bims-axbals 2026-06-07 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–06–07
33 papers selected by
TJ Krzystek

Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction.
Late Endosome Transport by RILP-RAB7A Promotes Dendrite Arborization Independently of Degradation.
miR-146a is a pleiotropic regulator of motor neuron degeneration.
A guide to selecting high-performing antibodies for ARID2 (UniProt ID: Q68CP9) for use in western blot, immunoprecipitation, and immunofluorescence.
Global transcriptional changes across multiple isogenic C9orf72 patient iPSC-derived neurons.
TDP-43 oxidation and PP1 crosstalk at RNA granule-mitochondria contact sites.
Kv7.2 loss-of-function causes early hyperexcitability and network remodelling.
Neuronal membrane organization by the submembranous spectrin-ankyrin scaffold: evolution, specialization and disease.
The retroelement-derived human protein PEG10 is a regulator of mRNA splicing in neurons.
Protocol for minimizing necrotic core formation in iPSC-derived cortical organoids.
LASER couples damage sensing to ESCRT assembly for lysosome repair.
Leucine-rich repeat kinase 2 (LRRK2): balancing cellular homeostasis and Parkinson's disease (PD) pathogenesis.
Fluid-based biomarkers of amyotrophic lateral sclerosis: recent advances and future prospects.
From autophagy-lysosomal deficits to neurodegeneration in Niemann-Pick type C1 disease: implications for age-related neurodegenerative disorders.
A cytosolic IF1 reporter enables real-time visualization of severe mitochondrial membrane damage.
How short and dense are axonal microtubules? Approaches for analyzing microtubule organization and dynamics.
Fos regulates age-dependent neuroinflammation in VAPBALS.
Harnessing Neuronal Autophagy: Bridging Mechanistic Breakthroughs to Therapeutic Interventions.
Streamlined isolation of enriched axoplasm from primary dissociated neuronal cultures.
Molecular mechanisms of autophagy-lysosomal pathway dysfunction in neurodegenerative diseases and therapeutic strategies for lysosomal repair: a review.
Disease-causing MFN2 mutants impair mitochondrial fission dynamics by distinct DRP1 dysregulation.
Live cell imaging reveals paclitaxel-induced lysosome motility and function disruption in DRG neurons.
Neuroprotective Effects of RNS60 in TDP-43 Pathology-Associated Amyotrophic Lateral Sclerosis.
Targeting mitophagy for neuroprotection: mechanisms and therapeutic opportunities.
An electrophysiological and proteomics roadmap for human induced glutamatergic neurons: fine-tuning of culture conditions for pathophysiological studies.
Iron Deficiency Impairs Mitochondrial Energetics and Early Axonal Growth and Branching in Developing Hippocampal Neurons.
High-resolution nuclear cell biology by cryo-electron tomography.
Multimodal analysis of cell-free DNA identifies epigenetic biomarkers for amyotrophic lateral sclerosis diagnosis and progression.
Site-Specific Raman Probes Reveal Droplet Aging and Residue-Level Fibril Polymorphism in TDP-43CTD.
High level dynein impairs mitochondrial distribution and differentiation of rhabdomyosarcoma cells.
Building the autophagosome: Molecular logic and membrane dynamics of autophagy.
Regulation of mitochondrial protein translocases.
HaloTag Fusion Enables Dynamic Analysis of Prion Protein Biosynthesis, Turnover, and Misfolding.

Sci Transl Med. 2026 Jun 03. 18(852): eaeb8517

Cryptic splicing in synaptic and membrane excitability genes links TDP-43 loss to neuronal dysfunction.

Caiwei Guo, Kuchuan Chen, Sarat Vatsavayai, Tetsuya Akiyama, Chang Liu, Yi Zeng, Odilia Sianto, Edith Yang, Juliane Bombosch, Rasheen Powell, Shannon Zhen, Shila Mekhoubad, Ryan D Morrie, Georgiana Miller, Dragana Ilic, Moritz Boll, Euan Parnell, Peter Penzes, Noa Lipstein, Eric M Green, Leonard Petrucelli, William W Seeley, Aaron D Gitler.

TAR DNA binding protein 43 (TDP-43) pathology is a defining pathological hallmark of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). A major feature of TDP-43 pathology is its nuclear depletion, leading to the aberrant inclusion of cryptic exons during RNA splicing. STMN2 and UNC13A have emerged as prominent TDP-43 splicing targets, but the broader impact of TDP-43-dependent cryptic splicing on neuronal function remains unclear. Here, we report previously unidentified TDP-43 splicing targets critical for membrane excitability and synaptic function, including KALRN, RAP1GAP, SYT7, and KCNQ2. Using human stem cell-derived neurons, we showed that TDP-43 reduction induces cryptic splicing and down-regulation of these genes, resulting in impaired excitability and synaptic transmission. In postmortem brains from patients with FTD, these cryptic splicing events occurred selectively in neurons with TDP-43 pathology. Suppressing individual cryptic splicing events using antisense oligonucleotides partially restored neuronal function, and combined targeting almost fully rescued the synaptic deficit caused by TDP-43 loss. Together, our findings provide evidence that cryptic splicing in these synaptic and membrane excitability genes is not only a downstream marker but instead a direct driver of neuronal dysfunction, establishing a mechanistic link between TDP-43 pathology and neurodegeneration in ALS and FTD.

DOI: https://doi.org/10.1126/scitranslmed.aeb8517
J Neurosci. 2026 Jun 04. pii: e2214252026. [Epub ahead of print]

Late Endosome Transport by RILP-RAB7A Promotes Dendrite Arborization Independently of Degradation.

Chan Choo Yap, Laura Digilio, Lloyd P McMahon, Ryan J Mulligan, Isabelle F Witteveen, Bettina Winckler.

Directional dendritic transport of late endosomes (LEs) retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of LEs and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of LEs and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of LEs and subsequent fusion with lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via the dynein-interacting RAB7A effector RILP. RILP also binds the LE-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in LE transport and fusion defects with lysosomes. In this work, we express a separation-of-function mutant of RAB7A (RAB7A-L8A) incapable of RILP binding. Based on the results in non-neuronal cells, we hypothesized that both endosome motility and degradation in neurons depended on RILP. Our data in cultured rat and mouse hippocampal neurons of both sexes suggest that endogenous RILP is a functional RAB7A-dependent dynein adaptor for LE motility in dendrites. In addition, it promotes endosome carrier formation. As a consequence of LE transport inhibition, degradative cargos are not cleared normally from dendrites in RAB7A-L8A. Surprisingly, lysosomal fusion and somatic degradation do not require RAB7A-RILP interactions. Despite the normal degradation, dendrite arborization is impaired in RAB7A-L8A expressing neurons, demonstrating that dendrite morphology defects are separable from degradation blockade. This indicates that normal dendrite growth/maintenance is dependent on sustained RAB7A/RILP-dependent LE transport.Significance Statement Dendrite growth requires membrane trafficking, but the roles of individual compartments and regulators are not well established. Stunted dendrite growth is often associated with endolysosomal traffic jams and degradation block. In contrast, our work reveals a requirement for transport of late endosomes via RAB7A-RILP to support dendrite growth independently of cargo transport to the lysosome for degradation.

DOI: https://doi.org/10.1523/JNEUROSCI.2214-25.2026
Proc Natl Acad Sci U S A. 2026 Jun 09. 123(23): e2526314123

miR-146a is a pleiotropic regulator of motor neuron degeneration.

Dylan A Galloway, Hunter L Patterson, Mariah L Hoye, Tao Shen, Mark Shabsovich, Kathleen M Schoch, Cindy V Ly, Timothy M Miller.

  Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease affecting motor neurons. Here, we have profiled motor neuron microRNAs (miRNAs) during motor neuron degeneration in vivo to gain a better understanding of ALS pathophysiology. We demonstrate that one miRNA, miR-146a, is downregulated in diseased motor neurons despite upregulation in bulk tissue. Genetic deletion of miR-146a significantly extended survival in SOD1G93A mice with heterozygous animals demonstrating the largest benefit. A corresponding reduction in spinal cord gliosis but not motor neuron loss was observed. Finally, we observed that a proportion of miR-146a knockout animals develop spontaneous paralysis, motor neuron loss and chronic neuroinflammation with advanced age. Together these findings demonstrate that a single miRNA influences multiple aspects of motor neuron disease and highlights the complex role for neuroinflammation in ALS pathogenesis.

Keywords:  amyotrophic lateral sclerosis; microRNA; neuroinflammation

DOI:  https://doi.org/10.1073/pnas.2526314123
F1000Res. 2026 ;15 614

A guide to selecting high-performing antibodies for ARID2 (UniProt ID: Q68CP9) for use in western blot, immunoprecipitation, and immunofluorescence.

Vera Ruíz Moleón, Charles Alende, Maryam Fothouhi, Sara González Bolívar, Riham Ayoubi, Vincent Francis, Peter S McPherson, Carl Laflamme.

  The AT-rich interactive domain-containing protein 2 (ARID2) is a core component of the chromatin-remodeling complex that regulates transcription by modulating nucleosome positioning. Here we have characterized six ARID2 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  ARID2; AT-Rich Interaction Domain 2; Q68CP9; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.179367.1
iScience. 2026 Jun 19. 29(6): 116054

Global transcriptional changes across multiple isogenic C9orf72 patient iPSC-derived neurons.

Aparna Sreeram, Desiree M Baron, Alberto Brusati, Karly Stallworth, Jack Humphrey, John E Landers.

  Hexanucleotide repeat expansions in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal degeneration (FTD); yet, mechanisms underlying selective neuronal vulnerability remain unclear. A major challenge in identifying consistent transcriptomic changes across C9orf72 patient-derived neuron lines has been heterogeneous differentiations, lack of isogenic controls and low sequencing depth. To overcome these challenges, we generated homogeneous cortical neuron (iCNs) cultures from multiple isogenic C9orf72 patient iPSC pairs and performed RNA deep sequencing. We identified robust and reproducible gene expression and splicing alterations in pathways related to cytoskeletal organization, extracellular matrix adhesion and synaptic signaling. Notably, we observed exon 30 skipping in the cytoskeletal regulator filamin B (FLNB), resulting in loss of its hinge domain. This was accompanied by altered FLNB localization, disrupted actin crosslinking, and mechanotransduction signaling. These findings reveal convergent transcriptomic and functional disruptions across multiple isogenic C9orf72 patient-derived iCNs offering insights into ALS/FTD pathogenesis.

Keywords:  Cell biology; Genomics; Neuroscience

DOI:  https://doi.org/10.1016/j.isci.2026.116054
Nat Commun. 2026 Jun 05.

TDP-43 oxidation and PP1 crosstalk at RNA granule-mitochondria contact sites.

Hannah E Ball, Abby C Woods, Yvette C Wong.

Inter-organelle contact sites are key hubs for organelle bidirectional crosstalk. However, how mitochondria and RNA granules interact at contact sites and its regulation by mitochondrial oxidative phosphorylation (OXPHOS) remain unclear. Here, using Super-Resolution live microscopy, we identify RNA granule-mitochondria contact site formation in OXPHOS conditions. Reactive oxygen species (ROS) generated by mitochondrial OXPHOS promotes TDP-43 localization to cytoplasmic RNA granules via TDP-43 cysteine oxidation at Cys173/Cys175. Mechanistically, RNA granule-mitochondria contact tethering is mediated by TDP-43 on RNA granules binding to GADD34 on mitochondria, while contact untethering is regulated by TDP-43 oxidation. Functionally, this allows for GADD34 and its binding partner PP1 to regulate TDP-43 RNA granule dynamics, and conversely, for TDP-43 oxidation to regulate the ability of the phosphatase PP1 to form granules. Finally, disease-associated mutant TDP-43 misregulates this pathway, ultimately leading to PP1 granules lacking TDP-43. This dynamic crosstalk between TDP-43 oxidation and PP1 has significant consequences for TDP-43-associated diseases including Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD).

DOI: https://doi.org/10.1038/s41467-026-74009-9
Brain. 2026 Jun 06. pii: awag199. [Epub ahead of print]

Kv7.2 loss-of-function causes early hyperexcitability and network remodelling.

Nina Dirkx, Marcus Kaji, Els De Vriendt, Giusy Carleo, Francesco Miceli, Bob Asselbergh, Peter Verstraelen, Noortje Zonnekein, Lidia Carotenuto, Louis T Dang, Vera Sommers, Eve Vlaemynck, Lieven Lagae, Berten Ceulemans, Peter De Jonghe, Winnok H De Vos, Maurizio Taglialatela, Sarah Weckhuysen.

  Loss-of-function (LOF) variants in KCNQ2, encoding the potassium channel subunit Kv7.2, cause a spectrum of neonatal epilepsies ranging from self-limiting familial neonatal epilepsy (SeLFNE) to severe developmental and epileptic encephalopathy (DEE). To dissect the developmental consequences of Kv7.2 LOF, we conducted a longitudinal and multimodal comparative analysis in a human neuronal model generated from patients with KCNQ2-DEE and KCNQ2-SeLFNE. KCNQ2-LOF variants induced a biphasic dysfunction at both single-cell and network levels, characterized by early Kv7-driven hyperexcitability accompanied by a clear reduction M-current density, which was rescued by acute Retigabine treatment. At later stages, intrinsic excitability and M-current normalized, yet network activity diverged further from control trajectories, indicating compensatory and ultimately maladaptive network remodeling. Transcriptomic analysis mirrored this biphasic dynamic trajectory, revealing an initial upregulation followed by a subsequent downregulation of synaptic genes. Structural analysis showed a steeper decline in presynaptic density alongside a distal shift in the axon initial segment (AIS) throughout maturation, and impaired AIS plasticity at later stages. Overall, KCNQ2-LOF variants disrupt human neuronal maturation through dynamic, biphasic changes in function, gene expression and structure, offering insights into disease mechanisms and therapeutic options.

Keywords:  Kv7.2 channel dysfunction; M-current; human iPSC-derived neurons; microelectrode array; neuronal network development; retigabine

DOI:  https://doi.org/10.1093/brain/awag199
Biol Chem. 2026 Jun 03.

Neuronal membrane organization by the submembranous spectrin-ankyrin scaffold: evolution, specialization and disease.

Maximilian Goy, Jan Pielage.

  Neuronal function relies on precise compartmentalization into dendritic, somatic, axonal, and synaptic domains that require specialized cellular architectures. Axons, in particular, can extend over extraordinary distances while preserving stable membrane composition, mechanical integrity, and reliable excitability. A submembranous scaffold composed of spectrin, ankyrin, actin, and associated proteins provides a conserved platform for coupling membrane proteins to cytoskeletal support and organizing membrane domains. In axons, this scaffold assembles into a membrane-associated periodic skeleton (MPS) with near-regular spacing that supports mechanical load, patterns membrane components, and contributes to compartmental boundaries. Comparative genetics indicates that core principles of spectrin-ankyrin organization are ancient, whereas vertebrate evolution expanded spectrin and ankyrin families and enabled specialized excitable domains such as the axon initial segment (AIS) and nodes of Ranvier. Pathogenic variants in spectrin and ankyrin genes disrupt neuronal development and excitability and cause a growing spectrum of neurodevelopmental and neurodegenerative disorders, underscoring scaffold integrity as a key determinant of circuit stability. Here, we examine how conserved spectrin-ankyrin scaffold principles were adapted to neuronal cell-type diversity and domain specialization, with emphasis on axons, synapses, and disease, and we discuss how the membrane-associated periodic skeleton may function not only as a stabilizing framework but also as a nanoscale organizer of neuronal membrane architecture.

Keywords:  ankyrin; membrane-associated periodic skeleton (MPS); molecular ruler; spectrin; spectrinopathy; synapse

DOI:  https://doi.org/10.1515/hsz-2026-0117
bioRxiv. 2026 May 24. pii: 2026.05.21.727000. [Epub ahead of print]

The retroelement-derived human protein PEG10 is a regulator of mRNA splicing in neurons.

A M Matthews, A M Whiteley.

Retroelements, including retrotransposons, endogenous retroviruses, and their fragments, as well as rare co-opted or domesticated retroelements, can contribute to neurodegenerative disorders and aging through modulation of gene expression and induction of neuroinflammation. Paternally Expressed Gene 10 (PEG10) is a retroelement-derived human gene that has recently been identified as a putative driver of Amyotrophic Lateral Sclerosis (ALS) and Angelman's Syndrome. PEG10 has been reported to bind nucleic acid and undergoes a complex self-processing pathway that results in gene expression changes when the protein accumulates in cells. Here, we report that PEG10 has selectivity for binding U/G-rich RNAs and influences widespread gene expression changes. PEG10 overexpression mimics the loss of TDP-43 in broad changes to gene expression, including dysregulation of mRNA splicing pathways. Specific changes to mRNA splicing were largely unique between TDP-43 knockdown and PEG10 overexpression, as classic TDP-43 targets including STMN2 were not altered by PEG10. Instead, we identified a unique role for PEG10 in regulating splicing of neuregulin 3 (NRG3) , a ligand for the neuronal receptor ERBB4. In SH-SY5Y cells and in human neurons overexpressing PEG10, NRG3 protein levels were decreased along cellular processes, suggesting that these cells are less competent at signaling through the NRG3/ERBB4 axis. Using human patient data, we observed similar changes to NRG3 splicing in UBQLN2 -mediated ALS, where PEG10 is accumulated, as well as in some cases of sporadic ALS. In conclusion, the retroelement-derived gene PEG10 plays an unexpected role in regulating splicing of neuronal transcripts, which mimics some of the transcript changes observed in human ALS patient samples. Ultimately, this work has implications for the study of PEG10, and mRNA splicing in neurological diseases associated with elevated PEG10 abundance.
Highlights: PEG10 NC expression influences abundance of transcripts implicated in ALS PEG10 NC expression leads to an exon skipping event in neuregulin 3 (NRG3) NRG3 expression is decreased along dendrites of PEG10 NC expressing human neuronsExpression of PEG10 NC mimics changes observed in human ALS.

DOI: https://doi.org/10.64898/2026.05.21.727000
STAR Protoc. 2026 Jun 04. pii: S2666-1667(26)00262-5. [Epub ahead of print]7(2): 104609

Protocol for minimizing necrotic core formation in iPSC-derived cortical organoids.

Md Mahfuz Al Mamun, Sundari Chetty.

  Cortical organoids (COs) derived from human induced pluripotent stem cells (hiPSCs) provide a unique opportunity to model human neurodevelopment in vitro. Here, we present a protocol to prevent necrotic core formation without requiring organoid slicing. We describe steps for hiPSC culturing, preparation of single-cell suspension, seeding, and embryoid body (EB) and neuroectoderm formation. We then detail procedures for CO formation and maintenance without necrotic core formation. This protocol sustains long-term viability and preserves structural integrity by enhancing oxygen and nutrient diffusion.

Keywords:  Neuroscience; Organoids; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2026.104609
Nature. 2026 Jun 03.

LASER couples damage sensing to ESCRT assembly for lysosome repair.

Claire S Goul, Aakriti Jain, Samira Yitiz, Zahra E Soltani, Serim Yang, Simon Rapp, Martina Spacci, Scot Federman, James Sacco, Huinan Li, Lauren D Enriquez, Nalan Liv, Laralynne Przybyla, Roberto Zoncu.

Lysosomal membrane integrity is essential for cell survival, but how damage sensing is spatiotemporally coupled to repair remains poorly understood. Recruitment and assembly of endosomal sorting complex required for transport (ESCRT) I-III rapidly counteracts membrane damage, but it is unclear how ESCRT-I recognizes defective lysosomal membranes. Here, leveraging genome-wide CRISPRi screens in a damage-sensitized genetic background, we identified LC3/GABARAP-assisted stimulator for ESCRT recruitment (LASER), a multicomponent protein assembly that forms rapidly upon calcium release from damaged lysosomes and couples sensing of lysosomal membrane damage to ESCRT-dependent repair. At the core of LASER is TFG, an endoplasmic reticulum exit-site-resident protein that translocates to damaged lysosomes by binding to ATG8 family proteins (LC3 and GABARAP) conjugated to lysosomal phospholipids. ATG8-bound TFG forms oligomeric assemblies that directly recruit the essential ESCRT-I subunit TSG101 via conserved motif recognition enhanced by avidity-driven interactions. TFG binding to TSG101 stimulates sequential ESCRT-I-II-III polymerization and promotes membrane repair. TFG mutations that drive hereditary spastic paraplegia disrupt its oligomerization and impair lysosomal ESCRT recruitment and membrane resealing, implicating defective repair as a driver of TFG-associated neurodegeneration. Thus, LASER promotes ESCRT polymerization at damaged lysosomes and couples damage sensing to membrane repair.

DOI: https://doi.org/10.1038/s41586-026-10604-6
Ann Med. 2026 Dec;58(1): 2677288

Leucine-rich repeat kinase 2 (LRRK2): balancing cellular homeostasis and Parkinson's disease (PD) pathogenesis.

Iman Aolymat, Diala Walid Abu-Hassan, Aya Khleaf Oleimat, Wasan Sameer, Ban Wreikat, Tala Iqilan, Hafez Al-Momani, Heba Ali, Lubna Tahtamouni, Mahmoud Iqsairi, Mohammed Albeik, Omar Debas.

   BACKGROUND: Leucine-rich repeat kinase 2 (LRRK2) is a kinase with multi-signalling function that regulates various processes essential for neuronal and systemic physiology. It is involved in autophagy, vesicular trafficking, mitochondrial dynamics, and immune response. Pathogenic mutations of LRRK2 can significantly interfere with these physiological pathways essential for neuronal homeostasis, inducing degeneration of dopaminergic neurons-a characteristic feature of Parkinson's disease (PD).
OBJECTIVE: This review comprehensively summarizes the normal cellular functions of LRRK2 and the potential impact of its dysregulation on various physiological pathways, predisposing individuals to familial and sporadic PD. The mechanistic connections between LRRK2's kinase hyperactivity, disturbances in vesicular trafficking and redox status, systemic and neuronal inflammation, and metabolic disorders will be thoroughly discussed.
RESULTS: Dysregulation of vesicular trafficking, mitochondrial redox balance, inflammatory pathways, and metabolism promotes α-synuclein accumulation and contributes to the degeneration of nigrostriatal dopaminergic neurons, a central pathological feature of PD. Understanding the physiological role of LRRK2 across neuronal and peripheral tissues uncovers its connection with multiple pathways to maintain homeostasis. Its dysfunction disseminates local stresses into broader neurodegenerative changes.
CONCLUSION: LRRK2 is implicated in multiple pathways that control neuronal integrity and neurodegeneration. Therefore, therapeutic targeting of LRRK2 could potentially help in restoring physiological function and management of PD.

Keywords:  Neurodegeneration; diagnostic innovations; molecular mechanisms; oxidative stress; therapeutic development

DOI:  https://doi.org/10.1080/07853890.2026.2677288
Brain Res. 2026 May 29. pii: S0006-8993(26)00255-6. [Epub ahead of print]1888 150395

Fluid-based biomarkers of amyotrophic lateral sclerosis: recent advances and future prospects.

Yiru Jiang, Shuqi Hu, Bingjie Yang, Lingling Zhang, Ying Wang, Gaoyi Yang, Hao Zhang.

  Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder with no definitive cure. The absence of specific diagnostic biomarkers leads to diagnostic delays, hindering early intervention and management. This review provides a critical appraisal of fluid-based biomarkers for ALS across multiple sources-cerebrospinal fluid (CSF), blood, urine, saliva, and tears-with emphasis on their diagnostic and prognostic potential, limitations, and readiness for clinical translation. While neurofilaments (NfL, pNfH) are well-established as sensitive indicators of neuroaxonal injury and are increasingly used as prognostic and pharmacodynamic markers in clinical trials, they lack disease specificity. Biomarkers reflecting ALS-specific pathology, such as TDP-43 species and C9orf72 dipeptide repeat proteins (DPRs), show promise but remain in early validation stages with limited multicenter data. Emerging markers from non-invasive sources (urine p75ECD, salivary chromogranin A, tear metabolomics) offer potential for repeated sampling but require rigorous external validation before clinical adoption. To address current gaps, we introduce a standardized evidence grading framework (Tier 1-3) and a comprehensive reporting template for biomarker studies, including explicit performance metrics (AUC, sensitivity, specificity, confidence intervals) and validation status. We also propose minimum reporting standards for study design, pre-analytical variables, and statistical rigor, modeled on REMARK guidelines. A roadmap for biomarker validation and a cross-fluid comparison matrix are provided to guide future research. Despite considerable progress, significant challenges remain, including biological heterogeneity, pre-analytical variability, and insufficient external validation. Future efforts should prioritize multicenter prospective studies, assay harmonization, ethical frameworks for early diagnosis, and integration of emerging technologies such as artificial intelligence and digital twins. Fluid-based biomarkers, while not yet replacing clinical evaluation, are essential tools for accelerating drug development, enabling patient stratification, and moving toward personalized medicine in ALS.

Keywords:  Amyotrophic lateral sclerosis; Digital twin; Fluid biomarkers; Saliva; Tears; Urine

DOI:  https://doi.org/10.1016/j.brainres.2026.150395
Front Neurosci. 2026 ;20 1857866

From autophagy-lysosomal deficits to neurodegeneration in Niemann-Pick type C1 disease: implications for age-related neurodegenerative disorders.

Yuki Kawachi, Gamze Kocak, Viktor I Korolchuk, Tetsushi Kataura, Sovan Sarkar.

  Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by loss-of-function mutations in the NPC1 gene. NPC1 deficit primarily disrupts lipid homeostasis and subsequently drives cellular degeneration through mechanisms involving impaired autophagy and mitophagy, mitochondrial dysfunction, and, recently demonstrated NAD depletion that links autophagy impairment to neuronal death. Emerging evidence also highlights the activation of innate immune signaling leading to neuroinflammation. In this review, we synthesize current mechanistic insights and describe how these molecular deficits are interconnected to drive neuronal death in NPC1 disease. We also discuss how these pathological processes parallel those observed in major age-related neurodegenerative pathologies such as Alzheimer's and Parkinson's disease. Finally, we highlight emerging therapeutic strategies that can potentially ameliorate these cellular deficits, offering avenues for mitigating neurodegeneration in NPC1 disease and other related neurodegenerative disorders.

Keywords:  NAD; NPC1; autophagy; cell death; lysosome; mitochondria; neurodegeneration; neuroinflammation

DOI:  https://doi.org/10.3389/fnins.2026.1857866
J Cell Biol. 2026 Aug 03. pii: e202511088. [Epub ahead of print]225(8):

A cytosolic IF1 reporter enables real-time visualization of severe mitochondrial membrane damage.

Erqing Gao, Liwei Guan, Kehang Zhang, Shuze Qi, Jingxiu Xu, Jie Zhang, Tianyi Zhu, Bing Yang, Chonglin Yang, Eric Miska, Mao Zhang, Suhong Xu.

Maintenance of mitochondrial integrity is fundamental for cellular survival, yet how cells recognize catastrophic mitochondrial membrane damage remains unknown. Here, we identify MAI-1 as the first genetically encoded reporter of severe mitochondrial membrane damage. MAI-1 is a Caenorhabditis elegans homolog of the ATP synthase inhibitor IF1 that lacks a mitochondrial targeting sequence, resides in the cytosol under basal conditions, but rapidly and irreversibly translocates to severely damaged mitochondria within milliseconds. We validate MAI-1 across diverse injury paradigms and demonstrate that cytosolic IF1 variants from other species exhibit conserved damage-induced recruitment. Mechanistically, MAI-1 recruitment requires the presence of an intact ATP synthase complex. Using MAI-1 as a sensor, we uncover that these severely damaged mitochondria are cleared through the LGG-1-mediated, PINK1/PARKIN-independent lysosomal pathway. Together, our findings establish a powerful tool for visualizing severe mitochondrial membrane damage and reveal a surveillance mechanism dedicated to structural integrity control.

DOI: https://doi.org/10.1083/jcb.202511088
Histol Histopathol. 2026 Jun 04. 25104

How short and dense are axonal microtubules? Approaches for analyzing microtubule organization and dynamics.

Lisha Singh, Laryssa Alves Borba, Kim Stuke, Roland Brandt.

Axons are cable-like extensions of neurons that conduct nerve impulses and relay information to subsequent cells at synapses. Their length varies dramatically, from micrometers in small invertebrates to over a meter in some neurons of large vertebrates. The cytoskeleton is the most important internal structure that determines neuronal morphology. Microtubules, in particular, play a crucial role in maintaining axonal structure, transport, and function. The classical view of axonal microtubules as long, continuous, and stable polymers extending over vast distances is being challenged by evidence suggesting that the axonal microtubule network is composed of overlapping microtubule fragments of varying lengths and is considerably dynamic. In this article, we describe how the unique organization of the axonal microtubule network has been elucidated in parallel with the development of novel microscopy techniques that provide high-resolution structural data and information on microtubule dynamics in neurons. We discuss how this unique microtubule architecture confers structural stability and adaptability to axons and hypothesize that the length distribution of microtubules reveals whether microtubule organization is more dynamic and adaptable or functions primarily as a stabilizing backbone for the axon shaft. Finally, we discuss potential future perspectives, both in terms of technological advances and open questions regarding the role of alterations in axonal microtubule arrangement in the aging process and neurodegenerative diseases.

DOI: https://doi.org/10.14670/HH-25-104
Dis Model Mech. 2026 Jun 04. pii: dmm.052810. [Epub ahead of print]

Fos regulates age-dependent neuroinflammation in VAPBALS.

Namrata Pramod Kulkarni, Aparna Thulasidharan, Amarendranath Soory, Pulkit Goel, Sanhita Sarkar, Vidyadheesh Kelkar, Girish S Ratnaparkhi.

  Amyotrophic Lateral Sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive loss of motor function. We have developed a Drosophila model of ALS8 (VAPBP58S) using CRISPR/Cas9 genome editing. VAPB is an ER-based adapter protein associated with and regulating intracellular membrane:membrane contact sites. VAPB P58S flies show progressive age-dependent motor deficits and a shortened lifespan, paralleling features of the human disease. VAPBP58S brains exhibit age-dependent neuroinflammation, as measured by whole-transcriptome quantitative mRNA sequencing, suggesting a broad, low-grade enhancement of signalling across multiple immune pathways (Toll, IMD, Jak-STAT, and Jun-kinase). We implicate glial cells in the brain as the site of brain inflammation and identify Drosophila Fos (Kayak) as a key modulator of age-dependent inflammation. In accordance, we find that overexpression of wild-type kayak or its dominant-active variant kayakK357R in glia reduces inflammation and, concomitantly, improves motor function. In contrast, knockdown of glial kayak accelerates age-dependent deterioration of motor function and enhances neuroinflammation. Our study underscores the roles of glial-modulated brain inflammation in dictating ALS8 progression and identifies kayak as a central negative regulator of neuroinflammation in disease.

Keywords:  Amyotrophic Lateral Sclerosis; CRISPR/Cas9; Kayak; Neuroinflammation; Vesicle-associated membrane protein-associated protein B

DOI:  https://doi.org/10.1242/dmm.052810
Aging Dis. 2026 May 19.

Harnessing Neuronal Autophagy: Bridging Mechanistic Breakthroughs to Therapeutic Interventions.

Nuzhat Ahsan, Farheen Badrealam Khan, Jhinuk Basu, Mohd Shariq, Nida Zaidi, Rizwan Hasan Khan, Reshmi Raj, Mohammed Akli Ayoub, Avadhesha Surolia, Ghulam Md Ashraf.

Autophagy, an essential cellular process that degrades and recycles misfolded proteins, damaged organelles, and intracellular pathogens, is vital for neurons due to their limited capacity for apoptosis. Dysregulation of autophagy and lysosomal pathways is closely linked to the onset and progression of major neurodegenerative diseases (NDDs), including Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis. In this manuscript, we offer a thorough examination of the molecular mechanisms that regulate autophagy, emphasizing both bulk and selective autophagy pathways and their roles in maintaining neuronal homeostasis. Genetic mutations in autophagy-related genes and endolysosomal genes are identified as significant risk factors, and the pathological roles of protein aggregation and mitochondrial dysfunction are also discussed. The therapeutic restoration of autophagic function represents a promising strategy for alleviating neurodegeneration. This manuscript examines potential interventions, including small molecules, gene therapy, and natural compounds, that enhance autophagic flux and facilitate protein clearance. Furthermore, the translational potential was underscored by including ongoing clinical trials that target autophagy pathways. This review artcile emphasizes the essential role of autophagy in neuronal health and disease, providing a framework for utilizing autophagic mechanisms to develop targeted therapeutic strategies for NDDs.

DOI: https://doi.org/10.14336/AD.2025.0804
Methods. 2026 Jun 04. pii: S1046-2023(26)00141-6. [Epub ahead of print]

Streamlined isolation of enriched axoplasm from primary dissociated neuronal cultures.

Sabta Alarcón, Andrés Fuentes, Elizabeth Carrazana, Karen Fabres, Paulina Bahamonde, Mario Sanhueza, Natalia Salvadores.

  Axons are highly specialized neuronal compartments that rely on local molecular regulation to support growth, maintenance, and synaptic function. The cytoplasmic contents of axons, collectively referred to as the axoplasm, constitute a biochemically distinct domain whose composition is central to neuronal development and disease. However, biochemical analysis of axoplasm has been technically challenging, particularly in dissociated neuronal cultures, where axons and somata are intermingled and axonal material is limited. As a result, most axoplasm-focused studies have relied on explant-based or peripheral neuron models, restricting direct biochemical access to axons from dissociated central nervous system neurons. Here, we describe a simple and robust culture-based approach that enables efficient physical separation of somata and axons from primary dissociated neuronal cultures and allows the recovery of enriched axoplasm fractions. Using a custom reaggregation device, neurons reorganize into compact somatic clusters that extend radially oriented axons, facilitating reliable manual separation of somatic and axonal material. Morphological and immunofluorescence analyses demonstrate robust neuronal polarization and effective spatial segregation of somatic and axonal components. Biochemical and molecular validation reveals that axonal fractions are enriched in axonal proteins and contain bona fide axon-associated transcripts, while being depleted of nuclear markers and genomic DNA, providing a practical and accessible method for axoplasm isolation from dissociated neuronal cultures. By enabling biochemical analysis of axonal material from neuron types that are otherwise difficult to access, this method expands the experimental toolkit for studying axon-specific molecular mechanisms in neuronal development, plasticity, and disease.

Keywords:  Axonal RNA; Axonal proteins; Axoplasm; Neuronal culture

DOI:  https://doi.org/10.1016/j.ymeth.2026.06.001
Front Neurosci. 2026 ;20 1819002

Molecular mechanisms of autophagy-lysosomal pathway dysfunction in neurodegenerative diseases and therapeutic strategies for lysosomal repair: a review.

Weilin Wang, Xianguo Meng.

  The autophagy-lysosomal pathway (ALP) is a critical intracellular protein degradation system responsible for maintaining proteostasis and metabolic balance within cells. Dysfunction of this pathway has been increasingly recognized as a key pathological basis underlying various neurodegenerative diseases (NDs). This review provides a comprehensive overview of the molecular mechanisms by which ALP impairment contributes to defective protein degradation in neurodegeneration. We focus on the impact of lysosomal structural integrity and functional imbalance on cellular fate, highlighting the interplay between protein oxidative damage and degradation system dysregulation. Furthermore, we summarize the current therapeutic strategies aimed at lysosomal repair, evaluating their potential clinical applications and efficacy. By integrating the latest research advances, this review aims to deepen the understanding of the pathological mechanisms of autophagy-lysosomal pathway dysfunction in neurodegenerative diseases, clarify the key molecular targets of lysosomal damage and repair, and provide theoretical basis for target screening and validation and practical reference for the development of targeted drugs for neurodegenerative diseases.

Keywords:  autophagy-lysosomal pathway; lysosomal repair; molecular mechanisms; neurodegenerative diseases; protein degradation; therapeutic strategies

DOI:  https://doi.org/10.3389/fnins.2026.1819002
Cell Death Dis. 2026 Jun 05.

Disease-causing MFN2 mutants impair mitochondrial fission dynamics by distinct DRP1 dysregulation.

Daniel Lagos, Pamela R de Santiago, Nicolás Pérez-Bravo, Benjamín Cartes-Saavedra, Josefa Vial-Brizzi, Diego Troncoso-Chandía, Oliver Podmanicky, Rita Horvath, Verónica Eisner.

Mitochondria undergo fusion and fission. While DRP1 regulates fission, fusion is controlled by OPA1, MFN1, and MFN2. The balance between these processes and the crosstalk between machineries remains poorly understood. MFN2 mutations cause Charcot-Marie-Tooth disease type 2 A (CMT2A), affecting mitochondrial fusion and morphology. However, their role in fission is unclear. Using skin fibroblasts from CMT2A patients (L248H and M376V MFN2 mutations) and wild-type mouse embryonic fibroblasts expressing these variants, we studied how MFN2 mutations impact mitochondrial dynamics beyond fusion. We analyzed mitochondrial morphology and dynamics by live-cell confocal microscopy and tested fusion/fission protein levels, oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and oxidative phosphorylation complex subunits. MFN2 mutations impaired mitochondrial fusion and displayed distinct effects on fission and cellular metabolism. L248H-expressing cells showed hyper-elongated mitochondria, impaired fission, and increased OCR, while M376V cells exhibited fragmentation, enhanced fission, and elevated ECAR. These effects correlated with differential Drp1 phosphorylation. Our findings demonstrate that MFN2 mutants differentially influence fission and metabolism, highlighting the need to consider these effects in therapies aimed at modulating mitochondrial dynamics.

DOI: https://doi.org/10.1038/s41419-026-08838-3
bioRxiv. 2026 May 21. pii: 2026.05.19.726221. [Epub ahead of print]

Live cell imaging reveals paclitaxel-induced lysosome motility and function disruption in DRG neurons.

Kathleen Cate Domalogdog, Ishwarya Sankaranarayan, Úrzula Franco-Enzástiga, Juliet M Mwirigi, Shani Mai Nguyen, Diana Tavares-Ferreira, Theodore J Price.

Lysosomal trafficking and homeostasis are biological functions that are pivotal for DRG neurons, given their metabolic demands and extremely long axons. Previous studies indicate that lysosomal signaling is altered in a mouse model of chemotherapy-induced peripheral neuropathy (CIPN) and that blocking mitogen activated protein kinase-associated kinase (MNK1/2) signaling can alleviate pain behaviors in CIPN. Here, we investigated lysosome dynamics and lysosome-associated signaling in a mouse model of CIPN induced by paclitaxel (PTX), a chemotherapeutic agent used for various types of cancer. Using spinning disk super-resolution microscope (SPINSR), we demonstrate that PTX treatment in vivo causes reduced lysosome motility observed in vitro. PTX likewise drives the accumulation of Sequestosome 1 (SQSTM1), also known as P62, in cultured mouse DRG neurons, indicating lysosomal dysfunction in DRG neurons. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, was also upregulated in the nucleus of cultured mouse DRG neurons treated with PTX. In line with this, increased lysosomal-associated membrane protein 1 (LAMP1) expression was observed in PTX-treated mice. Given that our previous work demonstrated PTX treatment increases MNK1/2-eIF4E signaling in DRG neurons, we examined whether MNK1/2 inhibition could rescue lysosomal dysfunction. Treatment with Tomivosertib (eFT508), a potent MNK1/2 inhibitor, restored P62 levels in DRG neurons of PTX-treated mice and reduced TFEB in DRG treated in vitro . To establish translation relevance, we further show that PTX elevates phosphorylated eiF4E (p-eIF4E) in human DRG neurons, and concurrent eFT508 administration attenuates this effect. Collectively, these findings indicated that PTX disrupts lysosome trafficking and biogenesis, and that MNK inhibition with eFT508 restores lysosomal signaling and can serve as a neuroprotective strategy for CIPN.

DOI: https://doi.org/10.64898/2026.05.19.726221
Muscle Nerve. 2026 Jun 04.

Neuroprotective Effects of RNS60 in TDP-43 Pathology-Associated Amyotrophic Lateral Sclerosis.

Danny R Vesevick, Supurna Ghosh, Andreas Kalmes, P Hande Ozdinler, Mukesh Gautam.

INTRODUCTION: TDP-43 pathology is broadly observed in the cerebral cortex of patients with amyotrophic lateral sclerosis (ALS). RNS60, an experimental treatment for acute ischemic stroke and ALS, enhanced mitochondrial biogenesis and function in other preclinical models. We investigated whether RNS60 improved mitochondrial stability and upper motor neuron (UMN) health in a TDP-43 mouse model of ALS.
METHODS: prpTDP-43A315T-UeGFP mice, in which UMNs express green fluorescent protein (eGFP), and WT-UeGFP mice were treated with RNS60 or placebo intraperitoneally every other day from post-natal day (P) 30 until P90. Astrogliosis and microgliosis in brain and spinal cord were quantified by immunocytochemistry. Mitochondrial ultrastructure was studied via electron microscopy, and mitochondrial function was assessed using flow cytometry. Neuromuscular junction (NMJ) integrity was assessed in gastrocnemius, tibialis, and diaphragm muscles.
RESULTS: RNS60 treatment reduced defective mitochondria in UMNs (prpTDP-43A315T + vehicle: 53.2% ± 0.71%; prpTDP-43A315T + RNS60: 19.6% ± 1.4%, p = 0.0001) and spinal motor neurons (prpTDP-43A315T + vehicle: 70.1% ± 0.4.48%; prpTDP-43A315T + RNS60: 33.5% ± 4.43%, p = 0.001). It increased mitochondrial membrane polarization (prpTDP-43A315T-UeGFP + vehicle: 7184 ± 1689 mean intensity; prpTDP-43A315T-UeGFP+RNS60: 22120 ± 4818 mean intensity, p = 0.032), reduced the extent of astrogliosis and microgliosis in motor cortex and spinal cord, protected UMNs compared to placebo, and enhanced the proportion of intact NMJs in leg and diaphragm muscles (prpTDP-43A315T-UeGFP + vehicle: 29.6% ± 3.6%; prpTDP-43A315T-UeGFP + RNS60: 64.3% ± 4.4%, p = 0.0002).
DISCUSSION: These results suggest that RNS60 treatment promotes motor neuron health in ALS by protecting mitochondrial structure and function, preserving NMJ integrity, and reducing gliosis.

DOI: https://doi.org/10.1002/mus.70289
NPJ Aging. 2026 Jun 03.

Targeting mitophagy for neuroprotection: mechanisms and therapeutic opportunities.

Jiahui Yang, Jianfeng Li, Xintong Hou, Yifei Zheng, Zeyu Zhao, Ting Zhou, Tongwei Jing, Jiming Kong, Guohui Zhang, Ying Guo.

Mitochondria are essential for neuronal energy production, cellular homeostasis, and overall neuronal function. Due to their high metabolic demands and limited regenerative capacity, neurons are particularly vulnerable to mitochondrial dysfunction, which leads to ATP depletion, excessive reactive oxygen species (ROS) production, and calcium imbalance-ultimately causing oxidative stress, metabolic disruption, and neuronal death. Mitophagy is a selective process that removes damaged mitochondria through the autophagy-lysosome pathway. As a key mechanism of mitochondrial quality control, mitophagy preserves energy production, limits oxidative damage, and maintains mitochondrial network integrity. This process is regulated by pathways such as PINK1-Parkin and receptor-mediated mechanisms involving BNIP3 and FUNDC1, all of which help sustain cellular health by preventing mitochondrial dysfunction. Impaired mitophagy is a common feature of several neurodegenerative diseases, including Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS), and Huntington's disease, exacerbating mitochondrial damage and neuronal stress. Emerging therapeutic strategies that target mitophagy-ranging from pharmacological agents and gene therapies to dietary interventions-show promise in restoring mitochondrial quality and protecting neurons from degeneration. Nevertheless, challenges remain in translating these findings into effective clinical treatments. Mitophagy represents a critical mechanism for preserving neuronal integrity and offers a compelling target for innovative therapies against neurodegenerative disorders.

DOI: https://doi.org/10.1038/s41514-026-00424-3
Cell Death Discov. 2026 Jun 05.

An electrophysiological and proteomics roadmap for human induced glutamatergic neurons: fine-tuning of culture conditions for pathophysiological studies.

Martina Servetti, Giulia Parodi, Martino Caramia, Ennio Nano, Martina Bartolucci, Antonella Marte, Giacomo Mazzoni, Simone Giubbolini, Farah Diab, Andrea Petretto, Pierluigi Valente, Sergio Martinoia, Simona Baldassari, Anna Fassio, Fabio Benfenati, Anna Corradi, Bruno Sterlini.

Induced glutamatergic neurons (iGluNeurons) generated by Neurogenin-2 (NGN2) overexpression in human pluripotent stem cells are a powerful model for studying human neuronal maturation and function; however, NGN2-based protocols still lack standardized culture conditions that critically affect neuronal development and function. Three key factors have been identified by previous literature, namely the composition of extracellular matrix coating, the initial plating density, and the choice of culture medium, but the differential effects of their combination have not been thoroughly analyzed. Here, we investigated the combinatorial effects of these three variables, testing eight distinct culture conditions resulting from the combinations of two coatings (poly-L-ornithine and polyethyleneimine), two media (BrainPhys and Neurobasal), and two cell densities (4800 and 1200 cells/mm²). We assessed electrophysiological properties at the single-cell and network levels, characterized morphofunctional and proteomic features across multiple developmental stages. Electrophysiological data indicate that medium composition and plating density, rather than substrate coating, determine neuronal maturation dynamics, with BrainPhys and high density promoting rapid but transient maturation while Neurobasal and low density supporting gradual and sustained network development. Morphofunctional analyzes of synapses and the axon initial segment, together with neuronal maturation markers, support an early BrainPhys-driven acceleration of development that is later exceeded by Neurobasal. To enable accurate proteome profiling of the iGluNeuron system-comprising human neurons and rat astrocytes-we developed a robust taxonomic filtering algorithm that selectively identifies human-specific proteins. This approach confirmed the presence of a conserved core of NGN2-driven differentiation pathways across all settings, in addition to condition-specific signatures. Finally, in the optimal conditions identified through our experimental analyzes, robust spontaneous and evoked synaptic activity was observed. These results provide a framework for optimizing iGluNeuron cultures, balancing rapid maturation and long-term functional stability, and establishing a benchmark for human neuronal models in disease research and drug screening.

DOI: https://doi.org/10.1038/s41420-026-03185-w
J Nutr. 2026 Jun 03. pii: S0022-3166(26)00279-8. [Epub ahead of print] 101630

Iron Deficiency Impairs Mitochondrial Energetics and Early Axonal Growth and Branching in Developing Hippocampal Neurons.

List Daniel C Mendez, Karishma Devgun, Luke H Carlson, Daniel J Mickelson, Timothy R Monko, Livia Reeves, Lorene M Lanier, Michael K Georgieff, Thomas W Bastian.

   BACKGROUND: Energy deficits underlie many neurodevelopmental, neuropsychiatric and neurodegenerative diseases implicating mitochondria as a potential therapeutic target. Iron is necessary for neuronal energy output through its direct role in mitochondrial oxidative phosphorylation. Iron deficiency (ID) reduces mitochondrial energetic capacity in developing hippocampal neurons and causes simplified dendritic arbors and impaired learning and memory.
OBJECTIVE: To determine the effect of ID on axonogenesis, which has not been previously explored.
METHODS: We used an embryonic mouse mixed-sex primary hippocampal neuron culture model of developmental ID, using iron chelation with low micromolar deferoxamine (DFO) from 3 days in vitro (DIV) to 7DIV compared to untreated control cultures. Mitochondrial respiration and dynamics, cytoskeletal and metabolic gene expression, and axonal and synaptic morphology were quantified and compared using t-test, ANOVA, and multivariate statistical analyses.
RESULTS: 7DIV DFO-treated neuron cultures (n=4-17) demonstrated moderate ID with significantly decreased mRNA levels for genes involved in axon cytoskeletal development (Gda, Pfn2, and Nuak1; ∼20-40% lower) and metabolic homeostasis (Ndufs1, Ddit4, and Slc2a3; ∼20-25% lower). DFO significantly reduced total ATP production rate and measures of mitochondrial oxidative phosphorylation by ∼25-50% compared to control cultures (n=11-14). DFO significantly reduced the length of the primary axon and axonal branches by ∼20%, without affecting branch number (n=100 neurons). Axonal mitochondrial motility was not altered by ID (n=11-12 neurons), suggesting that impaired mitochondrial energetics, and not trafficking, is the predominate mitochondrial contribution to axon morphological deficits. Ultimately, at 18DIV, DFO significantly reduced the density of post-synaptic density puncta, a measure of neuronal capacity for synapse formation, by 30% (n=26-32 neurons).
CONCLUSIONS: These findings provide the first link between iron-dependent neuronal energy production and early axon structural development and highlight the importance of maintaining sufficient iron during the embryonic period of rapid axonal growth to prevent the persistent negative consequences of ID on neuronal structure.

Keywords:  axon; axonogenesis; energy metabolism; gene expression; iron deficiency; mitochondria; mitochondrial motility; mitochondrial trafficking; neuron development

DOI:  https://doi.org/10.1016/j.tjnut.2026.101630
Nucleus. 2026 Dec;17(1): 2675754

High-resolution nuclear cell biology by cryo-electron tomography.

Zanetta Kechagia, Ohad Medalia.

  The nucleus is a structurally diverse and dynamic organelle that anchors chromatin and orchestrates a large number of essential processes, including transcription, replication, ribosome biogenesis, and nucleocytoplasmic transport. Understanding how nuclear macromolecular assemblies are organized and coordinate these processes requires high-resolution imaging methods, capable of achieving sub-molecular resolution while preserving native cellular structures. Cryo-electron tomography (cryo-ET) now provides unprecedented three-dimensional views of nuclear architecture in situ, up to sub-nanometer resolution. In this review, we discuss how cryo-ET has reshaped our understanding of nuclear biology including chromatin organization, nuclear pore complex (NPC) architecture and dynamics, and chromatin - lamina interactions. We highlight how these insights have resolved long-standing debates in biology, linked nuclear structure to function, and set the stage for future developments that will bridge molecular and cellular scales.

Keywords:  Cryo-ET; chromatin; nuclear lamina; nuclear pore complex; nucleus

DOI:  https://doi.org/10.1080/19491034.2026.2675754
J Clin Invest. 2026 Jun 01. pii: e191508. [Epub ahead of print]136(11):

Multimodal analysis of cell-free DNA identifies epigenetic biomarkers for amyotrophic lateral sclerosis diagnosis and progression.

Sebastian Michels, Chaorong Chen, Wolfgang P Ruf, M Madhy Garcia Garcia, Frederick J Arnold, Zhuoxing Wu, Craig L Bennett, Daniel Shams, Leslie M Thompson, Alyssa C Walker, Dennis W Dickson, Leonard Petrucelli, Johannes Dorst, Mercedes Prudencio, Wei Li, Albert R La Spada.

  The role of the epigenome in age-related neurodegenerative disorders remains understudied. Here, we analyzed circulating cell-free DNA (cfDNA) from blood to detect methylation changes as a liquid biopsy for Amyotrophic Lateral Sclerosis (ALS). Our study included 20 patients with sporadic ALS, 10 patients with C9orf72-associated ALS, 10 asymptomatic carriers of the C9orf72 repeat expansion mutation, and 21 nondisease control individuals. Following targeted enzymatic methyl-sequencing (EM-seq) of approximately 4 million CpG sites, we detected numerous differentially methylated genes, including several implicated in ALS disease risk and pathogenesis. By integrating multiple epigenetic features, we delineated a distinct epigenetic signature, which achieved an average area under the curve (AUC) of 0.91 ± 0.10 upon receiver operator characteristic (ROC) analysis, which enabled detection of approximately 70% of patients with ALS with close to 100% specificity. Furthermore, we also identified a set of genes whose methylation status significantly correlated with clinical disease progression and cerebrospinal fluid (CSF) neurofilament levels. Our results reveal the potential of cfDNA-based biomarkers to accurately diagnose ALS and potentially predict disease progression.

Keywords:  Biomarkers; Epigenetics; Genetics; Neurodegeneration; Neuroscience

DOI:  https://doi.org/10.1172/JCI191508
J Am Chem Soc. 2026 Jun 02.

Site-Specific Raman Probes Reveal Droplet Aging and Residue-Level Fibril Polymorphism in TDP-43CTD.

Matthew D Watson, Jennifer C Lee.

The C-terminal domain of TAR DNA-binding protein 43 (TDP-43CTD) drives both liquid-liquid phase separation (LLPS) and amyloid formation. Understanding how TDP-43CTD droplets convert into amyloid aggregates, a process implicated in amyotrophic lateral sclerosis and frontotemporal dementia, requires methodology capable of site-specific structural characterization with spatial resolution. Here, we used confocal Raman spectroscopy in conjunction with an alkyne-modified amino acid (4-ethynyl-l-phenylalanine, FCC) to probe aging in individual TDP-43CTD droplets at seven aromatic sites. While nascent droplets are composed of disordered proteins, β-sheet conformers develop in aged droplets and amyloid aggregates. All three states are spectrally distinct via the alkyne stretching band, with sensitivity that varies depending on the aromatic site probed. C-terminal sites (Y374FCC, W385FCC, and F397FCC) are highly sensitive amyloid probes, revealing multiple polymorphs at the single-residue level that are not resolvable by global secondary structure or morphological characterization alone. Strikingly, while W334FCC abolishes β-sheet formation in droplets, de novo aggregation still occurs, demonstrating that droplet aging is not required for amyloid formation. Given its broad applicability to other proteins and compatibility with cellular imaging, this work establishes a generalizable approach for investigating conformational changes underlying LLPS and amyloid formation in cellulo.

DOI: https://doi.org/10.1021/jacs.6c07727
iScience. 2026 Jun 19. 29(6): 116057

High level dynein impairs mitochondrial distribution and differentiation of rhabdomyosarcoma cells.

Ting-Ling Ke, Linyi Chen.

  Rare disease rhabdomyosarcoma-derived RD cells and RH30 cells are defective in myogenesis. In this study, we demonstrate that mitochondria in these cells are enlarged and display a perinuclear distribution. Given that impaired mitochondrial morphology, trafficking, and activity are implicated in many human diseases, characterizing the link between these phenotypes and their physiological outcomes is essential. We found that RD cells had reduced levels of the myosin motor MYO19 and elevated levels of the dynein motor and MIRO1/2 adaptors. Our findings indicate that impaired local actin-based anterograde transport, together with enhanced microtubule-based retrograde transport, drives this perinuclear mitochondrial clustering. Overexpression of MYO19 in RD cells partially rescued this phenotype, while dynein inhibition altered mitochondrial distribution and restored myogenic differentiation in both RD and RH30 cells. Collectively, these findings reveal an intricate interplay among mitochondrial morphology, distribution, and myoblast fusion that underlies both normal physiology and disease.

Keywords:  Cell biology; Molecular biology; Specialized functions of cells

DOI:  https://doi.org/10.1016/j.isci.2026.116057
Curr Opin Cell Biol. 2026 May 30. pii: S0955-0674(26)00039-6. [Epub ahead of print]101 102651

Building the autophagosome: Molecular logic and membrane dynamics of autophagy.

Shilpa Gopan, Sharon A Tooze.

Autophagy is initiated by the formation of a double-membrane autophagosome which is fine-tuned by the involvement of multiple protein machineries, organelles, and membrane pools. Autophagosome formation proceeds through steps requiring membrane nucleation, membrane expansion, and vesicle closure, initiated and coordinated by the cohort of ATG (Autophagy) proteins and lipids, such as PI(3)P and PE. Recent studies provide insights into how different molecular machineries act and interact to enable this complex vesicular pathway. Here, we review the current understanding of the steps that lead to autophagosome formation from a molecular perspective and, in this context, discuss the role of protein-membrane crosstalk in moulding the phagophore structure.

DOI: https://doi.org/10.1016/j.ceb.2026.102651
Protein Sci. 2026 Jul;35(7): e70662

Regulation of mitochondrial protein translocases.

Lea Bertgen, Agnieszka Chacinska.

  Mitochondria are essential for cellular health, and their function is underlain by the plasticity of the mitochondrial proteome. Most mitochondrial proteins are nuclear encoded, synthesized in the cytosol, and require precise import into mitochondrial subcompartments to fulfill their proper functions. Multimeric mitochondrial translocases ensure accurate protein localization and membrane integration. Recent work has begun to reveal how translocase activity and composition are dynamically regulated within mammalian cells. This review discusses regulatory mechanisms, including phosphorylation and protein degradation, that emerge as important players in adjusting the capacity and/or selectivity of the mitochondrial translocase to metabolic demands. Particular emphasis will be placed on the TIM23 complex as an emerging regulator of the inner membrane and matrix proteome composition.

Keywords:  TIM23 complex; TOM complex; mitochondria; mitochondrial biogenesis; proteases; protein translocases; protein turnover

DOI:  https://doi.org/10.1002/pro.70662
Chembiochem. 2026 Jun 15. 27(11): e202500831

HaloTag Fusion Enables Dynamic Analysis of Prion Protein Biosynthesis, Turnover, and Misfolding.

Antonio Masone, Eleonora Busani, Roberto Chiesa.

  Understanding the dynamics of prion protein (PrP) biosynthesis, misfolding, and degradation is essential for elucidating prion disease pathogenesis and exploring therapeutic strategies. Here, we developed a HaloTag-based PrP fusion (PrP-Halo) that enables real-time analysis of PrP biosynthesis, conformational changes, and turnover in living cells. The HaloTag moiety allows specific, covalent labeling with fluorescent ligands, facilitating pulse-chase imaging and biochemical tracking of distinct PrP subpopulations. PrP-Halo maintains physiological localization, trafficking, and proteolytic processing, faithfully recapitulating the behavior of native PrP. Using this system, we captured early misfolding events induced by pathogenic mutations. Moreover, we show that PrP-Halo can be used to probe the mechanisms of action of PrP-lowering compounds, distinguishing their effects on newly synthesized versus cell-surface PrP. Together these findings establish PrP-Halo as a versatile and sensitive tool for dissecting the molecular and cellular mechanisms underlying PrP misfolding and turnover.

Keywords:  drug screening; neurodegeneration; prion disease; prion protein degraders; protein misfolding

DOI:  https://doi.org/10.1002/cbic.202500831