bims-axbals 2025-10-05 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–10–05
twenty-one papers selected by
TJ Krzystek

Generation of C9orf72 repeat knock-in iPSC lines for modelling ALS and FTD.
Integrated profiling of iPSC-derived motor neurons carrying C9orf72, FUS, TARDBP, or SOD1 mutations.
Symptomatic treatment by a BBB-permeable AAV engineered to restore TDP-43 function slows motor neuron disease and prevents paralysis.
Muscle-derived miR-126 regulates TDP-43 axonal local synthesis and NMJ integrity in ALS models.
Proteomic analysis of brain and spinal cord tissue reveals distinct immune and mitochondrial processes between human and mouse ALS models.
Neuronal Damage Induced by Gradual Oxidative Stress in iPSC-Derived Neurons: Implications for Ferroptosis Involvement and ALS Drug Evaluation.
MAPT-isoform 0N3R is essential for human brain development: Loss-of-function for novel TAU-associated disease paradigms.
Development of a Human iPSC-Derived "Corticospinal Tract-on-a-Chip" for Neurodegenerative Disease Research.
Detecting MUNC18-1 related presynaptic dysfunction and rescue in human iPSC-derived neurons.
Utilization of Induced Pluripotent Stem Cell-Derived Neurons to Investigate the Splice-Modification Efficacy of Splice-Switching Drug Candidates.
Dynamic changes in chromosome and nuclear architecture during maturation of normal and ALS C9orf72 motor neurons.
C9orf72 related poly-Glycine-Alanine promotes tau phosphorylation and cell death via ERK1/2 interaction in cellular models.
Intraneuronal Nanotracers in Axonal Transport.
Expansion of lysosomal capacity in early adult neurons driven by TFEB/HLH-30 protects dendrite maintenance during aging in Caenorhabditis elegans.
SCFFBXO21-mediated ubiquitination and degradation of NMNAT2 regulates axon survival in nerve injury.
Munc13-1 restoration mitigates presynaptic pathology in spinal muscular atrophy.
Translation landscape of stress granules.
Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites.
Structural and molecular differentiation of cultured human neurons is accompanied by alterations of spontaneous and evoked calcium dynamics.
Establishment of human Leber's hereditary optic neuropathy model using iPSC-derived retinal organoids.
Uncovering cargo clients and accessory factors of AP-1 and AP-4 through vesicle proteomics.

bioRxiv. 2025 Feb 11. pii: 2025.02.10.637041. [Epub ahead of print]

Generation of C9orf72 repeat knock-in iPSC lines for modelling ALS and FTD.

Rachel Coneys, Alexander J Cammack, Remya R Nair, David Thompson, Jonas Mechtersheimer, Mireia Carcolé, Yashica Gupta, Gabriel E Rech, Michael Flower, Niamh O'Brien, Marc-David Ruepp, Sarah Mizielinska, Fiona Ducotterd, Sarah J Tabrizi, Elizabeth M C Fisher, Thomas J Cunningham, Michael Ward, William C Skarnes, Adrian M Isaacs.

Induced pluripotent stem cell (iPSC) models are powerful tools for neurodegenerative disease modelling, as they allow mechanistic studies in a human genetic environment and they can be differentiated into a range of neuronal and non-neuronal cells. However, these models come with inherent challenges due to line-to-line and clonal variability. To combat this issue, the iPSC Neurodegenerative Disease Initiative (iNDI) has generated an iPSC repository using a single clonal reference line, KOLF2.1J, into which disease-causing mutations and revertants are introduced via gene editing. Here we describe the generation and validation of lines carrying the most common causative mutation for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), a repeat expansion in the C9orf72 gene, for the iNDI collection of neurodegenerative iPSC models. We demonstrate that these C9orf72 knock-in lines differentiate efficiently into neurons and display characteristic C9orf72 -associated pathologies, including reduced C9orf72 levels and the presence of dipeptide repeat proteins (DPRs) and RNA foci, which increase in abundance over time in culture. These pathologies are not present in revertant cells lacking the repeat expansion. These repeat expansion and revertant cell lines are now available to academic and for-profit institutions through the JAX iPS cell repository and will help to facilitate and standardise iPSC-based ALS/FTD research.

DOI: https://doi.org/10.1101/2025.02.10.637041
Stem Cell Reports. 2025 Oct 02. pii: S2213-6711(25)00253-X. [Epub ahead of print] 102649

Integrated profiling of iPSC-derived motor neurons carrying C9orf72, FUS, TARDBP, or SOD1 mutations.

Guo-Ming Ma, Cong-Cong Xia, Bo-Yu Lyu, Jie Liu, Fang Luo, Ming-Feng Guan, Jun-Ying Wang, Li Sun, Lin Zhang, Yan Chen, Ying-Wei Mao, Guo-Qiang Yu, Wen-Yuan Wang.

  Here, we conducted temporal RNA sequencing (RNA-seq) profiling of human induced pluripotent stem cells (hiPSCs) and induced pluripotent stem cell (iPSC)-derived motor neurons (iMNs) carrying C9orf72, FUS, TARDBP, or SOD1 mutations in both patients with amyotrophic lateral sclerosis (ALS) and healthy individuals. We discovered dysregulated gene expression and alternative splicing (AS) throughout iMN development and maturation, and iMNs with mutations in ALS-associated genes displayed enrichment of cytoskeletal defects and synaptic alterations from the premature stage to mature iMNs. Our findings indicate that synaptic gene dysfunction is a common molecular hallmark of familial ALS, which may result in neuronal susceptibility and progressive motor neuron degeneration. Analysis of upstream splicing factors revealed that differentially expressed RNA-binding proteins (RBPs) in iMNs from patients with ALS may cause abnormal AS events. Overall, our research provides a comprehensive and valuable resource for gaining insights into the shared mechanisms of familial ALS pathogenesis during motor neuron development and maturation in iMN models.

Keywords:  ALS; RNA sequencing; gene expression alterations; synaptic dysfunction

DOI:  https://doi.org/10.1016/j.stemcr.2025.102649
bioRxiv. 2025 Aug 20. pii: 2025.08.14.670400. [Epub ahead of print]

Symptomatic treatment by a BBB-permeable AAV engineered to restore TDP-43 function slows motor neuron disease and prevents paralysis.

Aswathy Peethambaran Mallika, Jessica G Yu, Opal Sitzman, Meghraj Singh Baghel, Shruti Renganathan, Irika R Sinha, Tatiana Melnikova, Jonathan P Ling, Philip C Wong.

TAR DNA-binding protein 43kDa (TDP-43) dysfunction is an early pathogenic mechanism that underlies amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disorder that lacks disease modifying therapies. We previously developed a mouse model in which TDP-43 is selectively deleted from motor neurons ( ChAT-Cre;Tardbp f/f ) that mimics the early stages of ALS. Here, we demonstrate that intravenous delivery of a blood-brain-barrier (BBB) permeable AAV capsid expressing our rationally designed splicing repressor CTR (AAV-PHP.eB-CTR) in symptomatic ChAT-Cre;Tardbp f/f mice markedly slowed disease progression and prevented paralysis. Systemic delivery of AAV-PHP.eB-CTR led to transduction of ∼80% of spinal motor neurons, repression of TDP-43-associated cryptic exons within motor neurons expressing CTR, and attenuation of motor neuron loss. Notably, the addition of the TARDBP 3'UTR autoregulatory element to CTR maintained its expression within a physiological range. In control littermates that received AAV-PHP.eB-CTR and were monitored for >20 months, grip strength and body weight remained normal, and no histopathological abnormalities were observed, underscoring a favorable safety profile for this gene therapy. These results provide preclinical proof-of-concept that BBB-crossing AAV delivery of CTR can rescue motor neuron disease through the restoration of TDP-43 function, offering a promising mechanism-based therapeutic strategy for ALS.

DOI: https://doi.org/10.1101/2025.08.14.670400
Nat Neurosci. 2025 Oct 03.

Muscle-derived miR-126 regulates TDP-43 axonal local synthesis and NMJ integrity in ALS models.

Ariel Ionescu, Lior Ankol, Anand Ganapathy Subramaniam, Topaz Altman, Iddo Magen, Yahel Cohen, Yehuda Danino, Tal Gradus-Pery, Yoav Niv, Ori Bar Avi, Danielle Geller, Amjd Ibraheem, Ruilei Cheng, Noam Steinberg, Leenor Alfahel, Pauline Duc, Zeynep Ergul-Ulger, Doruk Arslan, Ersin Tan, Florence Rage, Nilo Riva, Angello Quattrini, Can Ebru Bekircan-Kurt, Adrian Israelson, Amir Dori, Eran Hornstein, Eran Perlson.

Amyotrophic lateral sclerosis (ALS) is characterized by neuromuscular junction (NMJ) disruption and neurodegeneration. Recent findings highlight a pivotal role for TAR DNA-binding protein 43 (TDP-43) in forming axonal pathological condensates and facilitating NMJ disruption through inhibition of local protein synthesis. However, the mechanisms that drive local TDP-43 accumulation remain unknown. Here we identify that the TDP-43 axonal accumulation in peripheral nerves of SOD1 patients and mice stems from its aberrant local synthesis. This is a non-cell-autonomous process driven by muscle-derived miR-126a-5p extracellular vesicles (EVs). Inhibiting muscle secretion of miR-126a-5p prompts presynaptic TDP-43 synthesis and accumulation, which disrupts axonal translation and causes NMJ degeneration. Introducing miR-126 to SOD1G93A mice, primary co-cultures and human induced pluripotent stem cell (iPSC)-derived co-cultures with ALS mutations exhibits neuroprotective effects and delays motor decline. These findings identify a transcellular communication axis between muscles and motor neurons that regulates axonal local synthesis and NMJ maintenance, offering insights into ALS onset and progression.

DOI: https://doi.org/10.1038/s41593-025-02062-6
Sci Rep. 2025 Sep 30. 15(1): 33959

Proteomic analysis of brain and spinal cord tissue reveals distinct immune and mitochondrial processes between human and mouse ALS models.

Alanna G Spiteri, Joel R Steele, Han-Chung Lee, Haijian Zhang, Jiaqi Sun, Ralf B Schittenhelm, Chien-Hsiung Yu, Catriona McLean, Colin L Masters, Benjamin Goudey, Liang Jin, Yijun Pan.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease resulting in the progressive loss of motor neurons in the brain and spine. More than 95% of cases are pathologically characterized by the cytoplasmic accumulation of hyperphosphorylated and ubiquitinated transactive response DNA-binding protein 43 (TDP-43). Multiple mouse models with TDP-43 accumulation have been developed, however, whether they recapitulate molecular features of ALS pathology is unclear. Given the lack of curative treatment for ALS, there is an urgent need to identify the precise biological processes contributing to disease pathogenesis for the development of effective therapeutic treatments. Thus, in this study we employed label-based untargeted proteomics to characterize the ALS proteome and related biological processes in the spinal cord and brain of TDP-43Q331K mice, a transgenic mouse model of ALS and the motor cortex and the cervical, thoracic, and lumbar spinal cord regions from humans. In humans, we observed highly overlapping responses across the four tissues examined, primarily related to the upregulation of immune processes and the downregulation of mitochondrial function. In contrast, TDP-43Q331K mice demonstrate a lack of enrichment for immune activation and the opposite regulation of mitochondrial processes. A meta-analysis of previously published mouse datasets identified the Ubqln2 knock-out mouse model as showing stronger parallels with our late-stage human ALS. Overall, this study provides in-depth analysis of the site-specific dysregulated proteomes and their associated functional processes across species. Thereby, identifying potential therapeutic targets while emphasizing the limitations of specific mouse models at certain timepoints in recapitulating ALS-related processes for future model development.

Keywords:  Amyotrophic lateral sclerosis; Immune-mediated pathology; Mitochondrial dysfunction; Motor neurone disease; Neurodegeneration; TMT-proteomics

DOI:  https://doi.org/10.1038/s41598-025-11466-0
J Neurochem. 2025 Oct;169(10): e70246

Neuronal Damage Induced by Gradual Oxidative Stress in iPSC-Derived Neurons: Implications for Ferroptosis Involvement and ALS Drug Evaluation.

Hayato Kobayashi, Hitoshi Suzuki-Masuyama, Hirokazu Tanabe, Hiroshi Kato, Setsu Endoh-Yamagami.

  The molecular mechanisms underlying neurodegenerative diseases are not fully understood, but oxidative stress is known to play a central role in the pathogenesis of neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and Alzheimer's disease (AD). In this study, we developed a method to induce gradual oxidative stress in induced pluripotent stem cell (iPSC)-derived motor neurons and cortical excitatory neurons by omitting antioxidants in the media, aiming to create a platform for studying oxidative stress-dependent neuronal damage in neurodegenerative diseases. Neuroprotective effects in this platform were observed with edaravone, an approved ALS medicine, in iPSC-derived motor neurons, suggesting its potential for ALS drug evaluation. The oxidative stress-induced neuronal damage was accompanied by increased lipid peroxidation, and it was suppressed by ferroptosis inhibitors and an iron-specific chelator, suggesting that neurons died through ferroptosis. Furthermore, through a compound screen, a cholesterol biosynthesis inhibitor, AY 9944, was identified as being capable of inhibiting neuronal damage induced by oxidative stress. Additionally, neuroprotective activity was observed with 7-dehydrocholesterol, an immediate precursor of cholesterol, while the efficacy of AY 9944 was compromised by knockout of the EBP gene, which encodes an enzyme involved in cholesterol biosynthesis. These findings suggest the involvement of ferroptosis in the progression of neurodegenerative diseases and the inhibition of ferroptosis by modulating the cholesterol biosynthesis pathway, providing potential insights for drug development.

Keywords:  ALS; cholesterol; edaravone; ferroptosis; motor neurons; oxidative stress

DOI:  https://doi.org/10.1111/jnc.70246
Neural Regen Res. 2025 Sep 29.

MAPT-isoform 0N3R is essential for human brain development: Loss-of-function for novel TAU-associated disease paradigms.

Hans Zempel.

   ABSTRACT: TAU, a microtubule-associated protein, encoded by the microtubule-associated protein tau (MAPT) gene, is a central regulator of microtubule stability and axonal function in the human brain, with its pathological aggregation representing a hallmark of Alzheimer's disease and related tauopathies. Despite extensive research into the role of TAU in neurodegeneration, its essentiality for human brain development has remained unclear. This perspective synthesizes recent genetic, molecular, and cellular evidence to demonstrate that the human brain-specific TAU isoform 0N3R is indispensable for proper neurodevelopment, pointing to loss-of-function of this isoform as a novel paradigm for TAU-associated disease. Alternative splicing of MAPT generates six brain-specific TAU isoforms, with 0N3R being exclusively expressed during fetal brain development. Analysis of large-scale human genetic datasets (gnomAD v4.0.0) reveals a high probability of loss-of-function intolerance (pLI = 0.96) for the 0N3R isoform. This is in stark contrast to the canonical Matched Annotation from the NCBI and EMBL-EBI (MANE) transcript and peripheral "Big TAU," both of which are tolerant to loss-offunction mutations. This intolerance is further supported by the scarcity of loss-of-function mutations in 0N3R-encoding exons and high missense constraint scores, suggesting strong evolutionary selection against disruption of this isoform. Functional studies using human induced pluripotent stem cell-derived cortical neurons with CRISPR-Cas9-mediated MAPT knockout reveal that, unlike in murine models where compensation by other microtubule-associated proteins occurs, loss of TAU in human neurons leads to deficits in neurite outgrowth, axon initial segment shortening, and a trend toward hyperexcitability, accompanied by broad transcriptomic changes affecting genes involved in microtubule organization and synaptic structure. Remarkably, re-expression of any of the six human brain-specific TAU isoforms rescues these phenotypes, underscoring their functional redundancy during development. These findings position the 0N3R isoform as essential for human brain development and suggest that loss-of-function mutations affecting this isoform likely result in neurodevelopmental impairment, potentially manifesting as intellectual disability without overt dysmorphic features. This contrasts with the apparent tolerance to MAPT loss-of-function in mice and peripheral tissues, highlighting a critical species- and isoform-specific requirement for TAU in human neurodevelopment. The hypothesis of 0N3R-TAU loss-of-function intolerance opens new avenues for understanding neurodevelopmental disorders and refines the conceptual framework of TAUassociated disease mechanisms beyond toxic gain-of-function.

Keywords:  0N3R isoform; Alzheimer’s disease; TAU protein; alternative splicing; intellectual disability; neurodevelopmental disorders; tauopathy

DOI:  https://doi.org/10.4103/NRR.NRR-D-25-00298
bioRxiv. 2025 Sep 25. pii: 2025.09.24.678092. [Epub ahead of print]

Development of a Human iPSC-Derived "Corticospinal Tract-on-a-Chip" for Neurodegenerative Disease Research.

Andriana Charalampopoulou, Arens Taga, Khalil Rust, Evelyn Luciani, Katherine Marshall, Elliot Montgomery, Anuradha Mansinghka, Richa Singh, Yang Zhao, Christine O'Keefe, Tza-Huei Wang, Arun Venkatesan, Christa W Habela, Nicholas J Maragakis.

Degeneration of the corticospinal tract is a central feature in a number of neurodegenerative disorders and leads to significant disability. However, modeling corticospinal neuron (CSN) pathology and corticospinal connectivity in neurological disorders is particularly challenging. While rodent models are important for understanding early degeneration of CSN, interspecies differences in corticospinal connectivity and challenges of in vivo study suggest that human in vitro models of corticospinal biology may be ripe for development. Human induced pluripotent stem cells (hiPSC) are promising tools for overcoming intrinsic limitations that arise from physiological differences between rodents and humans. We have developed an innovative hiPSC-based microfluidic platform for modeling human CSN and spinal motor neuron (SpMN) connectivity. The incorporation of regionally specific astrocyte subtypes (cortical and spinal) in addition to CSNs and SpMNs in this newly designed system allows for the modeling of both regional and neural cell-subtype interactions. Using this model, multielectrode array electrophysiology reveals the maturation of both cortical and spinal motor neurons over the time course of 12 weeks. Retrograde labeling methods demonstrate synaptic connectivity between corticospinal and spinal motor neurons. Optogenetic strategies to selectively activate excitatory CNs attenuated by glutamate receptor antagonism confirms the functional relevance of the model. Incorporating morphological, electrophysiological and physiological measures of corticospinal connectivity, this platform is a versatile model for use in neurodegenerative disease research and for the future development of targeted CSN therapies.
Significance Statement: Degeneration of the corticospinal tract is a key feature of numerous neurodegenerative diseases, yet current in vitro models lack the anatomical and functional fidelity to study this system. We developed a human iPSC-derived "Corticospinal Tract-on-a-Chip" using a multielectrode array platform that incorporates regionally patterned cortical and spinal neurons and astrocytes. This model demonstrates structural and functional synaptic connectivity and enables longitudinal electrophysiological recordings. Critically, it supports compartment-specific manipulation and real-time analysis of CST network dynamics, capabilities lacking in existing systems. By mimicking human corticospinal physiology in vitro , this platform offers a novel tool for mechanistic investigation and preclinical testing of CST-targeted therapies. It holds broad relevance for studying disorders such as ALS, hereditary spastic paraplegia, and primary lateral sclerosis.

DOI: https://doi.org/10.1101/2025.09.24.678092
Sci Rep. 2025 Sep 30. 15(1): 33958

Detecting MUNC18-1 related presynaptic dysfunction and rescue in human iPSC-derived neurons.

Manzhao Long, Nicholas B Gallo, Jennifer Zoll, Sharanya Unnikrishnan, Edward Guilmette, Davide Gianni, Sijia Wu, Christopher A Hinckley.

Human induced pluripotent stem cell (hiPSC) derived neurons are powerful tools to model disease biology in the drug development space. Here we leveraged a spectrum of neurophysiological tools to characterize iPSC-derived NGN2 neurons. Specifically, we applied these technologies to detect phenotypes associated with presynaptic dysfunction and rescue in NGN2 neurons lacking a synaptic vesicle associated protein MUNC18-1, encoded by syntaxin binding protein 1 gene (STXBP1). STXBP1 homozygous knock out NGN2 neurons lacked miniature post synaptic currents and demonstrated disrupted network bursting as assayed with multielectrode array and calcium imaging. Furthermore, knock out neurons released less glutamate into culture media, consistent with a presynaptic deficit. These synaptic phenotypes were rescued by reconstitution of STXBP1 protein by AAV transduction in a dose-dependent manner. Our results identify a complementary suite of physiological methods suitable to examine the modulation of synaptic transmission in human neurons.

DOI: https://doi.org/10.1038/s41598-025-11059-x
Methods Mol Biol. 2026 ;2963 173-184

Utilization of Induced Pluripotent Stem Cell-Derived Neurons to Investigate the Splice-Modification Efficacy of Splice-Switching Drug Candidates.

Yukiko Ueta, Hiroaki Ohara, Tomonari Awaya, Masatoshi Hagiwara.

  Modification of RNA splicing represents a promising therapeutic strategy for treating genetic diseases. Several splice-switching drugs have already been approved, notably for spinal muscular atrophy and specific genotypes of Duchenne muscular dystrophy. While peripheral blood mononuclear cells and fibroblasts are commonly used to evaluate the efficacy of these drug candidates in patient-derived samples, such approaches may lead to misleading conclusions due to the cell type-specific nature of RNA splicing.Induced pluripotent stem cells (iPSCs), which can be differentiated into various somatic cell types, offer a valuable tool to overcome this limitation. Once efficient differentiation protocols are established, iPSCs can give rise to neurons, retinal cells, cardiomyocytes, and other hard-to-obtain cell types. In this chapter, we describe a protocol for evaluating the splice-modification efficacy of drug candidates in iPSC-derived neurons, using the removal of a poison exon in SCN1A gene, associated with Dravet syndrome as a model.

Keywords:  Antisense oligonucleotides; Functional validation of splicing correction; Induced pluripotent stem cells (iPSCs); RNA splicing; Small-molecule compounds; Splice-switching oligonucleotides (SSOs)

DOI:  https://doi.org/10.1007/978-1-0716-4738-7_12
bioRxiv. 2025 Sep 22. pii: 2025.09.22.677835. [Epub ahead of print]

Dynamic changes in chromosome and nuclear architecture during maturation of normal and ALS C9orf72 motor neurons.

Özgün Uyan, Snehal Sambare, Marlies E Oomen, Nicholas Wightman, Allana Schooley, Joseph R Klim, Houda Belaghzal, Özkan Aydemir, Betul Akgol-Oksuz, Zeynep Sena Agim Uslu, Kevin Eggan, Robert H Brown, Job Dekker.

We have investigated changes in chromosome conformation, nuclear organization, and transcription during differentiation and maturation of control and mutant motor neurons harboring hexanucleotide expansions in the C9orf72 gene that cause amyotrophic lateral sclerosis (ALS). Using an in vitro reprogramming, differentiation and neural maturation protocol, we obtained highly purified populations of post-mitotic motor neurons for both normal and diseased cells. As expected, as fibroblasts are reprogrammed into iPSCs, and as iPSCs differentiate into motor neurons, chromatin accessibility, chromosome conformation, and nuclear organization change along with large-scale alterations in transcriptional profiles. We find that the transcriptome changes extensively during the first three weeks of post-mitotic neuronal maturation, with thousands of genes changing expression, but then is relatively stable for the next three weeks. In contrast, chromosome conformation and nuclear organization continue to change over the entire 6-week maturation period: chromosome territoriality increases, long-range interactions along chromosomes decrease, compartmentalization strength increases, and centromeres and telomeres increasingly cluster. In motor neurons derived from ALS patients such changes in chromosome conformation were much reduced. Chromatin accessibility changes also showed delayed maturation. The transcriptome in these cells matured relatively normally but with notable changes in expression of genes involved in lipid, sterol and mitochondrial function. We conclude that neural maturation is associated with large scale post-mitotic changes in gene expression, chromosome conformation and nuclear organization, and that these processes are defective in motor neurons derived from ALS patients carrying C9orf72 hexanucleotide repeat expansions.

DOI: https://doi.org/10.1101/2025.09.22.677835
Neuroscience. 2025 Oct 01. pii: S0306-4522(25)00983-2. [Epub ahead of print]

C9orf72 related poly-Glycine-Alanine promotes tau phosphorylation and cell death via ERK1/2 interaction in cellular models.

Jiahan Zhuang, Zixuan Zhang, Hongfu Jin, Ji Qi, Yuanyuan Chen, Lin Ding, Chenglai Fu, Weiwei Cheng.

  Frontotemporal lobar degeneration (FTLD), particularly cases linked to the C9ORF72 GGGGCC repeat expansion (r(G4C2)exp), is closely associated with TAR DNA-binding protein 43 (TDP-43) pathology but also exhibits concurrent tau pathology characterized by hyperphosphorylation and neurofibrillary tangles (NFTs). Despite evidence suggesting heightened tau pathology severity in C9ORF72 mutation carriers compared to other FTLD subtypes, the mechanistic interplay between r(G4C2)exp and tau dysregulation remains poorly understood. Using a cellular model, we demonstrated that (GA)50 causes significant neuronal cell death. We found that (GA)50 was shown to specifically bind to extracellular-regulated kinase 1/2 (ERK1/2) protein, leading to its hyperphosphorylation. This activation of ERK1/2 was associated with increased tau phosphorylation and aggregation. Importantly, inhibiting ERK1/2 activity with U0126 significantly reduced tau phosphorylation, aggregation, and cell death in cells overexpressing (GA)50. These in vitro findings suggest that (GA)50-driven ERK1/2 hyperphosphorylation may represent potential driver of tau pathology in C9ORF72-related FTLD, highlighting the ERK1/2 signaling or its interaction with poly-glycine-alanine (GA) as a potential therapeutic target.

Keywords:  C9ORF72 ALS/FTLD; ERK1/2; Tau aggregation; Tau phosphorylation; poly-Glycine-Alanine

DOI:  https://doi.org/10.1016/j.neuroscience.2025.09.053
ACS Nano. 2025 Sep 30.

Intraneuronal Nanotracers in Axonal Transport.

Zhigao Yi, Angelo H All, Yuxia Liu, Xiaogang Liu.

  Axonal transport is fundamental to neuronal function, ensuring the movement of organelles, proteins, and other cellular components necessary for growth, signaling, and repair. Disruptions in this transport system are implicated in various neurodegenerative and neurodevelopmental disorders. Recent advances in tracking methods, both in vivo and in vitro, have deepened our understanding of transport defects, their molecular mechanisms, and their potential applications in diagnostics and therapeutics. This review discusses key scientific and technical breakthroughs in axonal transport visualization, revisiting the core principles of transport machinery. We examine critical components involved in axonal transport, including microtubules, motor proteins and their adaptors, and organelle cargoes. Additionally, we highlight recent developments in nanomaterial-based tracing methods, present notable examples, and outline future challenges and research directions.

Keywords:  axonal tracking; cargo transport; microscopic imaging; microtubule; motor protein; nanotracer; neurodegenerative disease; photoluminescence; upconversion nanoparticle

DOI:  https://doi.org/10.1021/acsnano.5c13559
PLoS Biol. 2025 Sep 30. 23(9): e3002957

Expansion of lysosomal capacity in early adult neurons driven by TFEB/HLH-30 protects dendrite maintenance during aging in Caenorhabditis elegans.

Ruiling Zhong, Claire E Richardson.

Lysosomes are essential for neuronal homeostasis, providing degradation and recycling functions necessary to support neurons' complex operations and long lifespans. However, the regulation of lysosomal degradative capacity in healthy neurons is poorly understood. Here, we investigate the role of HLH-30, the sole Caenorhabditis elegans homolog of Transcription Factor EB (TFEB), a master regulator of lysosome biogenesis and autophagy that is thought to predominantly function in the context of starvation or stress. We demonstrate that HLH-30 is dispensable for neuronal development but acts cell-intrinsically to expand lysosomal degradative capacity during early adulthood. Loss of HLH-30 leads to lysosomal dysfunction and delayed turnover of synaptic vesicle proteins from the synapse. Notably, we show that basal HLH-30 activity is sufficient to expand neuronal lysosomal capacity without nuclear enrichment, in contrast to the nuclear translocation associated with starvation- and stress-induced activation of TFEB and HLH-30. Furthermore, we show that neuronal lysosomal function declines with age in wild-type animals, and this corresponds to a decrease in basal HLH-30-mediated transcription. We further demonstrate that basal HLH-30 activity is crucial for neuron maintenance: lysosomal dysfunction due to inadequate HLH-30 activity leads to dendrite degeneration and aberrant outgrowths. In summary, our study establishes a critical role for HLH-30/TFEB in promoting lysosomal capacity to preserve neuronal homeostasis and structural integrity of mature neurons in vivo.

DOI: https://doi.org/10.1371/journal.pbio.3002957
J Cell Biol. 2025 Nov 03. pii: e202501072. [Epub ahead of print]224(11):

SCFFBXO21-mediated ubiquitination and degradation of NMNAT2 regulates axon survival in nerve injury.

Wenjing Long, Shunyi Li, Qiangqiang Wang, Wenkai Yue, Yanbin Fu, Haiqiong Wang, Mingsheng Jiang, Xianyan Hu, Yunxia Li, Jihong Cui, Ang Li, Yaoyang Zhang, Zairong Zhang, Yanshan Fang.

NMNAT2 is an essential but labile protein required for axon integrity. It is rapidly degraded after nerve injury, promoting axon degeneration. However, the mechanisms regulating NMNAT2 ubiquitination and turnover in neurons remain unclear. In this study, we identify the F-box protein FBXO21 as an NMNAT2-binding protein, and its deficiency confers axonal protection via increasing NMNAT2 abundance. FBXO21 recruits SKP1, CUL1, and RBX1 to form an SCFFBXO21 complex, which promotes NMNAT2 ubiquitination in vivo and in vitro. SCFFBXO21 ubiquitinates NMNAT2 at K155 within an isoform-specific targeting and interaction domain of the family of NMNATs, which underlies the unique labile nature of NMNAT2. The ubiquitination-deficient NMNAT2-K155R exhibits substantially reduced protein turnover and enhanced axon-protective capacity. Finally, in Fbxo21 knockout mice, NMNAT2 levels are markedly increased and the survival of injured sciatic nerves is significantly prolonged. Collectively, our findings reveal a crucial role of FBXO21 in axon degeneration, highlighting the SCFFBXO21 complex as a potential target for modulating NMNAT2-dependent axon survival.

DOI: https://doi.org/10.1083/jcb.202501072
Nat Commun. 2025 Sep 30. 16(1): 8724

Munc13-1 restoration mitigates presynaptic pathology in spinal muscular atrophy.

Mehri Moradi, Julia Weingart, Chunchu Deng, Mahoor Nasouti, Michael Briese, Sibylle Jablonka, Markus Sauer, Michael Sendtner.

Degeneration of neuromuscular synapses is a key pathological feature of spinal muscular atrophy (SMA), yet cellular mechanisms underlying synapse dysfunction remain elusive. Here, we show that pharmacological stimulation with Roscovitine triggers the assembly of Munc13-1 release sites that relies on its local translation. Our findings show that presynaptic mRNA levels and local synthesis of Munc13-1 are diminished in motoneurons from SMA mice and hiPSC-derived motoneurons from SMA patients. Replacement of the Munc13-1 3'UTR with that of Synaptophysin1 rescues Munc13-1 mRNA transport in SMA motoneurons and restores the nanoscale architecture of presynaptic Munc13-1 release sites. Restoration of Munc13-1 levels leads to functional synaptic recovery in cultured SMA motoneurons. Furthermore, SMA mice cross-bred with a conditional knock-in mouse expressing modified Munc13-1 with a heterologous 3'UTR display attenuated synapse and neurodegeneration and improved motor function. Identifying Munc13-1 as an SMA modifier underscores the potential of targeting synapses to mitigate neuromuscular dysfunction in SMA.

DOI: https://doi.org/10.1038/s41467-025-64164-w
Sci Adv. 2025 Oct 03. 11(40): eady6859

Translation landscape of stress granules.

Yichun Wu, Xing Wang, Lingyu Meng, Zhizhao Liao, Wei Ji, Peipei Zhang, Jie Lin, Qiang Guo.

Stress granules, cytoplasmic assemblies of RNA binding proteins and messenger RNAs formed during cellular stress, are implicated in translational control. However, their exact functions remain elusive. Here, we used cryogenic correlative light and electron microscopy to visualize stress granules in their native environment and reconstructed them in three dimensions using tomography. This approach provided the first quantitative and spatial analysis of the translational machinery within stress granules. Our findings suggest that stress granules have a limited impact on global translation regulation but serve to protect small ribosomal subunits and preinitiation complexes from degradation. Numerical simulations based on a phase-field model accurately reproduced the spatial distribution of ribosomal components inside and outside the stress granules, shedding light on the thermodynamic principles governing this process.

DOI: https://doi.org/10.1126/sciadv.ady6859
bioRxiv. 2025 Sep 26. pii: 2025.09.25.678303. [Epub ahead of print]

Atlas of Lysosomal Aging Reveals a Molecular Clock of Storage Disorder-Associated Metabolites.

Anna M Puszynska, Thao P Nguyen, Andrew L Cangelosi, Andrea Armani, Justin M Roberts, Kristin A Singh, James C Cameron, Tenzin Tseyang, Grace Y Liu, Steven Lai, Hans-Georg Sprenger, Jason Yang, William N Colgan, Jibril F Kedir, Kathrin M Kajderowicz, Theodore K Esantsi, Yuancheng Ryan Lu, Millenia Waite, Tenzin Kunchok, Caroline A Lewis, Fabian Schulte, George W Bell, David M Sabatini, Jonathan S Weissman.

Lysosomal dysfunction is a well-recognized feature of aging, yet its systematic molecular investigation remains limited. Here, we employ a suite of tools for rapid lysosomal isolation to construct a multi-tissue atlas of the metabolite changes that murine lysosomes undergo during aging. Aged lysosomes in brain, heart, muscle and adipose accumulate glycerophosphodiesters and cystine, metabolites that are causally linked to juvenile lysosomal storage disorders like Batten disease. Levels of these metabolites increase linearly with age, preceding organismal decline. Caloric restriction, a lifespan-extending intervention, mitigates these changes in the heart but not the brain. Our findings link lysosomal storage disorders to aging-related dysfunction, uncover a metabolic lysosomal "aging clock," and open avenues for the mechanistic investigation of how lysosomal functions deteriorate during aging and in age-associated diseases.
One-Sentence Summary: Aging in mice is tracked by a lysosomal "clock", where glycerophosphodiesters and cystine - metabolites causally linked to juvenile lysosomal storage disorders - gradually accumulate in lysosomes of the brain, heart, skeletal muscle and adipose tissue.

DOI: https://doi.org/10.1101/2025.09.25.678303
Sci Rep. 2025 Oct 01. 15(1): 34196

Structural and molecular differentiation of cultured human neurons is accompanied by alterations of spontaneous and evoked calcium dynamics.

Deepika Negi, Susan Shorter, Iain Goodhall, Daniel Razansky, Sukhi Shergil, Saak V Ovsepian.

  During development, neuronal precursors transform from a pluripotent state into specialized neurons. While much research has been conducted into morphological and molecular changes, there is a pressing need to define accompanying functional alterations. We used immunofluorescence microscopy and live imaging in SH-SY5Y-derived human neurons to elucidate the relationship between structural and molecular differentiation with evoked and spontaneous Ca2+ dynamics. In the undifferentiated state expressing trace amounts of neuronal markers, SH-SY5Y cells maintain spontaneous high-amplitude slow Ca2+ oscillations, with their stimulation by carbochol activating low-amplitude Ca2+ transients. Driving SH-SY5Y cells into the 2CL state by retinoic acid facilitated the outgrowth of neurites and expression of neuron-specific proteins. These changes are accompanied by the abolition of Ca2+ oscillations. Differentiating SH-SY5Y cells into definitive neurons by a cocktail of retinoic acid and BDNF induced their polarization and enrichment with specific neuronal markers, accompanied by a resurgence of spontaneous Ca2+ oscillations but with faster kinetics. The carbachol-induced rise of Ca2+ in these cells showed a higher peak and biphasic decay. At all developmental stages, Ca2+ transients in response to ionomycin were indistinguishable. These findings lead us to conclude that a switch of Ca2+ dynamics accompanies structural and molecular differentiation of SH-SY5Y cell-derived human neurons, contributing to the developmental process.

Keywords:  BDNF; Calcium imaging; Molecular polarization; Neuroblastoma-derived neurons; SH-SY5Y cells; Spontaneous activity

DOI:  https://doi.org/10.1038/s41598-025-15561-0
Front Cell Neurosci. 2025 ;19 1635775

Establishment of human Leber's hereditary optic neuropathy model using iPSC-derived retinal organoids.

Kota Aoshima, Yuya Takagi, Michinori Funato, Yoshiki Kuse, Shinsuke Nakamura, Masamitsu Shimazawa.

  Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease caused by mitochondrial DNA mutations, leading to central vision loss and retinal ganglion cell (RGC) degeneration. Progress in understanding LHON and developing treatments has been limited by the lack of human-like models. In this study, we aimed to establish a human retinal model of LHON using retinal organoids (ROs) from LHON patient-derived induced pluripotent stem cells (LHON-iPSCs). We first confirmed LHON-iPSCs were successfully differentiated into ROs (LHON-ROs). LHON-RO showed a reduction in RGC numbers and the density of neural axons. Additionally, both mitochondrial membrane potential and ATP production were decreased in LHON-RO. Finally, treatment with idebenone, the only approved therapeutic agent for LHON, improved RGC numbers in LHON-RO. This model replicates key clinical features of LHON, including RGC and axonal loss, and demonstrates idebenone's therapeutic potential. Furthermore, a comprehensive analysis of the LHON-RO model revealed impaired mitophagy, suggesting novel therapeutic targets for LHON. Thus, the LHON-RO model offers a valuable platform for studying LHON pathogenesis and evaluating treatments.

Keywords:  Leber’s hereditary optic neuropathy; in vitro disease modeling; mitochondrial disease; mitophagy; retinal organoid

DOI:  https://doi.org/10.3389/fncel.2025.1635775
Proc Natl Acad Sci U S A. 2025 Oct 07. 122(40): e2508961122

Uncovering cargo clients and accessory factors of AP-1 and AP-4 through vesicle proteomics.

Ziqing Peng, Jingran Fan, Yang Liu, Qinyu Jia, Junkun An, Jianying Wang, Yan Huang, Zhong-Ping Yao, Yusong Guo.

  The trans-Golgi network (TGN) is a crucial sorting station in the secretory pathway, where adaptor protein (AP) complexes ensure selective cargo packaging into transport vesicles. However, the complete repertoire of cargoes and regulators associated with individual AP complexes remains poorly defined. Intriguingly, AP-4-mediated TGN export operates independently of clathrin, suggesting the involvement of uncharacterized accessory factors in vesicle biogenesis. To address these gaps, we developed an in vitro vesicle formation assay using wild-type HeLa cells or cells deficient in AP1γ1 or AP4ε, reconstituting their roles in packaging their known clients, Vangl2 and ATG9A, respectively. Coupling this assay with label-free quantitative mass spectrometry, we mapped distinct cargo profiles for AP-1 (which buds from the TGN and ARF1-positive endosomes) and AP-4, identifying the 45 kDa calcium-binding protein (CAB45) as an AP-1-dependent cargo and the Type-1 angiotensin II receptor-associated protein (ATRAP) as an AP-4-dependent cargo. Additionally, we uncovered PRRC1 and WDR44 as cytosolic regulators essential for AP-4-mediated TGN export. Our study advances the mechanistic understanding of AP-1 and AP-4 in secretory trafficking and provides a robust strategy to systematically identify cargo clients and accessory factors for specific adaptor complexes.

Keywords:  adaptor protein complexes; cargo sorting; the secretory pathway; trans-Golgi network; vesicular trafficking

DOI:  https://doi.org/10.1073/pnas.2508961122