs has been described in sporadic ALS cases and animal models of ALS^18,19,20, implying that the impairment of autophagy may be an inevitable outcome of TDP-43 loss, as the expression of some autophagy genes can be influenced by TDP-43^44,58. In such cases, the upregulation of autophagy and the UPS likely represents an early response of SMNs to a decline in TDP-43 function, which serves as the first line of defense to maintain proteostasis. This defense mechanism may eventually become overwhelmed by the increasing catabolic load over time, as autophagy gradually declines owing to the loss of TDP-43 function. We observed a decline in the UPS activity in a small population of CaP cells in TDP-43 DKO fish, which may reflect the sequential bidirectional effect of TDP-43 loss. In these scenarios, mitigating catabolic stress may be more fundamental and effective in preventing disease initiation than merely enhancing the degradation machinery. Another non-exclusive possibility is that the further enhancement of intracellular degradation in SMNs due to TDP-43 DKO fish might be driven by certain pronounced non-cell-autonomous phenotypes, such as circulatory alterations and muscle degeneration observed upon TDP-43 loss⁶⁶.

A key question arising from the present study is why the catabolic load accumulates more in large SMNs than in small SMNs. Autophagy flux, UPS, and UPR were found to positively correlate with motor neuron soma size. However, it is noteworthy that the activity of these pathways shows considerable variability among large SMNs. These findings imply that although soma size, a canonical proxy for motor neuron cell size^21,67, shows a significant positive correlation with cellular degradation activity, it may not directly determine the level of this activity. While this study suggests that MiT/TFE transcription factors are unlikely to regulate basal levels of autophagic flux in SMNs, the upstream regulatory mechanisms of autophagic flux, UPS, and UPR in SMNs remain open questions. Future studies should aim to identify physiological parameters that more directly define the level of cellular degradation activity. In addition, long-term live imaging may help determine whether fluctuations in cellular degradation activity underlie this variability. Given that both the autophagy and the UPS are accelerated in large SMNs and can be further activated by the loss of TDP-43, dysfunction of the degradation system is unlikely to be the primary driver of degradation substrate accumulation. Instead, an increase in production may be a plausible explanation. SMNs are among the largest cell types in the body, allowing them to connect distant tissues, namely, the CNS and peripheral muscles, and transmit information over long distances. As a general characteristic of large-sized cells⁶⁸, high protein synthesis rates are assumed to increase the likelihood of misfolding, while oxidative stress from active metabolism disrupts protein folding, and a large cell volume dilutes protein quality control factors^69,70. Clarifying how these vulnerabilities intrinsic to large cells are compensated for large SMNs is crucial for understanding the proteostatic control of both innate and disease-induced catabolic loads. The mechanism also provides insights into the design of therapeutic strategies to protect large SMNs from degeneration by alleviating the burden of cellular degradation.

Even though this study benefits from the ability to monitor proteostasis dynamics with cellular resolution in real time within the intact CNS, the approach is limited to zebrafish at the larval stage, when their tissues are highly translucent, allowing non-invasive and wide-field optical access to the CNS. Given that aging is a risk factor for ALS, further technological advances are needed to analyze the dynamics of autophagic flux, UPS, and UPR across the adult spinal cord segment to further validate the findings of this study. Nonetheless, this study provides important in vivo evidence of the proteostatic stress experienced by motor neurons under physiological conditions, which may drive the transition to pathological states.

Methods

Animals

This study was performed in accordance with the Guide for the Care and Use of Laboratory Animals of the Institutional Animal Care and Use Committee (IACUC) of National Institute of Genetics (NIG, approval numbers: 30-15, 31-14, R2-6, R3-1, R4-7, R5-1, R6-9, R6-10, R6-22). All experimental protocols were approved by the IACUC of NIG. All authors complied with the ARRIVE guidelines. Wild-type and transgenic zebrafish, hybrids of the AB and Tübingen strains, were used in all experiments. The fish were raised at 28 °C under a 12-h light/12-h dark cycle (L/D) for the first five days post-fertilization. At these developmental stages, sex is not yet determined. Transgenic zebrafish lines used in this study were described in detail in Supplementary information.

Mutant fish lines used in this study

For the generation of tfeb knockout fish, target sequences for Cas9-mediated cleavage were searched by CRISPRscan⁷¹. The target sequences CGGTTTGAGCCTGGACCATGggg and TGGAGAGTGCATGTTCGGTGggg, where the protospacer adjacent motifs (PAMs) are indicated by lower cases, were chosen for the generation of tfeb-n115 allele. A mixture of these two crRNAs and Streptococcus pyogenes Cas9 Nuclease V3 (Alt-R^® CRISPR-Cas9 System, IDT) was injected into one-cell stage zebrafish embryos. For the generation of tfe3a knockout fish, a mixture of two crDNAs (CGGAGCCGGTTGGGGTCACAggg and GGGCTTTGCGGGCAGGTTGGagg) was used. For the generation of tfe3b knockout fish, a crRNA (TAATCTAGGCTCCAACAAAGagg) was used.

MG132 treatment

Zebrafish embryos at 30 hpf were treated with 100 µM MG132 or DMSO in E3 buffer at 28 ˚C for 18 hpf and analyzed by confocal microscopy at 48 hpf. The final concentration of DMSO was 1% in both treated and control conditions.

Microscopic analyses

All images were acquired from live fish embedded in 0.8–1% low-melting agarose (NuSieve GTG Agarose, Lonza) on a Glass Base dish (IWAKI, 3010-035) using an Olympus FV1200 laser confocal microscope with a 20x water immersion objective (NA1.0). For confocal imaging, the fish were raised in an embryonic buffer containing 0.003% (w/v) N-phenylthiourea (SIGMA, P7629) to inhibit melanogenesis. The hemicord at the level of spinal segments 13–16 was scanned, where the cloaca was typically located around the boundary between segments 16 and 17 along the rostrocaudal axis. To compare with ABNs, SMNs from spinal segments 1–5 were analyzed at 72 hpf. Confocal images were acquired as serial sections along the z-axis, analyzed using Olympus Fluoview Ver2.1b Viewer and Image J (Version 2.16.0/1.54p), and processed for presentation using Adobe Photoshop (Version 22.5.9) and Illustrator (Version 27.0). The axon lengths were measured by Imaris Filament Tracer (Imaris 9.2.0).

Measurement of soma size and fluorescence signal intensity

To measure soma size and fluorescence signal intensity, we first generated a maximum intensity projection from confocal z-stack images using ImageJ. We then outlined the soma using the Freehand Selection tool to define regions of interest (ROIs) and measured the area. Cells whose somatic boundaries could not be clearly distinguished due to overlap with neighboring cells were excluded from analysis. For fluorescence intensity measurements, we used the same ROIs (defined on the maximum projection) and applied them to the corresponding z-stacks created using the “Sum Slices” function in ImageJ.

Cell type classification

Tg[SAGFF213A] line specifically labels only CaP among SMNs at 48 hpf⁴². In addition, at 30 hpf, the primary motor neuron VaP, which is destined to undergo cell death by 48 hpf, is occasionally labeled alongside CaP. Because it is difficult to distinguish CaP from VaP somas prior to VaP cell death at 30 hpf, we excluded spinal hemisegments in which both CaP and VaP were present from the analysis. RB neurons were identified based on several distinguishing features, such as large, round somas, dorsal positioning of the cell body in the spinal cord, and the presence of both ascending and descending central axons. In spinal hemisegments where only CaPs were labeled as motor neurons, we identified the non-RB neurons as interneurons.

Statistics and reproducibility

Data for statistical analysis were obtained from at least three biologically independent larvae across at least three independent experiments. Statistical analyses were performed using GraphPad Prism Software (Version 10.4.1). First, the normality of each dataset within the groups to be compared was assessed using the Shapiro-Wilk test. If all datasets within the groups showed normal distribution, a t test was performed. If any dataset deviated from normality, the Mann–Whitney test was used. Statistical evaluation of correlations was performed using Pearson’s correlation analysis when the dataset followed a normal distribution, and Spearman’s rank correlation analysis when it deviated from normality. Exact p values are reported unless p < 0.0001, which is reported as p < 0.0001. Error bars indicate standard deviation in all figures.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

All data supporting the findings of this study are available within the article and Supplementary Information files. Source data are provided with this paper.

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Acknowledgements

We are grateful to Drs Noboru Mizushima and Hideaki Morishita for sharing the GFP-LC3-RFP-LC3∆G plasmid and instructing autophagic flux measurements and to Drs Haruhisa Inoue and Keiko Imamura for valuable discussions. This work was supported by The Nakabayashi Trust for ALS Research (K.A.), The Kato Memorial Trust For Nambyo Research (K.A.), Daiichi-Sankyo Foundation of Life Science (K.A.), Takeda Science Foundation (K.A.), SERIKA FUND (K.A.), ALS Foundation, Japan ALS Association (K.A.), KAKENHI Grant numbers JP22H04657 (K.A.), JP19K06933 (K.A.), JP22H02958 (K.A.), JP23H04266 (K.A.), JP21H02463 (K.K.), JP23H00375 (Y.S.) and AMED-PRIME under Grant Number JP23gm6410011h0003 (K.A.) and the National BioResource Project (NBRP) of the MEXT, Japan.

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Authors and Affiliations

Neurobiology and Pathology Laboratory, Department of Gene Function and Phenomics, National Institute of Genetics, Mishima, Shizuoka, Japan
Kazuhide Asakawa & Shinobu Shioya
The Graduate University for Advanced Studies (SOKENDAI), Department of Genetics, Mishima, Shizuoka, Japan
Kazuhide Asakawa & Koichi Kawakami
Division of Protein Metabolism, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
Takuya Tomita & Yasushi Saeki
Department of Molecular Pharmacology, Center for Future Medical Research, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan
Hiroshi Handa
Laboratory of Molecular and Developmental Biology, Department of Gene Function and Phenomics, National Institute of Genetics, Mishima, Shizuoka, Japan
Koichi Kawakami

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Kazuhide Asakawa
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Takuya Tomita
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Shinobu Shioya
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Hiroshi Handa
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Yasushi Saeki
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Koichi Kawakami
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Contributions

K.A. conceived and designed the research. K.A. and S.S. performed the experiments. T.T. and Y.S. provided the UPS probe. K.A., Y.S., T.T. and K.K. analyzed the data. K.A. and Y.S. wrote the manuscript with support from K.K. and H.H. All authors discussed the results and commented on the manuscript.

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Correspondence to Kazuhide Asakawa.

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Asakawa, K., Tomita, T., Shioya, S. et al. Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model. Nat Commun 16, 9213 (2025). https://doi.org/10.1038/s41467-025-65097-0

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Received: 11 March 2025
Accepted: 02 October 2025
Published: 27 October 2025
Version of record: 27 October 2025
DOI: https://doi.org/10.1038/s41467-025-65097-0