bims-axbals 2026-05-17 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–05–17
27 papers selected by
TJ Krzystek

Investigating how changes in the levels of kinesins impact neuronal health in Drosophila and human iPSC-derived neurons AD model.
TDP-43: [GU]-ardian of the transcriptome.
Cytoskeletal Imbalance and Axonal Vulnerability in Sporadic PSP-RS: Early Changes in a Human iPSC-Derived Neuronal Model with Altered mTOR Signaling.
Generation of spinal cord organoids from human induced pluripotent stem cells caudalised to a lumbar fate.
Distinct Rab11-associated membrane trafficking pathways bidirectionally control neuronal extracellular vesicle cargo traffic.
Maintenance and disruption of the physiological dimer structure of TDP-43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration.
Methodology for human-induced pluripotent stem cell-derived excitatory and inhibitory neuron coculture with astrocytes for Alzheimer's disease modelling.
Induced pluripotent stem cells from a transgenic minipig model of Huntington's disease reveal early metabolic changes.
S-acylation of TDP43 regulates its condensation in amyotrophic lateral sclerosis.
Distinct mitochondrial-derived vesicle pathways connect mitochondria with peroxisomes and lysosomes.
APOE ε4 influences the widespread TDP-43 pathological subtype in sporadic amyotrophic lateral sclerosis.
Axonal dying back of upper motor neurons in human ALS.
NaV1.7 mRNA and Protein Expression in Resident Neurons of the Human Spinal Dorsal Horn.
Ultrastructural studies reveal a new role for JIP3/MAPK8IP3 in endo-lysosomal maturation within the neuronal cell body.
Disruption of the SYNGAP1 PDZ ligand motif accelerates differentiation of human iPSC-derived GABAergic neurons.
ER-driven Contact Sites in Autophagosome Biogenesis Sequence.
Regulation between LRRK2 and PP2A signaling in cellular models of Parkinsons disease.
Early α-synuclein-mediated mitochondrial dysfunction in a human cell model of Parkinson's disease dementia.
Single-molecule mitochondrial DNA imaging reveals heteroplasmy dynamics shaped by developmental bottlenecks and selection in vivo.
A Cell-Based Protocol to Assess Manganese Content and Relative Transport Activity of Manganese Transporters.
Automated stem cell-derived organoid platforms for disease modeling.
Peroxisome calcium uptake is dependent on ER-peroxisome membrane contact.
Dissecting acute neuronal responses to glioblastoma using a dual-interface human iPSC neuronal culture platform.
Methods for Autophagy Detection by Fluorescence Microscopy.
Quantitative FRET FLIM imaging reveals multiple interactions between proteins of the ESYT, ORP and VAP families at the endoplasmic reticulum membrane.
In the absence of mitochondrial fusion unequal segregation of mitochondria drives mtDNA loss.
Identification of small-molecule autophagy activators via GFP-LC3 high-throughput screening and a cargo-based autophagy flux and processing assay.

Open Biol. 2026 May 13. pii: 250319. [Epub ahead of print]16(5):

Investigating how changes in the levels of kinesins impact neuronal health in Drosophila and human iPSC-derived neurons AD model.

Deepthy Francis, Francesco Paonessa, Victoria L Hewitt, Maria Southall, Isabel Peset, Alexander J Whitworth, Frederick J Livesey, Caroline C G Fabre, Isabel M Palacios.

  Alzheimer's disease (AD) is the leading cause of dementia and the most common neurodegenerative disorder. Understanding the molecular pathology of AD may help identify new ways to reduce neuronal damage. In the past decades, Drosophila has become a powerful tool in modelling mechanisms underlying human diseases. Here, we investigate how the expression of the human 42-residue β-amyloid (Aβ) carrying the E22G pathogenic 'Arctic' mutation (Aβ42Arc) affects axonal health and behaviour in Drosophila. We find that Aβ42Arc flies present aberrant neurons, with altered axonal transport of mitochondria and aberrant terminal boutons at neuromuscular junctions. We demonstrate that the motor proteins kinesin-1 and kinesin-3 are essential for the correct development of neurons in Drosophila larvae and in human induced pluripotent stem cell-derived cortical neurons. We then show that the overexpression of kinesin-1 or kinesin-3 restores the correct number and morphology of boutons in Aβ42Arc-expressing neurons and rescues neuronal function measured by negative geotaxis locomotor behavioural assay. We therefore provide new evidence towards understanding the mechanisms of axonal transport defects in AD, and our results support the idea that kinesins should be considered as potential drug targets to help reduce dementia-associated disorders.

Keywords:   Drosophila neurons; Alzheimer’s disease; amyloid beta; axonal transport; human neurons; induced pluripotent stem cell; motor proteins; neurodegeneration

DOI:  https://doi.org/10.1098/rsob.250319
Mol Neurodegener. 2026 May 14.

TDP-43: [GU]-ardian of the transcriptome.

Irika R Sinha, Abigail L Atkinson, Katherine E Irwin, Jonathan P Ling, Philip C Wong.

TDP-43 is a ubiquitously expressed, primarily nuclear DNA/RNA-binding protein implicated in neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD). In this review, we examine the structure and regulation of TDP-43, how these features influence its localization and functional activity, and how their disruption may contribute to disease. Among TDP-43's diverse functions, splicing repression of nonconserved RNA sequences termed cryptic exons has emerged as especially central to human disease. TDP-43 nuclear depletion and cytoplasmic aggregation are well-established pathological features in affected neurons and glia of neurodegenerative diseases, and accumulating evidence suggests that loss of TDP-43-mediated splicing repression occurs presymptomatically in disease. Advances in RNA-sequencing have enabled systematic identification of cryptic exon inclusion as a sensitive marker of TDP-43 dysfunction. Here, we synthesize current knowledge of TDP-43 biology and curate datasets from human tissues and experimental models, focusing on cryptic splicing to provide a resource for leveraging cryptic exon biology to better understand, detect, and target TDP-43 dysfunction.

DOI: https://doi.org/10.1186/s13024-026-00944-2
Cells. 2026 Apr 23. pii: 754. [Epub ahead of print]15(9):

Cytoskeletal Imbalance and Axonal Vulnerability in Sporadic PSP-RS: Early Changes in a Human iPSC-Derived Neuronal Model with Altered mTOR Signaling.

Raffaele Covello, Giorgia Lucia Benedetto, Stefania Scalise, Caterina Gabriele, Desirèe Valente, Clara Zannino, Barbara Puccio, Andrea Quattrone, Pietro Hiram Guzzi, Marco Gaspari, Aldo Quattrone, Giovanni Cuda, Elvira Immacolata Parrotta.

  Progressive supranuclear palsy-Richardson's syndrome (PSP-RS) is a primary 4R tauopathy in which early axonal dysfunction may precede overt neurodegeneration; however, the mechanisms linking Tau dysregulation to cytoskeletal vulnerability remain poorly defined. Here, we generated induced pluripotent stem cell (iPSC)-derived midbrain dopaminergic neurons from individuals with sporadic PSP-RS and matched healthy controls and performed integrated transcriptomic and proteomic analyses. PSP-RS neurons exhibited coordinated suppression of dopaminergic and synaptic programs alongside activation of cytoskeletal remodeling and stress-related pathways. These changes were accompanied by increased Tau phosphorylation, neurofilament accumulation, and structural alterations of the axonal compartment, consistent with an early axonopathic phenotype. Notably, mechanistic target of rapamycin (mTOR) signaling significantly increased. Pharmacological inhibition of mTOR reduced Tau phosphorylation and neurofilament levels, indicating that mTOR activity contributes to the maintenance of cytoskeletal imbalance. In conclusion, our findings support a model in which early cytoskeletal dysfunction in PSP-RS arises from the convergence of Tau dysregulation, impaired structural homeostasis, and altered signaling pathways. Rather than acting as a primary driver, mTOR appears to function as a pathogenic amplifier that sustains axonal stress. This study provides a human cellular framework to investigate early axonopathic mechanisms in sporadic PSP-RS.

Keywords:  Tau pathology; axonal degeneration; cytoskeletal dysregulation; dopaminergic neurons; mTOR signaling; progressive supranuclear palsy

DOI:  https://doi.org/10.3390/cells15090754
Sci Rep. 2026 May 14.

Generation of spinal cord organoids from human induced pluripotent stem cells caudalised to a lumbar fate.

Li Jun Loh, Pratibha Panwar, Shila Ghazanfar, Kwaku Dad Abu-Bonsrah, Forough Habibollahi, Joel Mason, Brett J Kagan, Bradley J Turner, Samantha K Barton.

  Organoids offer a powerful platform to model human development and disease in vitro, while preserving key features of in vivo tissue architecture and complexity. In this study, we developed a protocol to generate human induced pluripotent stem cell (iPSC)-derived spinal cord organoids patterned to the lumbar region. Through immunofluorescent labelling and single-cell RNA sequencing analyses of these lumbar spinal cord organoids, we identified an enriched neuronal population complemented by a diverse array of glial subtypes that successfully recapitulate the ventral spinal cord, demonstrating greater anatomical relevance than conventional 2D motor neuron cultures. Notably, these organoids displayed functional neuronal properties, including spontaneous activity, indicative of integrated neural networks. This spinal cord organoid platform provides a physiologically relevant model for investigating human spinal cord development and presents a promising tool for studying neurodegenerative diseases and spinal cord injury in a controlled, human-specific context.

Keywords:  Differentiation; Lumbar; Motor neurons; Organoid; Pluripotent stem cell; Spinal cord

DOI:  https://doi.org/10.1038/s41598-026-45679-8
bioRxiv. 2026 Feb 25. pii: 2026.02.25.708063. [Epub ahead of print]

Distinct Rab11-associated membrane trafficking pathways bidirectionally control neuronal extracellular vesicle cargo traffic.

Amy L Scalera, Cassandra R Blanchette, Erica C Dresselhaus, Eric Gomez, Jack Y Cheng, Avital A Rodal.

Neuronal extracellular vesicles (EVs) are released from synapses, and play roles in cellular communication, proteostasis, and the spread of toxic proteins in disease. The small GTPase Rab11 is required to maintain a reservoir of EV cargoes at presynaptic terminals, but how its diverse effector proteins contribute to this function and where Rab11 acts in neurons remains unclear. Using Drosophila motor neurons as a model, we show that EV cargoes redistribute from synapses to axons and cell bodies in rab11 mutants, concomitant with reduced release from synapses. We conducted a directed genetic screen of Rab11-associated factors and found that they have distinct roles in EV trafficking. Tethering and sorting factors are required to maintain levels of presynaptic EV precursors, supporting the hypothesis that Rab11 regulates EV cargo pools through recycling flux rather than by directly mediating EV release. Unexpectedly, we found that different classes of Rab11-associated proteins have opposite functions: the motor protein MyoV and the PI4KIIIα component Rbo sustain cargo levels at synapses, while the motor adaptor Nuf/Rab11FIP4 and the PI4KIIIβ homolog Fwd restrict cargo levels. Together, these results indicate that Rab11 regulates multiple distinct organelle transport trajectories and PI(4)P populations to direct EV cargoes toward different cellular fates.

DOI: https://doi.org/10.64898/2026.02.25.708063
BMC Med. 2026 May 14.

Maintenance and disruption of the physiological dimer structure of TDP-43 in amyotrophic lateral sclerosis and frontotemporal lobar degeneration.

Yoshitaka Tamaki, Shunya Kaneko, Makoto Urushitani.

   BACKGROUND: Transactive response DNA-binding protein of 43 kDa (TDP-43) is an essential regulator of RNA metabolism, playing a pivotal role in splicing, transport, and stability. While its cytoplasmic aggregation is the pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), recent evidence suggests that the earliest pathogenic event is the disruption of its physiological homodimeric structure. Under healthy conditions, TDP-43 forms dimers via its N-terminal domain, a configuration that is crucial for its nuclear solubility and cooperative RNA binding. In this review, we propose the "Molecular Zipper" hypothesis to describe the maintenance of TDP-43 structural homeostasis. In this framework, the N-terminal domain acts as a stabilizing "NTD-mediated anchor" that keeps the protein in a functional, "zipped" dimeric state, effectively sequestering its aggregation-prone C-terminal regions. Pathogenic triggers-including genetic mutations, aberrant post-translational modifications such as phosphorylation and acetylation, and environmental stressors-can "unzip" this structure, leading to the formation of pathogenic monomers. These pathogenic monomers show increased propensity for cytoplasmic mislocalization and recruit wild-type protein into aggregates through a prion-like seeded aggregation mechanism, culminating in nuclear functional loss and cytoplasmic gain-of-toxicity. We further evaluate the emerging diagnostic landscape, focusing on methods to monitor the dimer-to-monomer ratio.
SHORT CONCLUSION: Integrating prior biochemical data on TDP-43 dimerization with structural modeling enables a more coherent account of the transition from the physiological dimer to pathological conformers. The Molecular Zipper framework offers a conceptual foundation for reconciling existing experimental findings and for guiding future studies on early structural changes in TDP-43 proteinopathy.

Keywords:  Molecular Zipper hypothesis; NTD; RRM; TDP-43; dimerization

DOI:  https://doi.org/10.1186/s12916-026-04935-4
Brain Commun. 2026 ;8(3): fcag135

Methodology for human-induced pluripotent stem cell-derived excitatory and inhibitory neuron coculture with astrocytes for Alzheimer's disease modelling.

Jialin Li, Lucy E Brown, Naiomi Rambarack, Andi Chan, Priya Prakash, Charles Arber, Selina Wray, Afia B Ali.

  Alzheimer's disease symptoms include gradual cognitive decline and memory loss that is correlated with progressive loss of neuronal connections due to an imbalance of excitatory and inhibitory synaptic functions. These have been shown in various rodent models but direct measurements of excitatory-inhibitory changes have yet to be performed in human neurons. Therefore, our project aims to construct a human-induced pluripotent stem cell co-culture model representing important brain circuitry which captures synaptic dysfunction. Familial Alzheimer's disease patient induced pluripotent stem cells carrying mutant APP V717I and their isogenic controls were differentiated into cortical glutamatergic neurons and astrocytes using dual-SMAD inhibition followed by in vitro corticogenesis. Building upon this, we differentiated inhibitory interneurons expressing parvalbumin and somatostatin via ventral patterning with sonic hedgehog activation. Then, we co-cultured these cells with differentiated cortical neurons and astrocytes. The properties of the co-culture model were validated using immunohistochemistry, confocal microscopy combined with electrophysiological whole-cell recordings. Confocal microscopy validated the presence of excitatory cortical neurons, astrocytes, and two inhibitory interneuron types, parvalbumin and somatostatin expressing interneurons within the co-culture. Whole-cell recordings revealed intrinsic membrane properties from individual excitatory and inhibitory neurons in this co-culture from day 70 onwards. Spontaneous synaptic activity recorded from the APP V717I-induced pluripotent stem cell model showed synaptic hyperexcitability correlated with altered morphological changes, which was expected in contrast to the isogenic control co-culture. Our novel co-culture model, including astrocytes, excitatory and inhibitory neurons, represents a strong model of brain circuitry in Alzheimer's disease. These models will enable investigation of Alzheimer's disease causative mutations on neuronal connectivity in human neurons allowing for confirmation of network dysfunction in Alzheimer's disease in human neurons. It also has the potential of becoming a valuable preclinical tool to screen novel targeted therapies. This is a methods paper validating a human-induced pluripotent stem cell-derived excitatory-inhibitory neuron-astrocyte co culture with electrophysiological readouts and immunostaining, focusing on Alzheimer's disease relevant network physiology.

Keywords:  Alzheimer's disease; astrocytes; electrophysiology; induced pluripotent stem cell; interneurons

DOI:  https://doi.org/10.1093/braincomms/fcag135
Dis Model Mech. 2026 06 01. pii: dmm052585. [Epub ahead of print]19(6):

Induced pluripotent stem cells from a transgenic minipig model of Huntington's disease reveal early metabolic changes.

Irena Rysankova, David Sekac, Hana Hansikova, Katerina Vodickova Kepkova, Petr Vodicka, Michaela Vaskovicova, Marie Altmanova, Stefan Juhas, Jana Juhasova, Eliska Taborska, Jiri Klempir, Jan Motlik, Jiri Klima, Lars Eide, Zdenka Ellederova.

  Huntington's disease (HD) is a neurodegenerative autosomal dominant hereditary disease caused by a CAG triplet repeat expansion mutation in the gene encoding the huntingtin (HTT) protein. The main feature of HD is the loss of striatal neurons, accompanied by metabolic and transcriptional alterations in both neural and peripheral tissues. Induced pluripotent stem cells (iPSCs) derived from a transgenic HD (TgHD) minipig model expressing a mutant HTT construct were generated to investigate early metabolic, antioxidant and DNA integrity changes associated with HD development. Gene expression analysis showed increased expression of vascular endothelial growth factor (VEGF), pyruvate dehydrogenase kinase 1 (PDK1) and glutamine-oxaloacetic transaminase 1 (GOT1), implying early metabolic alteration in TgHD iPSCs. Moreover, upregulated FANCD2/FANCI-associated nuclease 1 (FAN1) expression indicated genotoxic stress linked to early HD development. These findings suggest metabolic shifts and putative genotoxic events in the pluripotent stem cell state of the TgHD model and point to early effect of the HD mutation. The model may be suitable for evaluating potential cell therapy and in vitro differentiation of iPSCs to neurons and other cells affected in HD.

Keywords:  DNA damage; Gene expression; Huntington's disease; Induced pluripotent stem cells; Mitochondria; Transgenic minipig model

DOI:  https://doi.org/10.1242/dmm.052585
Mol Cell. 2026 May 12. pii: S1097-2765(26)00270-4. [Epub ahead of print]

S-acylation of TDP43 regulates its condensation in amyotrophic lateral sclerosis.

Wentao Xu, Huina Li, Wei Zhang, Ge Bai, Chengyong Shen, Kejing Zhang.

  TDP43 inclusion bodies are widely present in the majority of patients with familial and sporadic amyotrophic lateral sclerosis (ALS). The mechanisms regulating TDP43 solubility remain incompletely understood. Here, we report that TDP43 undergoes S-acylation primarily at the Cys244 residue by the S-acyltransferase zDHHC23. This S-acylation maintains the liquid-like properties of TDP43 by reducing the aberrant interaction with poly(ADP-ribose) polymerase 1 (PARP1) and PARylated proteins, thereby countering the pathological condensation of TDP43. S-acylation-deficient TDP43 inclusions sequester the translational machinery and inhibit cytoplasmic protein translation, ultimately resulting in neurotoxicity. Importantly, TDP43 S-acylation is decreased in the familial ALS-associated TDP43 mutants as well as in SOD1-G93A mice and C9orf72-ALS induced pluripotent stem cell (iPSC)-derived neurons, suggesting the widespread involvement of TDP43 S-acylation in ALS pathogenesis. Our findings reveal an undescribed modification of TDP43 and provide deeper insight into the regulation of TDP43 pathological condensation in ALS.

Keywords:  S-acylation; TDP43; aggregation; amyotrophic lateral sclerosis; condensation

DOI:  https://doi.org/10.1016/j.molcel.2026.04.016
Mitochondrion. 2026 May 12. pii: S1567-7249(26)00057-7. [Epub ahead of print]90 102167

Distinct mitochondrial-derived vesicle pathways connect mitochondria with peroxisomes and lysosomes.

Ayumu Sugiura, Kohta Nakamura, Heidi M McBride, Yasushi Okazaki.

  Mitochondrial-derived vesicles (MDVs) mediate selective trafficking of mitochondrial proteins and lipids to other organelles and contribute to organelle communication and mitochondrial quality control. While MDVs that deliver mitochondrial cargo to lysosomes have been extensively studied, the diversity of MDV pathways linking mitochondria to peroxisomes remains poorly understood. In particular, it is unclear how MDV pathways targeting peroxisomes relate to those delivering cargo to lysosomes, and whether cargos targeted to pre-existing peroxisomes utilize the same vesicular intermediates that participate in de novo peroxisome biogenesis. Here we examined MAPL trafficking using a peroxisome reconstitution system in PEX3-deficient fibroblasts. We found that MAPL is excluded from PEX3-positive pre-peroxisomal vesicles and instead is delivered to pre-existing peroxisomes, indicating that MAPL trafficking occurs through a pathway distinct from vesicles that initiate peroxisome formation. Structure-function analysis further revealed that a C-terminal amphipathic helix within MAPL is required for efficient targeting to peroxisomes. SNX9 depletion impaired both MAPL delivery to pre-existing peroxisomes and stress-induced lysosomal MDV pathways, whereas VPS35 depletion selectively reduced MAPL delivery without affecting lysosomal MDV pathways. In contrast, Parkin depletion impaired lysosomal MDV pathways but did not influence MAPL trafficking. Together, these findings demonstrate that mitochondria generate multiple classes of MDVs that are sorted into mechanistically distinct trafficking routes linking mitochondria with peroxisomes and lysosomes.

Keywords:  Lysosomes; Mitochondria; Mitochondrial-derived vesicles; Peroxisomes

DOI:  https://doi.org/10.1016/j.mito.2026.102167
Acta Neuropathol. 2026 May 15. pii: 57. [Epub ahead of print]151(1):

APOE ε4 influences the widespread TDP-43 pathological subtype in sporadic amyotrophic lateral sclerosis.

Yuya Hatano, Asa Nakahara, Mari Tada, Akiyoshi Kakita, Osamu Onodera, Tomohiko Ishihara.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder, most sporadic cases exhibiting TAR DNA-binding protein 43 (TDP-43) pathology. The anatomical distribution of TDP-43 pathology varies among patients; however, factors contributing to this heterogeneity remain unclear. Apolipoprotein E (APOE) ε4 is known to influence the spread of pathological protein in several neurodegenerative diseases, raising the possibility that it also modulates the pathological distribution of TDP-43 inclusions in ALS. We investigated this hypothesis in a cohort of 145 autopsy-confirmed sporadic ALS cases. ALS-associated TDP-43 pathology was classified into two subtypes: type 1 - largely restricted to motor regions - and type 2 - characterized by widespread cortical involvement. APOE genotypes and rare variants in known ALS-associated genes were determined by exome sequencing. Amyloid-β and tau pathologies were assessed neuropathologically using established staging systems. Structural equation modeling (SEM) was applied to disentangle direct and indirect relationships among APOE ε4, temporal clinical parameters, Alzheimer's disease-related pathologies, and ALS TDP-43 subtype. Furthermore, we also performed an unbiased evaluation using random forest model. APOE ε4 carriers showed a significantly higher proportion of type 2 pathology than non-carriers. Bayesian SEM demonstrated that APOE ε4 was directly associated with the type 2, widespread TDP-43 subtype, independent of amyloid-β and tau pathology, while also reproducing the canonical cascade linking APOE ε4 to amyloid-β and tau. Rare variants in ALS-associated genes showed no clear effect on TDP-43 subtype. These findings indicate that APOE ε4 modifies the anatomical distribution of TDP-43 pathology in sporadic ALS through mechanisms independent of classical Alzheimer's disease pathology. Incorporation of APOE genotype into ALS stratification may be informative for biologically grounded subtype-specific therapeutic approaches.

Keywords:  APOE; Amyloid-β; Amyotrophic lateral sclerosis; Structural equation modeling; TDP-43 pathology; Tau

DOI:  https://doi.org/10.1007/s00401-026-03029-y
Sci Rep. 2026 May 15.

Axonal dying back of upper motor neurons in human ALS.

Haley C Cropper, Fozia Mir, Jianguo Liu, Fabien Dachet, Vidushi R Srivastava, Mohammed Ramizuddin, Kylie Kopecky, Ebony Mocanu, Qin Li Jiang, Madhu Soni, Tibor Valyi-Nagy, Diana Mnatsakanova, Charles K Abrams, Fei Song, Jeffrey A Loeb.

  Patients with amyotrophic lateral sclerosis (ALS) typically present with arm, leg, or bulbar weakness. While genetics plays a clear role, it cannot explain why symptoms start focally or how upper (UMN) and lower motor neuron (LMN) systems are linked. In this clinicopathological case series, we examined the relationships between UMN/LMN disease in ten ALS patients. Detailed clinical assessments and motor cortex, brainstem, and spinal cord tissues were collected via rapid autopsy. Tissues were stained for UMN/LMN, myelin, axons, microglia, and pTDP43, and RNA-sequencing was performed. None of the patients had symptoms of frontotemporal dementia (FTD), but all had focal sites of clinical onset and both UMN/LMN involvement. LMN degeneration and microglial activation were highest at disease onset sites. UMN degeneration was present at all spinal cord levels through the medulla, regardless of onset site. Surprisingly, there was no evidence of UMN axonal degeneration above the brainstem. While extensive pTDP43 aggregates were seen in degenerating LMNs, no pTDP43 aggregates were seen in UMN cell bodies or their axons. RNA-sequencing implicated inflammatory pathways at sites of disease onset. Our findings suggest that some ALS patients without FTD have a dying back of UMN axons rather than a primary upper neuronopathy of neurons.

Keywords:  amyotrophic lateral sclerosis; corticospinal tract; lower motor neuron; neuroinflammation; upper motor neuron

DOI:  https://doi.org/10.1038/s41598-026-52496-6
J Comp Neurol. 2026 May;534(5): e70168

NaV1.7 mRNA and Protein Expression in Resident Neurons of the Human Spinal Dorsal Horn.

Stephanie Shiers, Nwasinachi A Ezeji, Carla Lima-Truax, Jeffrey L Krajewski, Geoffrey Funk, Anna Cervantes, Peter Horton, Gregory Dussor, Stephanie Hennen, Theodore J Price.

  The voltage-gated sodium channel NaV1.7 is a pain target supported by human genetics, and many compounds have been developed to inhibit NaV1.7 but have disappointed in clinical trials due to cardiovascular effects. Because some recent reports suggest that pharmacological inhibition of NaV1.7 in the rodent spinal dorsal horn can achieve pain relief, we sought to better understand NaV1.7 expression in the human spinal cord. We report that NaV1.7 mRNA is expressed in putative projection neurons (NK1R+, GPR83+) in the human spinal dorsal horn, predominantly in lamina I and II, as well as in deep dorsal horn neurons. NaV1.7 mRNA was also detected in preganglionic parasympathetic and sympathetic neurons, motor neurons in the ventral horn, and ependymal cells lining the central canal. NaV1.7 protein was predominantly found in the central axons of sensory neurons terminating in lamina I-II and colocalized, in part, with presynaptic markers like Bassoon and CGRP. Postsynaptically, NaV1.7 protein was detectable in the soma of motor neurons but was more elusive in dorsal horn populations due to the abundance of presynaptic signal. However, NaV1.7 protein was detected in the axon initial segment of some resident dorsal horn neurons and in axons entering the anterior commissure. Given that projection neurons are critical for conveying nociceptive information from the dorsal horn to the brain, these data support that dorsal horn NaV1.7 expression may play an unappreciated role in pain phenotypes observed in humans with genetic SCN9A mutations.

Keywords:  NaV1.7; dorsal horn; spinal cord

DOI:  https://doi.org/10.1002/cne.70168
Mol Biol Cell. 2026 May 13. mbcE25050248T

Ultrastructural studies reveal a new role for JIP3/MAPK8IP3 in endo-lysosomal maturation within the neuronal cell body.

Mia Krout, Swetha Gowrishankar.

In recent years, substantial heterogeneity in lysosomes, in terms of their composition, function and positioning has come to be recognized. Despite this, there are gaps in knowledge of our understanding of the molecular basis of lysosome heterogeneity, especially in neurons. To this end, we used electron microscopy to define endo-lysosomal organelles of human iPSC-derived neurons, at the ultrastructural level. Through this, we identify endolysosomal maturation defects within neuronal cell bodies of iNeurons lacking JNK-Interacting Protein 3 (JIP3), a lysosome adaptor previously known to primarily regulate axonal lysosome movement. Loss of JIP3 results in an expansion of immature lysosomes within neuronal soma, along with a concomitant decrease in mature lysosomes. JIP3 loss also leads to delayed trafficking of endocytic cargo through these compartments, implicating JIP3 in regulation of endolysosomal maturation within neuronal cell bodies. Our studies highlight the utility of electron microscopy in understanding neuronal lysosomal heterogeneity. Additionally, given recent links between JIP3 and a neurodevelopmental disorder, our findings here could provide new insight into mechanisms underlying neurodevelopmental pathology.

DOI: https://doi.org/10.1091/mbc.E25-05-0248-T
bioRxiv. 2026 Feb 25. pii: 2026.02.24.707848. [Epub ahead of print]

Disruption of the SYNGAP1 PDZ ligand motif accelerates differentiation of human iPSC-derived GABAergic neurons.

Jianzhi Jiang, Ruslan Rust, Ilse Flores, Yaowen Feng, Peyman Nouri, Veronica A Clementel, Amir Arya, Ali Basirattalab, Ivy Y Yang, Antigoni Manousopoulou, Spiros D Garbis, Nicholas A Graham, Marcelo P Coba.

SYNGAP1 haploinsufficiency is a leading genetic cause of neurodevelopmental disorders (NDD), including intellectual disability and epileptic encephalopathy. While most studies on SYNGAP1 function have focused on glutamatergic neurons, its role in GABAergic neurons and during early neuronal development is unclear. Using human iPSC-derived GABAergic neurons, we demonstrate that SYNGAP1 haploinsufficiency accelerates neuronal maturation, characterized by increased dendritic length, synaptic density, and maturation of synaptic structures. Disruption of the isoform-specific SYNGAP1 PDZ binding motif reproduces these phenotypes, highlighting the critical role of PDZ-mediated interactions in regulating GABAergic neuronal differentiation. Proteomic and phosphoproteomic analyses reveal significant dysregulation of synaptic proteins, RNA processing, and transcriptional control, with a significant increase in postsynaptic density proteins content. RNA-seq analysis suggest that the acceleration in neuronal differentiation starts few hours after neuronal induction setting a path to a faster neuronal and synapse maturation. These findings establish that SYNGAP1 acts as a key regulator of neuronal differentiation across both excitatory and inhibitory neurons. Our work underscores the importance of the SYNGAP1 PDZ ligand motif for normal neuronal development and suggests translational strategies targeting SYNGAP1 alpha1 isoform levels to mitigate SYNGAP1-related NDD.

DOI: https://doi.org/10.64898/2026.02.24.707848
Contact (Thousand Oaks). 2026 Jan-Dec;9:9 25152564261451671

ER-driven Contact Sites in Autophagosome Biogenesis Sequence.

Juliane Da Graça, Dobrawa Amiri Czajkowski, Etienne Morel.

  Autophagosome biogenesis is a highly coordinated membrane remodeling process that relies on the de novo formation and expansion of the phagophore, yet the cellular principles governing its spatial and temporal organization remain incompletely understood. Accumulating evidence now places the endoplasmic reticulum (ER) at the center of this process, not merely as a membrane source, but as a dynamic scaffold that organizes phagophore assembly through extensive membrane contact sites with multiple organelles. ER-mediated contacts with endosomes, mitochondria, the plasma membrane, and ER-Golgi intermediates create specialized microenvironments that integrate signaling, lipid transfer, vesicle formation and trafficking, and biophysical constraints to drive phagophore nucleation and growth. These contact sites enable the coordinated mobilization of diverse membrane carriers and autophagy regulators in a stress- and context-dependent manner. In this review, we discuss how ER-driven membrane contact sites orchestrate autophagosome biogenesis, highlight emerging mechanistic and biophysical concepts, and consider their broader implications for cellular stress adaptation and disease.

Keywords:  ER-contact sites; autophagosome biogenesis; lipid transfer proteins

DOI:  https://doi.org/10.1177/25152564261451671
Biochem J. 2026 May 14. pii: BCJ20260194. [Epub ahead of print]

Regulation between LRRK2 and PP2A signaling in cellular models of Parkinsons disease.

Panagiotis S Athanasopoulos, Anna Memou, Franz Y Ho, Ahmed Soliman, Henderikus Pots, Vassiliki Papadopoulou, Felix von Zweydorf, Saiganesh Sriraman, Alexander Marc Thouin, Laurine Vandewynckel, William Sibran, Marie-Christine Chartier-Harlin, R Jeremy Nichols, Elisa Greggio, Jean-Marc Taymans, Christian Johannes Gloeckner, Hardy J Rideout, Arjan Kortholt.

  Mutations in Leucine-rich repeat kinase 2 (LRRK2) are the most frequent cause of late-onset familial and idiopathic Parkinson's disease (PD), known to date. Importantly, recent data from post-mortem tissue as well as biomarker studies suggest that independent of mutations, increased kinase activity of LRRK2 plays an essential role in idiopathic PD pathogenesis. Despite extensive research on LRRK2, its activation     mechanism(s) and how the various mutations result in increased kinase activity and neuronal death is still not completely understood. Accumulating evidence points to LRRK2 phospho-regulation, both auto-phosphorylation and phosphorylation by other kinases, as one potential molecular trigger of its activation. LRRK2 activation and localization is regulated by phosphatases such as Protein phosphatase 1 (PP1) and Protein phosphatase 2A (PP2A), however the exact mechanism of this phospho-regulation is not known. Our data reveal that in vitro PP2A dephosphorylates sites within the RocCOR-GTPase domain of LRRK2 and as a result de-stabilizes LRRK2 dimers, with consequent reduction of its kinase activity. Strikingly, our data further highlight that LRRK2 in turn phosphorylates the catalytic subunit of the PP2A holoenzyme PPP2CA at its critical residue T304. Furthermore, LRRK2-mediated phosphorylation of PP2CA T304 alters the methylation of the C-terminus, which is crucial for both holoenzyme formation and catalytic activity. Importantly, expression of WT-PPP2CA protects from LRRK2-G2019S induced neuronal cell death, while PPP2CA-T304 mutants fail to do so, suggesting that impaired PP2A holoenzyme formation might be detrimental for LRRK2-PD.

Keywords:  LRRK2; PP2A; Parkinsons disease; Phosphatase; kinase

DOI:  https://doi.org/10.1042/BCJ20260194
Commun Biol. 2026 May 12.

Early α-synuclein-mediated mitochondrial dysfunction in a human cell model of Parkinson's disease dementia.

Maha S Alfaidi, Léa M D Wenger, Kornélia Szebényi, Thomas B Stoker, Xiaoling He, Shaline V Fazal, Jana Sebestikova, Amir Jassim, Richard J Gilbertson, Raquel Garza, Johan Jakobsson, Maria Grazia Spillantini, Roger A Barker, Wei-Li Kuan.

Parkinson's disease (PD) is a progressive neurodegenerative disorder characterised by the misfolding and accumulation of α-synuclein (α-syn) into pathological aggregates known as Lewy bodies. PD remains incurable, partly due to limited physiologically relevant models that recapitulate human pathology to enable therapeutic development. We developed a novel in vitro PD dementia model using fetal human cortical neurons seeded with α-syn preformed fibrils (PFFs). This model successfully replicates key PD features, including α-syn aggregation and mitochondrial gene dysregulation. Importantly, RNA sequencing revealed significant transcriptomic concordance between our model and PD postmortem tissue, particularly in the downregulation of mitochondrial genes linked to oxidative phosphorylation. We then evaluated two peptide inhibitors, β-syn36D (B36D) and S62. Both peptides demonstrated effective disaggregation of α-syn fibrils, with B36D showing particular promise by reversing PFF-induced functional and transcriptional changes to baseline levels. This human-relevant model captures essential pathological and transcriptomic disease hallmarks as well as demonstrating utility for therapeutic screening of drugs that block α-syn aggregation.

DOI: https://doi.org/10.1038/s42003-026-10134-x
Dev Cell. 2026 May 13. pii: S1534-5807(26)00123-1. [Epub ahead of print]61(5): 1146-1161.e8

Single-molecule mitochondrial DNA imaging reveals heteroplasmy dynamics shaped by developmental bottlenecks and selection in vivo.

Rajini Chandrasegaram, Sara Gottardo, Abhilesh Dhawanjewar, Antony M Hynes-Allen, Beitong Gao, Suvagata Roy Chowdhury, Luis-Carlos Tábara, Michele Frison, Stavroula Petridi, Patrick F Chinnery, Julien Prudent, Hansong Ma, Jelle van den Ameele.

  Mitochondrial DNA (mtDNA) exists in many copies per cell, with cell-to-cell variability in mutation load, which is known as heteroplasmy. Developmental and age-related expansion of heteroplasmic mtDNA mutations contributes to the pathogenesis of mitochondrial and neurodegenerative diseases. Here, we describe an approach for in situ sequence-specific detection of single mtDNA molecules (mtDNA-single-molecule fluorescent in situ hybridization [smFISH]). We apply this method to visualize and measure mtDNA and heteroplasmy levels in situ at single-cell resolution in whole-mount Drosophila tissue and cultured human cells. In Drosophila, we identify a somatic mtDNA bottleneck during neurogenesis. This amplifies heteroplasmy variability between neurons, as predicted by a mathematical bottleneck model, predisposing individual neurons to a high mutation load. However, both during neurogenesis and oogenesis, mtDNA segregation is accompanied by purifying selection, promoting wild-type (WT) over pathogenic mtDNA. mtDNA-smFISH thus elucidates how developmental cell-fate transitions, accompanied by changes in cell morphology, behavior, and metabolism, can shape the transmission and selection of deleterious mtDNA variants.

Keywords:  Drosophila; bottleneck; heteroplasmy; mitochondria; mitochondrial DNA; mitochondrial disease; neurogenesis; oogenesis; purifying selection; single-molecule fluorescent in situ hybridization

DOI:  https://doi.org/10.1016/j.devcel.2026.03.011
Bio Protoc. 2026 May 05. 16(9): e5680

A Cell-Based Protocol to Assess Manganese Content and Relative Transport Activity of Manganese Transporters.

Huiwei Zhong, Xurui Shen, Hanting Yang.

  Manganese (Mn) is an essential trace element whose intracellular homeostasis is tightly controlled by specialized membrane transporters. Dysregulation of Mn transport leads to pathological Mn accumulation and severe human disease; however, efficient and quantitative cell-based methods for assessing Mn2+ transporter activity remain limited. Here, we present an optimized cellular Fura-2 manganese extraction assay (CFMEA) that enables robust quantification of cellular Mn content and provides a normalized framework for assessing relative Mn2+ transport activity in a high-throughput format. This protocol integrates Fura-2-based fluorescence detection of Mn2+ at the Ca2+ isosbestic excitation wavelength with dsDNA quantification to normalize dsDNA levels in cell extracts and immunoblotting to account for transporter protein expression levels. Cells expressing Mn2+ transporters are exposed to MnCl2 in 96-well plates, washed to remove extracellular Mn2+, and lysed in a Fura-2-containing extraction buffer. Fluorescence quenched by Mn2+ is quantified and converted to cellular Mn content using a cell-free Mn-Fura-2 standard curve and then normalized to dsDNA content and protein abundance to determine relative transporter activity. This workflow provides a relatively sensitive, reproducible, and low-cost approach for comparative analysis of Mn2+ transporters and their variants across multiple cell types. The protocol is demonstrated using the Mn2+ efflux transporter SLC30A10 in HEK293T cells and is readily adaptable for studying other Mn2+ transport pathways. Key features • High-throughput, cell-based assay for quantifying cellular manganese content and assessing relative Mn2+ transporter function. • Enhanced accuracy and reproducibility by integrating double-stranded DNA quantification and protein normalization into the cellular Fura-2 manganese extraction assay (CFMEA) workflow. • Workflow compatible with diverse cell types and Mn2+ transporters, including systems overexpressing SLC30A10 in HEK293Tcells.

Keywords:  CFMEA; Mn content; Mn2+ transporters; Relative Mn2+ transport activity; dsDNA quantitative assay

DOI:  https://doi.org/10.21769/BioProtoc.5680
Trends Biotechnol. 2026 May 14. pii: S0167-7799(26)00148-4. [Epub ahead of print]

Automated stem cell-derived organoid platforms for disease modeling.

Xingrui Mou, Nathan Dale, Kiran Ramnarine, Filippo Cipriani.

  Complex diseases arise from genetic, environmental, and lifestyle factors, the combination of which is difficult to model. Conventional animal and 2D cell culture models have limitations in scalability, reproducibility, or human relevance. Human-induced pluripotent stem cells (iPSCs) can be differentiated into 3D organoids that better mimic human biology. However, organoid protocols can be lengthy, variable, and labor-intensive, limiting high-throughput applications. Suspension bioreactors and multilineage differentiation have improved yield and function, but challenges remain in tissue maturity, vascularization, and consistency. Automated high-throughput liquid handling systems are emerging as a solution, enabling large-scale, reproducible production. Here, we discuss how combining iPSC-derived organoids with automation is poised to transform disease modeling and drug development.

Keywords:  automation and high-throughput systems; complex disease modeling; iPSC-derived organoids

DOI:  https://doi.org/10.1016/j.tibtech.2026.04.013
Cell Mol Life Sci. 2026 May 12. pii: 207. [Epub ahead of print]83(1):

Peroxisome calcium uptake is dependent on ER-peroxisome membrane contact.

Julia Kalinowski, Yelena Hartmann, Alexander Lütkemeyer, Sven Thoms.

  Peroxisomes are small, highly dynamic organelles involved in a plethora of metabolic pathways. They are essential for the efficient exchange of metabolites and cellular messengers orchestrating intracellular signaling. Calcium (Ca2+) is one of the most prominent physiological signaling elements and regulates a wide variety of processes in cellular homeostasis and function. Recently, we showed that peroxisomes participate in cellular Ca2+ dynamics by taking up and releasing Ca2+ following store-operated calcium entry (SOCE), however, the mechanism of peroxisomal Ca2+ uptake and its modulators remained unknown. Using live cell imaging in combination with genetically encoded calcium indicators (GECI), we show that peroxisomal calcium dynamics are independent of PEX11β and the pore protein PXMP2. Instead, we find that the ACBD5-dependent membrane contact site between peroxisomes and the endoplasmic reticulum (ER) is necessary for efficient peroxisomal Ca2+ uptake. Further, we identify the ACBD5-dependent peroxisome-ER contact site as the major factor restricting peroxisome motility within the cell. Microtubules and SOCE stimulation exert smaller and independent effects on peroxisome motility. This work expands the range of known functions of the peroxisome-ER contact site.

Keywords:  Calcium signaling; FRET sensor; MCS; Membrane contact site; Peroxisomal disorders

DOI:  https://doi.org/10.1007/s00018-026-06191-4
Acta Neuropathol Commun. 2026 May 16.

Dissecting acute neuronal responses to glioblastoma using a dual-interface human iPSC neuronal culture platform.

Ouada Nebie, Niyi Adelakun, Brian Fries, Luke Kollin, Liwen Zhang, Akhil Medikonda, Monica Venere, Pierre Giglio, Nam Chu, Nhat Le.

  Glioblastoma (GB) hijacks neuronal circuits to promote tumor progression, but the earliest neuronal responses remain poorly defined. We developed a dual-interface human iPSC-derived neuronal model to study acute paracrine signaling triggered by GB cells from two sources: serum-adapted U-87MG and serum-free NU-757. Within 24 h, exposed neurons displayed synaptic remodeling and activation of GB-related signaling cascades. Neurons exposed to U-87MG showed decreased dendritic spine number alongside increased total ERK and phospho-p38α at spines (1). In the soma, total ERK accumulated in the nucleus while phospho-ERK was primarily cytoplasmic; nuclear p38 and cytoplasmic MLK2 also increased (2). Conversely, NU-757 exposure enhanced spine growth but reduced postsynaptic density, NMDAR, and synaptophysin levels (3). Both total and phospho-ERK showed increased nuclear localization with NU-757, while total MLK2 and p38α levels remained stable but exhibited elevated nuclear (4) and spine localization. Pharmacological MEK/ERK inhibition reduced U-87MG proliferation and migration and restored neuronal spine numbers. This model system reveals source-dependent, compartment-specific signaling dynamics that govern synaptic vulnerability and provides a platform for investigating GB initiation, recurrence, and progression, as well as therapies targeting tumor growth and neural circuit remodeling.

Keywords:  ERK1/2; Glioblastoma; MAPK signaling; Patient-derived glioblastoma cells; Synaptic remodeling; Tumor-neuron interaction; iPSC-derived neurons; p38α MAPK

DOI:  https://doi.org/10.1186/s40478-026-02312-z
Methods Mol Biol. 2026 ;3039 173-179

Methods for Autophagy Detection by Fluorescence Microscopy.

Xiaoye Ke, Xianglong Zhang, Xiangxiang Zhang.

  Fluorescence microscopy is pivotal for investigating autophagy's role in plant antiviral immunity. Here, we present a standardized procedure using complementary probes, CFP-ATG8f for autophagosomal structures and monodansylcadaverine (MDC) for autophagic vacuoles, to assess autophagy during viral infection. This combined CFP-ATG8f and MDC staining system provides a powerful, reproducible method for evaluating autophagic activity in plant-virus interactions.

Keywords:  Autophagy; CFP-ATG8f; Confocal microscopy; MDC staining

DOI:  https://doi.org/10.1007/978-1-0716-5300-5_20
J Biol Chem. 2026 May 13. pii: S0021-9258(26)02016-8. [Epub ahead of print] 113144

Quantitative FRET FLIM imaging reveals multiple interactions between proteins of the ESYT, ORP and VAP families at the endoplasmic reticulum membrane.

Dounia Zamiati, Mouna Abdesselem, Oliver Nüsse, Marie Erard.

  The endoplasmic reticulum (ER) is a highly dynamic intracellular organelle that forms close contact sites with other organelles and the plasma membrane. These membrane contact sites play essential roles in lipid exchange and calcium homeostasis. ER membrane proteins from the VAP, ORP, and ESYT families are key players in the formation and function of these contacts. Numerous interactions between these proteins are likely critical for their activity. We investigate the interactome between these protein families in live cells by analyzing two members from each family, using Förster Resonance Energy Transfer (FRET) between pairs of proteins labeled with fluorescent proteins (FP). FRET is detected by Fluorescence Lifetime Imaging Microscopy (FLIM). Our quantitative approach shows that all tested proteins clusterize and sometimes interact within their respective families, and that their organization at the nanometer scale can be disrupted by specific mutations or domain deletions. In particular, we show that the coiled-coil domains of ORP5 and ORP8 are required for the formation of both homomeric and heteromeric complexes. Moreover, we demonstrate that FRET-FLIM can detect intra-molecular conformational changes in response to alterations in the cellular environment, such as variations in Ca2+ concentration, as observed for ESYT1. We also identify novel inter-family organization, including clustering between VAPB and ORP8, and between ESYT2 and ORP5/8. Finally, our approach highlights the broad interaction network (interactome) of VAPA/B, and shows how various potential binding partners can influence FRET efficiency.

Keywords:  Endoplasmic Reticulum; Extended Synaptotagmin; FRET FLIM microscopy; Membrane Contact Sites; Oxysterol-binding protein-Related Proteins; Vesicle-Associated membrane protein-associated Proteins; live-cell imaging; protein interactions

DOI:  https://doi.org/10.1016/j.jbc.2026.113144
EMBO Rep. 2026 May 14.

In the absence of mitochondrial fusion unequal segregation of mitochondria drives mtDNA loss.

Lisa Dengler, Francesco Padovani, Bianca Lemke, Rebecca Brugger, Alissa Benedikt, Benedikt Westermann, Boris Maček, Kurt M Schmoller, Jennifer C Ewald.

Mitochondrial biogenesis and inheritance must be tightly coordinated with cell division to maintain mitochondrial function and cell survival. The dynamics of the mitochondrial network, including fusion and fission, are essential for mitochondrial inheritance and quality control. In budding yeast, simultaneous inhibition of both processes compromises mitochondrial DNA (mtDNA) integrity, increasing the frequency of petite cells. Loss of fusion alone completely eliminates mtDNA. Although this has been known for decades, why mtDNA is lost remained unclear. Here, we examine the effects of impaired mitochondrial fusion by depleting the mitofusin Fzo1. By analyzing over thirty thousand single cells across their cell cycles, we show that Fzo1-depletion induces rapid mitochondrial fragmentation and loss of membrane potential, followed by progressive declines in mtDNA content and growth rate. During division, Fzo1-depleted daughters inherit disproportionately large mitochondrial amounts, leaving mothers with too little. This imbalance, combined with an inability to upregulate compensatory mtDNA synthesis, drives rapid mtDNA loss. Our results reveal how fusion defects cause mtDNA loss and mitochondrial dysfunction, which might have implications for diseases linked to impaired fusion.

DOI: https://doi.org/10.1038/s44319-026-00794-5
Eur J Pharmacol. 2026 May 09. pii: S0014-2999(26)00440-1. [Epub ahead of print]1026 178958

Identification of small-molecule autophagy activators via GFP-LC3 high-throughput screening and a cargo-based autophagy flux and processing assay.

Freke Mertens, Farnaz Sedigheh Takhsha, Mélissa Lallier, Nikolai Engedal, Vera Goossens, Dominique Audenaert, Pieter-Jan Guns, Lynn Roth, Peter Vandenabeele, Erik Fransen, Cécile Vindis, Pieter Van der Veken, Vincent Timmerman, Guido R Y De Meyer, Wim Martinet.

   BACKGROUND AND AIM: Autophagy maintains cellular homeostasis by recycling macromolecules and nutrients. It involves the sequestration of superfluous or damaged cellular components into autophagosomes, which fuse with lysosomes for degradation. Reduced autophagy is implicated in numerous diseases, which may be treatable with autophagy-inducing drugs. However, most clinically available inducers act through mTORC1 inhibition, causing off-target effects that limit their therapeutic use. This study aimed to identify novel autophagy-inducing compounds that act independently of mTORC1, thereby offering greater translational potential.
METHODS: A high-throughput imaging assay was optimised to quantify autophagosome-like structures in L929 fibroblasts expressing GFP-LC3, a fluorescent autophagosome membrane marker. Hits were validated alongside several reference autophagy modulators in retinal epithelial hTERT RPE-1 cells expressing the LDHB-mKeima autophagy cargo reporter. This assay distinguishes functional autophagy flux inducers from compounds that merely increase autophagosome accumulation by blocking late-stage autophagy. Western blotting was used to investigate the mechanism of autophagy initiation.
RESULTS: High-throughput screening identified 30 hits that increased autophagosome-like structures more than fourfold. Ten compounds were confirmed to induce autophagic flux of bulk cargo. Nine of these acted independently of mTORC1, while elevating autophagic flux to a similar extent as the mTORC1 inhibitor rapamycin.
CONCLUSION: While GFP-LC3-based assays enabled efficient high-throughput screening, incorporation of the LDHB-mKeima cargo-based assay was essential for identifying functional autophagy flux activators. Nine compounds were identified that promoted autophagic cargo flux via mTORC1-independent mechanisms, providing promising leads for discovering new molecular targets and developing safer, more effective autophagy-based interventions to treat human disease.

Keywords:  Autophagic cargo flux; Autophagy; GFP-LC3; High-throughput screening; LHDB-mKeima

DOI:  https://doi.org/10.1016/j.ejphar.2026.178958