bims-axbals 2025-12-21 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–12–21
thirty-two papers selected by
TJ Krzystek

A system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i 3 Neurons.
TDP-43 dysfunction compromises UPF1-dependent mRNA metabolism in ALS.
GPNMB, LRRK2, and lysosome exocytosis in Parkinson's.
Detection of TDP-43 seeds in CSF of presymptomatic and symptomatic genetic FTD/ALS.
The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.
Pharmacological Activation of Mitophagy Confers Neuroprotective Benefits for Amyotrophic Lateral Sclerosis.
Endoplasmic Reticulum Stress Mediates Axon Initial Segment Shortening: Implications for Diabetic Brain Complications.
Protocol for screening of small molecules in a CLN3 disease patient-specific iPSC-derived neuronal progenitor cell model.
Age-dependent removal of Atg9-containing vesicle accumulations in motoneuron disease models by physical exercise.
PKA orchestrates long-range lysosomal vesicle transport during synaptic maintenance.
Proteomic Identification of ALDOA as a Pathogenic TDP-43 Interaction Partner in ALS.
TDP-43 directly inhibits mRNA accumulation in neurites through modulation of mRNA stability.
Late endosome transport by RILP-RAB7A promotes dendrite arborization.
Positive modulation of sigma-1 receptor: a new weapon to mitigate disease progression in amyotrophic lateral sclerosis.
Hydrogen Sulfide Signaling in Neurodegenerative Movement Disorders.
Reversing Autophagy Inhibition Ameliorates Neurodegeneration in Hereditary Spastic Paraplegia Caused by a Degradation-Resistant SPAST Mutation.
CRISPR screens in human neural organoids and assembloids.
VPS13B recruits lipid vesicles to promote mitochondrial fission and quality control.
Stable isotope labelling kinetics of neurofilament light.
Molecular Repositioning of Celecoxib as a Neurotherapeutic Agent in Fragile X‑Associated Tremor/Ataxia Syndrome (FXTAS).
Systematic Characterization of LUHMES Cell-Based Parkinson's Disease Models Reveals Potential Novel Drug Targets.
Phosphorylated Ubiquitin as a Clinical Biomarker for Mitochondrial Damage in Neurodegenerative Diseases.
Immuno-Regulation of Brain Region-Specific Organoids Containing Isogenic Microglia-Like Cells.
Mitochondrial position responds to glucose stimulation in a model of the pancreatic beta cell.
Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).
Mitochondrial membrane chromatography: Discovery of mitochondrial targeting modulators.
Proteomic analysis links truncated tau to lysosome motility, autophagy, and endo-lysosomal dysfunction.
RHOA regulates mitochondria-ER contact sites through modulation of the VAPB/PTPIP51 tether.
Maspardin/SPG21 controls lysosome motility and TFEB phosphorylation through RAB7 positioning.
Dual role of complement in neuronal repair.
A human iPSC-based neural spheroid platform for modelling glioblastoma infiltration using high-content imaging.
Vesicle-driven endomembrane systems in fungi.

bioRxiv. 2025 Nov 25. pii: 2025.11.22.689945. [Epub ahead of print]

A system for high-throughput axonal imaging of induced pluripotent stem cell-derived human i 3 Neurons.

Stella A C Whittaker, Elizabeth D McKenna, Stephanie L Sarbanes, Michael S Fernandopulle, Michael E Ward, Antonina Roll-Mecak.

Neurons have long, thin axons and branched dendritic processes which rely on an extensive microtubule network that functions as a cellular scaffold and substrate for cargo transport. Microtubule defects are a defining pathological feature of neurological disorders. The highly arborized, long, polarized neuronal processes pose challenges for imaging-based assays. Available methods use either dispersed cultures, which are inefficient for compartment-specific analyses, or microfluidic chambers, which allow clear separation of somatodendritic and axonal compartments but are expensive and difficult to maintain. Here, we introduce an "i 3 Neurosphere" culture model of induced pluripotent stem cell (iPSC)-derived human cortical i 3 Neurons that enables high-throughput imaging of hundreds of axons without specialized equipment. We characterize neurite outgrowth, polarization, microtubule dynamics, and motility of diverse cargo, providing a reference for future work on microtubule processes in this system. The high-throughput compartment-specific imaging we present, combined with facile genetic engineering in i 3 Neurons provides a powerful tool to study human neurons.
SIGNIFICANCE STATEMENT: Human neurons are difficult to study due to limited access to tissue and technical challenges in existing in vitro models of axonal transport. We developed i 3 Neurospheres , a simple and scalable 3D culture system of human iPSC-derived neurons that enables high-throughput imaging of axonal outgrowth, microtubule dynamics, and intracellular transport. This platform provides an accessible, reproducible method for investigating neuronal function and disease mechanisms, offering broad utility for neuroscience research and preclinical drug screening.

DOI: https://doi.org/10.1101/2025.11.22.689945
Neuron. 2025 Dec 12. pii: S0896-6273(25)00848-7. [Epub ahead of print]

TDP-43 dysfunction compromises UPF1-dependent mRNA metabolism in ALS.

Francesco Alessandrini, Matthew Wright, Tatsuaki Kurosaki, Olga S Perloff, Marilyn Ngo, Julia K Kofler, Christopher J Donnelly, Lynne E Maquat, Evangelos Kiskinis.

  Up-frameshift protein 1 (UPF1)-mediated mRNA decay maintains transcriptome integrity and cellular homeostasis. However, its role in amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by TAR DNA-binding protein 43 (TDP-43) pathology and disrupted mRNA metabolism in motor neurons (MNs), remains unresolved. Here, we integrated RNA sequencing (RNA-seq) after UPF1 knockdown with RNA immunoprecipitation (RIP)-seq of phosphorylated UPF1 to delineate direct UPF1 targets in induced pluripotent stem cell (iPSC)-derived MNs. These transcripts are enriched for autophagy and structurally characterized by GC-rich, long 3' untranslated regions (3' UTRs). UPF1 activity, measured by this transcript signature, is diminished in TDP-43-depleted and ALS patient MNs. Mechanistically, TDP-43 depletion impairs UPF1 phosphorylation; the two proteins interact in an RNA-dependent manner and co-aggregate in pathological inclusions in ALS tissue. Transcriptomic analyses reveal convergent regulation of alternative polyadenylation and 3' UTR length by UPF1 and TDP-43, processes disrupted in ALS models and patient neurons. Our study defines the mRNA surveillance network of UPF1 in MNs and uncovers a link between RNA decay, TDP-43 dysfunction, and ALS neurodegeneration.

Keywords:  3′ UTR; ALS; APA; NMD; TDP-43; UPF1; alternative polyadenylation; amyotrophic lateral sclerosis; iPSC-derived motor neurons; nonsense-mediated mRNA decay

DOI:  https://doi.org/10.1016/j.neuron.2025.11.001
Sci Adv. 2025 Dec 19. 11(51): eaed8002

GPNMB, LRRK2, and lysosome exocytosis in Parkinson's.

Suzanne R Pfeffer.

When two genes linked to increased Parkinson's risk converge on a lysosome, LRRK2 mutation enhances lysosomal release of soluble GPNMB potentially contributing to synuclein pathology.

DOI: https://doi.org/10.1126/sciadv.aed8002
Alzheimers Dement. 2025 Dec;21(12): e70989

Detection of TDP-43 seeds in CSF of presymptomatic and symptomatic genetic FTD/ALS.

Ilaria Linda Dellarole, Vittoria Aprea, Marcella Catania, Claudia Battipaglia, Aurora Romeo, Cristina Villa, Anna Burato, Luigi Celauro, Eleonora Dalla Bella, Nilo Riva, Erika Salvi, Giacomina Rossi, Giuseppe Di Fede, Giuseppe Legname, Julie F H De Houwer, Antonella Alberici, Barbara Borroni, Harro Seelaar, John C van Swieten, Fabio Moda, Paola Caroppo.

   INTRODUCTION: Seed amplification assays (SAAs) have shown promising results in detecting misfolded transactive response (TAR) DNA-binding protein 43 (TDP-43) in cerebrospinal fluid (CSF) of genetic frontotemporal dementia (FTD). To date, the use of SAA has yet to be evaluated in presymptomatic individuals.
METHODS: Thirty patients carrying GRN or C9orf72 mutations, 2 microtubule-associated protein tau (MAPT) carriers, 14 presymptomatic subjects, and 27 controls underwent CSF collection. We used SAA for detecting misfolded TDP-43 (TDP-43_SAA) and single molecule array (SIMOA) technology for neurofilament light chain (NfL) dosage.
RESULTS: TDP-43 seeding activity was detected in 67% of TDP-43-linked symptomatic patients, with a specificity of 93%. Almost half of presymptomatic subjects tested positive, mostly GRN carriers. Interestingly, among TDP-43_SAA positive presymptomatic individuals, two GRN carriers underwent phenoconversion.
DISCUSSION: TDP-43_SAA can also detect misfolded TDP-43 in the CSF of presymptomatic individuals. A possible link exists between positive TDP-43_SAA and conversion to the symptomatic phase.
HIGHLIGHTS: Seed amplification assay of transactive response (TAR) DNA-binding protein 43 (TDP-43_SAA) can detect misfolded TDP-43 in the cerebrospinal fluid (CSF) of patients with genetic frontotemporal dementia (FTD), linked to GRN and C9orf72 mutations. TDP-43_SAA can detect misfolded TDP-43 also in the CSF of presymptomatic individuals. In both groups, most TDP-43_SAA positive cases were carriers of GRN mutation. Two GRN carriers that resulted TDP-43_SAA positive converted to the symptomatic phase of the disease.

Keywords:  CSF; SAA; TDP‐43; amyotrophic lateral sclerosis; frontotemporal dementia; seed amplification assays

DOI:  https://doi.org/10.1002/alz.70989
bioRxiv. 2025 Nov 28. pii: 2025.11.25.690484. [Epub ahead of print]

The molecular mechanism of uptake and cell-to-cell transmission of arginine-containing dipeptide repeat proteins.

Alexandra B Sutter, Benito F Buksh, Jelena Mojsilovic-Petrovic, Casey Dalton, Nicholas A Till, Danielle C Morgan, David W C MacMillan, Robert G Kalb.

Micro-satellite repeat expansion of the 5' GGGGCC 3' sequence in the C9orf72 gene is the most common monogenic form of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dipeptide repeat proteins (DPRs) translated from the mutant allele can be detected in postmortem brains of afflicted individuals. The arginine containing peptides, poly-PR and poly-GR, are particularly noxious to cells. Both have been shown to undergo cell-cell transmission, but the underlying mechanisms are not understood. We found rapid internalization and nucleolar localization of bath-applied hemagglutinin (HA) tagged poly-PR with twenty repeats (HA-PR 20 ) in cell lines and neurons. Small molecule and RNAi approaches implicated a temperature-dependent, fluid phase endocytosis mechanism in HA-PR 20 uptake. We sought to identify DPR-related cell surface uptake factors using a high-resolution proximity labeling technique developed in the MacMillan group, termed µMap. DPR-iridium conjugates identified candidate cell-surface proteins which were interrogated in an RNAi screen. Focusing on our strongest candidate, chondroitin sulfate proteoglycan 4 (CSPG4), we showed that cellular uptake of HA-PR 20 is blocked by inhibition of glycosaminoglycan chain synthesis (using drugs or RNAi) and knockdown or ablation of CSPG4 (using RNAi or CRISPR editing). Reduction of CSPG4 protected PR 20 -induced neuronal toxicity. We used a dual reporter system to interrogate in vitro neuron-to-neuron transmission of PR 50 and found that PR 50 synthesized by one neuron readily spread to neighboring neurons. Transmission was significantly reduced when CSPG4 was knocked down. These results suggest CSPG4 is an important factor in poly-PR internalization and transmission and therefore may be a therapeutic target to slow DPR transmission and disease progression.
Significance Statement: A GGGGCC hexanucleotide repeat expansion in the C9orf72 gene is the most common monogenic form of ALS/FTD. This expansion leads to dipeptide repeat protein (DPR) production through non-canonical translation of repeat-containing RNA. DPRs have been shown to transmit between cells, but how this occurs is not well understood. We identified the cell surface protein chondroitin sulfate proteoglycan 4 (CSPG4) as a mediator of the uptake and intercellular spread of toxic arginine-rich DPRs. Targeting CSPG4 may provide a strategy to block DPR transmission and slow disease progression.

DOI: https://doi.org/10.1101/2025.11.25.690484
Aging Dis. 2025 Dec 14.

Pharmacological Activation of Mitophagy Confers Neuroprotective Benefits for Amyotrophic Lateral Sclerosis.

Sen Huang, Ang Li, Yixia Ling, Xinyu Yang, Jiayu Wang, Jing Yuan, Dajiang Qin, Xiaoli Yao.

Amyotrophic lateral sclerosis (ALS) is a rare and devastating neurodegenerative disease characterized by the progressive degeneration of motor neurons in the brain and spinal cord, for which no cure currently exists. Previous studies have shown that abnormal mitochondrial homeostasis and defective mitophagy occur in neurodegenerative diseases, including ALS. Here, we provide evidence that PINK1-Parkin-dependent mitophagy is impaired in multiple ALS mouse models, including the SOD1G93A, TDP43A315T, and rNLS8 strains, leading to the accumulation of damaged mitochondria in affected motor neurons. These findings suggest that mitophagy may be a druggable target for ALS treatment. A classical mitophagy agonist, urolithin A (UA) was used in this study. UA-induced mitophagy antagonizes ALS pathologies in the ALS SOD1G93A transgenic C. elegans model in a pink-1 (PTEN-induced kinase 1)- and pdr-1 (Parkinson's disease-related 1)-dependent manner. Furthermore, pharmacological activation of mitophagy by UA improves locomotor behavior, delays motor neuron degeneration and reduces neuroinflammation in ALS SOD1G93A transgenic mice. In conclusion, our results establish impaired mitophagy as a hallmark of ALS motor neuron degeneration and demonstrate that its pharmacological activation offers a neuroprotective strategy with therapeutic potential.

DOI: https://doi.org/10.14336/AD.2025.1224
J Mol Neurosci. 2025 Dec 19. 76(1): 1

Endoplasmic Reticulum Stress Mediates Axon Initial Segment Shortening: Implications for Diabetic Brain Complications.

Jennae N Shelby, Amanda M Chisholm, Islam Akhmedov, Nathan Sheriff, Ryan B Griggs, Keiichiro Susuki.

  Endoplasmic reticulum (ER) stress and activation of the three unfolded protein response pathways, in particular the protein kinase RNA-like ER kinase (PERK) pathway, contribute to the pathophysiology of various neurodegenerative conditions including type 2 diabetes mellitus (T2DM). T2DM is an increasingly prevalent metabolic disorder affecting millions. Even with strict glucose control, patients with T2DM frequently experience mild cognitive impairment and exhibit a significantly increased risk of developing dementia. We previously demonstrated that impaired cognitive flexibility is associated with shortening of axon initial segment (AIS) length in the prefrontal cortex in the T2DM model db/db mice. The AIS plays the crucial roles of regulation of action potential initiation and neuronal output. Even subtle shortening of AIS length can reduce excitability of neurons. In this study, we hypothesized that ER stress mediates AIS shortening in diabetic conditions. Utilizing primary mouse cortical cultures, we show that sodium 4-phenylbutyrate, a well-documented ER stress inhibitor, prevents AIS shortening and PERK activation induced by the T2DM factor methylglyoxal. Exposure of cortical cultures to an established ER stress inducer tunicamycin caused dose-dependent reduction of AIS length in the generalized population of the neurons without affecting neuronal viability. Co-exposure to a PERK-specific inhibitor GSK2606414 prevented AIS shortening induced by tunicamycin. These results demonstrate ER stress is sufficient and necessary for AIS shortening in vitro. Our findings identify ER stress and AIS shortening as potential therapeutic targets in T2DM-related cognitive impairment.

Keywords:  Axon initial segment; Endoplasmic reticulum stress; Methylglyoxal; Protein kinase RNA-like ER kinase; Type 2 diabetes mellitus

DOI:  https://doi.org/10.1007/s12031-025-02448-y
STAR Protoc. 2025 Dec 11. pii: S2666-1667(25)00675-6. [Epub ahead of print]6(4): 104269

Protocol for screening of small molecules in a CLN3 disease patient-specific iPSC-derived neuronal progenitor cell model.

Ramu Venkatesan, Paul C Trippier.

  Although rare, CLN3 disease is the most common neurodegenerative disorder of childhood. Here, we present a protocol for screening small molecules in a CLN3 disease patient-specific induced pluripotent stem cell (iPSC)-derived neuronal progenitor cell (NPC) model. We describe steps for converting human iPSCs (hiPSCs) to neural stem cells (NSCs) and developing NPCs using feeder-free medium and cytokine-based differentiation. We then detail procedures for achieving access to mature neurons. For complete details on the use and execution of this protocol, please refer to Simeon et al.1.

Keywords:  Cell differentiation; High-throughput screening; Stem cells

DOI:  https://doi.org/10.1016/j.xpro.2025.104269
Transl Neurodegener. 2025 Dec 16. 14(1): 69

Age-dependent removal of Atg9-containing vesicle accumulations in motoneuron disease models by physical exercise.

Alexander Veh, Melissa Ewald, Vinicius da Cruz Neris Geßner, Neha Jadhav Giridhar, Amy-Jayne Hutchings, Christian Stigloher, Beyenech Binotti, Katrin Gertrud Heinze, Patrick Lüningschrör.

   BACKGROUND: Atg9-containing vesicles are enriched in synapses and undergo cycles of exo- and endocytosis similarly to synaptic vesicles, thereby linking presynaptic autophagy to neuronal activity. Dysfunction of presynaptic autophagy is a pathophysiological mechanism in motoneuron disease (MND), which leads to impaired synaptic integrity and function. Here, we asked whether boosting neuronal activity by physical exercise modulates the cellular and motor phenotypes of Plekhg5-deficient mice, an MND model with defective presynaptic autophagy.
METHODS: To characterize the vesicle accumulations in Plekhg5-deficient mice, we performed immunohistochemical staining, electron microscopy, and super-resolution imaging. Following voluntary running wheel exercise, we quantified the histopathological changes within the spinal cord and at neuromuscular junctions using an unbiased machine-learning approach. Additionally, we analyzed the motor performance of the animals by measuring their grip strength. To assess changes in the autophagic flux upon physical exercise in vivo, we utilized mRFP-GFP-LC3 expressing mice. The presence of Atg9-containing vesicle clusters in SOD1G93A was analyzed to examine the relevance of this pathological feature in a second MND model.
RESULTS: We found marked accumulations of Atg9-containing vesicles at presynaptic sites of Plekhg5-deficient mice, which could be cleared by four weeks of voluntary running wheel exercise in young but surprisingly not in aged Plekhg5-deficient mice. However, physical exercise in aged mice led to synaptic vesicle sorting into the Atg9-containing vesicle accumulations without their removal. In line with these findings, short-term voluntary exercise triggered motoneuron autophagy in young but not old mice. Pointing to a broader role of Atg9-containing vesicles in the pathophysiology of MND, we also found Atg9-containing vesicle accumulations in SOD1G93A mice, a well-established ALS model. Strikingly, physical exercise in presymptomatic SOD1G93A mice resulted in a reduction of the vesicle accumulations.
CONCLUSIONS: Our data highlight the essential role of Atg9 in presynaptic autophagy and suggest that boosting autophagy by physical exercise provides a tool to maintain presynaptic function at the early but not late stages of Plekhg5-associated MND and possibly amyotrophic lateral sclerosis.

Keywords:  Atg9; Autophagy; Axon; Motoneuron disease; Physical exercise; Plekhg5

DOI:  https://doi.org/10.1186/s40035-025-00524-2
iScience. 2025 Dec 19. 28(12): 113878

PKA orchestrates long-range lysosomal vesicle transport during synaptic maintenance.

Kerriann K Badal, Yibo Zhao, Bindu L Raveendra, Sebastian Lozano-Villada, Kyle E Miller, Sathyanarayanan V Puthanveettil.

  Bidirectional long-distance transport of organelles is crucial for communication between the cell body and synapses, yet how this transport is regulated during synapse formation, maintenance, and plasticity remains unclear. In the Aplysia sensory neuron (SN)-motor neuron system, which mediates the gill-siphon withdrawal reflex, we demonstrate that synapse maintenance coincides with a sustained decrease in retrograde lysosomal vesicle (LV) transport in SNs. This reduction becomes evident three days after synapse formation, in contrast to anterograde mitochondrial transport, which increases within 12 h. Serotonin-induced, learning-related synapse formation further suppresses retrograde lysosomal transport within 24 h, while boosting anterograde mitochondrial transport as early as 1 h post-exposure. A pharmacological screen implicates PKA as a key regulator of retrograde LV transport during synapse maintenance. These results reveal organelle-specific, temporally distinct regulation of long-distance transport in synapse formation, maintenance, and plasticity.

Keywords:  cell biology; molecular biology; neuroscience

DOI:  https://doi.org/10.1016/j.isci.2025.113878
Degener Neurol Neuromuscul Dis. 2025 ;15 123-132

Proteomic Identification of ALDOA as a Pathogenic TDP-43 Interaction Partner in ALS.

Kaixin Yan, Jinfeng Deng, Yuxuan Yong, Fangfang Bi.

   Objective: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease affecting both upper and lower motor neurons, and its pathogenesis has not been fully elucidated. TAR DNA-binding protein 43 (TDP-43), as one of the key pathogenic genes in ALS, participates in the disease process through interactions with various proteins. This study aims to investigate the interaction mechanism between TDP-43 and aldolase A (ALDOA) in ALS.
Methods: HEK293T cell models transfected with wild-type and mutant TDP-43 (TDP-43M337V) plasmids were constructed. The interaction between TDP-43 and ALDOA was analyzed through proteomic screening of specific peptides and co-immunoprecipitation, and the co-localization of the two in cells was detected by immunofluorescence. Changes in ALDOA expression levels after intervention with mutant TDP-43 were detected by Western blot and quantitative real-time PCR.
Results: Proteomic analysis identified ALDOA as a potential interacting protein of TDP-43. Protein-protein interaction (PPI) analysis, co-immunoprecipitation, and immunofluorescence experiments further confirmed that both wild-type and mutant TDP-43 interact with ALDOA. Western blot and quantitative real-time PCR results showed that, compared with the wild-type TDP-43 group, the ALDOA expression was significantly increased in the TDP-43M337V mutant group.
Conclusion: TDP-43 interacts with ALDOA in ALS, and the TDP-43M337V mutation significantly promotes ALDOA expression, suggesting that ALDOA may be involved in the pathogenesis of TDP-43-mediated ALS. These findings provide new insights into the pathogenesis of ALS and highlight a potential therapeutic target.

Keywords:  ALDOA; TDP-43; amyotrophic lateral sclerosis; neurodegenerative diseases; proteomics

DOI:  https://doi.org/10.2147/DNND.S535954
EMBO J. 2025 Dec 15.

TDP-43 directly inhibits mRNA accumulation in neurites through modulation of mRNA stability.

Charlie Moffatt, Ankita Arora, Katherine F Vaeth, Bryan B Guzman, Gurprit Bhardwaj, Audrey Hoelscher, Levi B Gifford, Holger A Russ, Daniel Dominguez, J Matthew Taliaferro.

  The subcellular localization of many mRNAs to neuronal projections allows neurons to efficiently and rapidly react to spatially restricted external cues. However, for most of these RNAs, the mechanisms that govern their localization are unknown. Here, using subcellular fractionation and single-molecule RNA FISH, we found that loss of TDP-43 results in increased accumulation of hundreds of mRNAs in neurites. Using high-throughput functional assays in cells and high-throughput binding assays in vitro, we subsequently identified specific regions within these mRNAs that mediate their TDP-43-dependent localization and interaction with TDP-43. We found that the same regions also mediated TDP-43-dependent mRNA instability, suggesting a mechanism by which TDP-43 regulates mRNA localization. ALS-associated mutations in TDP-43 resulted in similar mRNA mislocalization phenotypes as did TDP-43 loss in mouse dorsal root ganglia and human iPS-derived motor neurons. These findings establish TDP-43 as a direct negative regulator of mRNA abundance in neurites and suggest that mislocalization of specific transcripts may occur in ALS patients.

Keywords:  ALS; RNA Localization; RNA Stability; RNA Trafficking; TDP-43

DOI:  https://doi.org/10.1038/s44318-025-00653-4
bioRxiv. 2025 Dec 04. pii: 2025.09.03.673267. [Epub ahead of print]

Late endosome transport by RILP-RAB7A promotes dendrite arborization.

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

Directional dendritic transport of late endosomes retrogradely towards the soma is required for fusion with lysosomes and for degradation in the soma. Both dendritic motility of late endosomes and somatic degradation require RAB7A. Similarly, interference with dynein function reduces motility of late endosomes and results in degradative failure. Blocking dynein function also impairs normal dendrite growth, suggesting that motility of late endosomes and/or lysosomes might be required for dendrite growth. RAB7A and dynein are mechanistically linked via RILP which is a dynein-interacting RAB7A effector. RILP also binds the late endosome-lysosome fusion tether HOPS. In non-neuronal cells, downregulation of RILP leads to impaired degradation due to deficiencies in late endosome 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 late endosome motility in dendrites. Interestingly, it also promotes endosome carrier formation. As a consequence of late endosome 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.

DOI: https://doi.org/10.1101/2025.09.03.673267
Transl Neurodegener. 2025 Dec 15. 14(1): 68

Positive modulation of sigma-1 receptor: a new weapon to mitigate disease progression in amyotrophic lateral sclerosis.

Julien Le Friec, Hugo Mourier, Simon Couly, Nicolas Cubedo, Kevin Dubois, Johann Meunier, Benjamin Delprat, Arnaud De Zordo-Banliat, Tahar Ayad, David Virieux, Tsung-Ping Su, Christelle Lasbleiz, Tangui Maurice, Jean-Charles Liévens.

   BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterised by degeneration of motor neurons, leading to muscle weakness and progressive paralysis. Currently, no treatment is available to halt or reverse the progression of the disease. Oxidative stress, mitochondrial dysfunction, accumulation of unfolded proteins and inflammation are interconnected key actors involved in ALS. A potent therapeutic strategy would be to find molecules that break this vicious circle leading to neuronal dysfunction and death. Targeting sigma-1 receptor (S1R) could meet this objective, as this chaperone protein modulates many cell survival mechanisms. So far, the impact of S1R activation in ALS has been studied using specific agonists and mostly on the SOD1 mutation that represents only 2% of patients. In the present study, the impact of two different S1R activators, the reference agonist PRE-084 and the positive modulator OZP002, was compared on two key ALS genes: TDP43 and C9orf72.
METHODS: The dissociation of S1R from Binding immunoglobulin Protein (BiP) was determined using ELISA. OZP002 toxicity was compared to PRE-084 on zebrafish larvae with increasing concentrations. The efficacy of OZP002 and PRE-084 was evaluated on the locomotor escape response of zebrafish expressing mutant TDP43 or one C9orf72 toxic dipeptide. Their effects on NRF2 target gene expression were studied by qPCR. The beneficial effect was further examined on the locomotor performances of TDP43A315T mice using rotarod and beam walking tests. We also performed analysis on motor neuron loss and glial reactivity.
RESULTS: OZP002 is a positive modulator of S1R, that increases the dissociation of the S1R-BiP complex induced by orthosteric agonists. S1R activation by both OZP002 and PRE-084 restored the locomotor response of ALS zebrafish expressing either TDP43 or one C9orf72 toxic dipeptide. The neuroprotection was due at least in part to the NRF2 cascade stimulation but not with a direct interaction. More importantly, OZP002 and PRE-084 prevented locomotor defects and degeneration of spinal motor neurons in TDP43A315T transgenic mice. Astroglial and microglial reactivities were also reduced by both activators.
CONCLUSIONS: We here emphasize the therapeutic value of S1R activation in mitigating ALS pathology. Additionally, we show that the positive modulators pave the way for the development of new S1R-activating compounds for ALS treatment.

Keywords:  Amyotrophic lateral sclerosis; C9orf72; NRF2 signalling; Sigma-1 receptor; Tar-DNA binding protein 43 kDa

DOI:  https://doi.org/10.1186/s40035-025-00527-z
Handb Exp Pharmacol. 2025 Dec 16.

Hydrogen Sulfide Signaling in Neurodegenerative Movement Disorders.

Andrew A Pieper, Bindu D Paul.

  Hydrogen sulfide (H2S) is a gaseous signaling molecule, also known as a gasotransmitter, present in nearly all mammalian organs. It plays crucial roles in regulating various physiological processes in both the brain and peripheral systems. The body maintains tight control over H2S levels, as both excessive and deficient levels can disrupt normal physiological functions and lead to disease. H2S has a significant impact on cognitive and motor functions, which are often compromised in neurodegenerative disorders. It modulates signaling and metabolism primarily by post-translationally modifying reactive cysteine residues on proteins through sulfhydration, also known as persulfidation. This chapter reviews the signaling mechanisms regulated by H2S in neurodegenerative diseases that significantly affect motor function, specifically focusing on Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), spinocerebellar ataxia (SCA), and Leigh syndrome (LS), as well as other mitochondrial disorders. While PD, HD, and SCA are linked to decreased levels of H2S, elevated levels of H2S are associated with ALS, DS, and LS. We also explore potential therapeutic applications of modulating H2S levels in the brain.

Keywords:  Amyotrophic lateral sclerosis; Cysteine; Huntington’s disease; Hydrogen sulfide; Parkinson’s disease; Spinocerebellar ataxia; Sulfhydration/persulfidation

DOI:  https://doi.org/10.1007/164_2025_757
Mov Disord. 2025 Dec 17.

Reversing Autophagy Inhibition Ameliorates Neurodegeneration in Hereditary Spastic Paraplegia Caused by a Degradation-Resistant SPAST Mutation.

Dingyi Zhang, Yang Hu, Jianhe Guo, Dongmei Wu, Yifan Zhang, Lingzi Yang, Yuxi Wang, Nong Xiao, Jinhua Fan, Yanling Dong, Min Zhong, Tongfei Liu.

   BACKGROUND: Hereditary spastic paraplegias (HSPs) are monogenic neurodegenerative disorders, and SPAST mutations causing spastic paraplegia type 4 (SPG4) represent the most common form of HSP. SPAST mutations elevate SPASTIN protein levels beyond haploinsufficiency, but the mechanisms and downstream consequences are unclear.
METHODS: We identified a de novo SPAST missense variant (p.I344E) in a young Chinese female with SPG4. Wild-type (WT) and mutant (I344E/K) SPAST constructs were expressed in HEK293 for biochemical and functional assays including cycloheximide chase, ubiquitination analysis, and immunofluorescence. Leveraging somatic cell reprogramming and CRISPR-based gene editing, we generated patient-derived induced pluripotent stem cells (iPSCs) and their isogenic controls. Both lines were differentiated into cerebral organoids.
RESULTS: I344E/K-SPASTIN exhibited markedly higher steady-state levels than WT-SPASTIN owing to impaired ubiquitin-proteasome-mediated degradation; the I344E variant showed the greatest accumulation. Mutant SPASTIN mislocalized in cells, displayed diminished microtubule-severing activity, and elevated acetylated tubulin-phenotypes that were rescued by overexpression of WT-SPASTIN. In patient cerebral organoids, the I344E mutation led to increased p62/SQSTM1 aggregates, reduced autophagic flux, and enhanced neuronal death. Rapamycin restored autophagy, decreased p62 levels, and reduced cell death.
CONCLUSIONS: Our study provides evidence linking autophagy dysfunction to SPG4 pathogenesis and demonstrates that the I344E mutation acts through a gain-of-function mechanism. These findings challenge the prevailing haploinsufficiency model and implicate autophagy modulation as a potential therapeutic strategy for SPG4 and possibly other HSPs. © 2025 International Parkinson and Movement Disorder Society.

Keywords:  SPAST; autophagy; hereditary spastic paraplegia; iPSC; organoid

DOI:  https://doi.org/10.1002/mds.70150
Nat Protoc. 2025 Dec 19.

CRISPR screens in human neural organoids and assembloids.

Xiangling Meng, Noah Reis, Michael C Bassik, Sergiu P Pașca.

Studying the molecular mechanisms underlying the assembly of the human nervous system remains a significant challenge. The ability to generate neural cells from pluripotent stem cells, combined with advanced genome-editing techniques, provides unprecedented opportunities to uncover the biology of human neurodevelopment and disease. Organoids and assembloids enable the in vitro modeling of previously inaccessible developmental processes, such as the specification and migration of human neurons, including the integration of cortical interneurons from the ventral into the dorsal forebrain. Here, we present a detailed protocol that combines pooled CRISPR-Cas9 screening with neural organoid and assembloid models and illustrate how it can be applied to map hundreds of disease genes onto cellular pathways and specific aspects of human neural development. Our protocol outlines key steps, from planning and optimizing genetic perturbations to designing readouts for neuronal generation and migration, conducting the screening and validating candidate genes. The screening experiments take ~3 months to complete and require expertise in stem cell culture and neural differentiation, genetic engineering of human induced pluripotent stem cell lines, fluorescence-activated cell sorting and next-generation sequencing and analyses. The integration of genetic screening and human cellular models constitutes a powerful platform for investigating the mechanisms of human brain development and disease, paving the way for the discovery of novel therapeutics.

DOI: https://doi.org/10.1038/s41596-025-01299-6
Nat Commun. 2025 Dec 16.

VPS13B recruits lipid vesicles to promote mitochondrial fission and quality control.

Soo-Kyeong Lee, Hyun-Ji Ham, Semin Park, Hye Eun Lee, Ji Young Mun, Deok-Jin Jang, Jin-A Lee.

Mutations in the gene VPS13B, which encodes a Golgi-associated protein, cause the neurodevelopmental disorder Cohen syndrome, but the protein's function is unclear. Here we show that this protein is essential for mitochondrial morphology and quality control. Cells lacking VPS13B, including neurons derived from Cohen syndrome patients, exhibit abnormally elongated and fused mitochondria with reduced membrane potential and impaired mitophagy. Mechanistically, the protein localizes to Mitofusin 2-positive mitochondria via its C-terminal region and recruits phosphatidylinositol-4-phosphate-rich Golgi vesicles to mitochondrial fission sites. Loss of VPS13B or depletion of phosphatidylinositol-4-phosphate results in incomplete mitochondrial fission despite normal recruitment of Dynamin-related protein 1, indicating that lipid transfer by VPS13B is required for membrane fission. VPS13B links Golgi-derived lipid vesicles to the mitochondrial fission machinery, ensuring proper mitochondrial fission and quality control and potentially explaining the mitochondrial defects in Cohen syndrome.

DOI: https://doi.org/10.1038/s41467-025-67445-6
Brain Commun. 2025 ;7(6): fcaf468

Stable isotope labelling kinetics of neurofilament light.

Claire A Leckey, Tatiana A Giovannucci, John B Coulton, Yingxin He, Chihiro Sato, Nupur Ghoshal, Tharini Vignarajah, Zane Jaunmuktane, Nicolas R Barthélemy, Henrik Zetterberg, Donald L Elbert, Kevin Mills, Selina Wray, Randall J Bateman, Ross W Paterson.

  This study provides the first quantification of neurofilament light chain (NfL) kinetics in the human CNS using stable isotope labelling kinetics. NfL is elevated in CSF and blood across a range of traumatic, inflammatory and neurodegenerative diseases of the CNS, and has been increasingly included in clinical trials as a secondary or exploratory outcome measure of target engagement. Interpreting trajectories of NfL post-treatment has been challenging, prompting a greater need and focus on understanding its pathophysiology. We set out to measure NfL kinetics in the human CNS using stable isotope labelling kinetics. In human neurons derived from induced pluripotent stem cells, we show that NfL turnover is relatively slow, comparable to other long-lived proteins such as tau. We detected a delay of 3 to 6 days in the release of NfL into the media, unexpected for a passively released protein and supporting that controlled mechanisms of release could contribute to the appearance of NfL in the extracellular milieu. We optimized the kinetic NfL assay to measure the turnover of NfL in the human CNS. Participants with diagnosed primary tauopathies (n = 10) were recruited to the Human CNS Tau Kinetics in Tauopathies study and a cohort of cognitively unimpaired or with mild cognitive impairment (Clinical Dementia Rating score ≤0.5; n = 22) to the Tau Stable Isotope Labelling Kinetics study. Patients with suspected normal pressure hydrocephalus (n = 3) and primary tauopathy cases (n = 3) were examined to assess labelling in the brain parenchyma and ventricular CSF. In brain tissue, isotopically labelled in vivo and sampled ex-vivo and post-mortem, NfL is rapidly labelled but remains stable 18 months after, indicating very slow turnover and likely incorporation into very stable NfL networks. In line with a controlled mechanism of release in vivo, appearance of labelled NfL in CSF was detectable between 53 and 162 days post-labelling, during which NfL labelling did not reach its peak, suggestive of a half-life in CSF >3 months. These findings support the interpretation that acute rises in CSF NfL concentration are likely to be related to passive release or CSF clearance failure. We also show that active but delayed release of newly translated NfL can contribute to the concentration of NfL in CSF, but this would not be expected for at least 8 weeks. Clinical trials using NfL as an outcome measure will benefit from substantially longer follow-up periods and isotopic labelling to understand the NfL response to therapeutic intervention.

Keywords:  human CNS; iPSC-derived neurons; neurodegeneration; neurofilament light chain; stable isotope labelling kinetics

DOI:  https://doi.org/10.1093/braincomms/fcaf468
ACS Pharmacol Transl Sci. 2025 Dec 12. 8(12): 4264-4284

Molecular Repositioning of Celecoxib as a Neurotherapeutic Agent in Fragile X‑Associated Tremor/Ataxia Syndrome (FXTAS).

Soumalya Das, Aditi Pramod Kumari, Krishna Singh, Sakshi Shukla, Shubhi Khandelwal, Amit Kumar.

  Fragile X-associated tremor/ataxia syndrome (FXTAS), a nucleotide repeat expansion disorder, arises from CGG repeat expansions in the 5' untranslated region (UTR) of the fragile X messenger ribonucleoprotein 1 (FMR1) gene, leading to RNA foci formation and toxic protein aggregation via repeat-associated non-AUG (RAN) translation. These fundamental mechanisms often lead to a series of consequences, including splicing defects, neuroinflammation, mitochondrial dysfunction, impaired autophagy, and cell death. Targeting toxic RNA repeats offers a promising therapeutic strategy. In this study, we identified Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, as a potential treatment for FXTAS. At first, we utilized various biophysical assays and molecular docking to confirm Celecoxib's strong binding affinity toward the r-(CGG)exp RNA. Further studies in the cellular model demonstrated the potency of Celecoxib in reducing toxic protein aggregates and improving splicing defects. Notably, it significantly reduces FMR1PolyG aggregates in the Drosophila FXTAS model, leading to improved locomotor impairments and the mitigation of associated downstream pathological consequences. Moreover, Celecoxib treatment significantly extends the lifespan of the flies. Thus, these results collectively support the therapeutic potential of repurposing Celecoxib for the treatment of FXTAS.

Keywords:  Celecoxib; FMR1PolyG; FXTAS; drug repurposing; r(CGG)exp; trinucleotide repeats

DOI:  https://doi.org/10.1021/acsptsci.5c00326
Mol Neurobiol. 2025 Dec 17. 63(1): 304

Systematic Characterization of LUHMES Cell-Based Parkinson's Disease Models Reveals Potential Novel Drug Targets.

Esra Nur Yiğit, Ekin Sönmez, Regan Odongo, Özgenur Akdaş, Şeyma Çimen, Gürkan Öztürk, Tunahan Çakır, Işıl Aksan Kurnaz.

  I t is established that Parkinson's disease (PD) results from the death of dopaminergic neurons in substantia nigra, and many of the genetic and molecular causes underlying this phenomenon are identified. Despite much progress in the understanding of mechanisms of the disease and existing treatments that manage certain symptoms and improve life quality, PD still encompasses incurable and progressive loss of neurons in patients, and the research for effective disease-modifying treatments is still ongoing. In this study, using a systems biology-based approach, we show that 6-OHDA treatment of alpha-synuclein-overexpressing LUHMES cells can be used as a physiologically relevant model of PD. RNA-seq analysis in these LUHMES-based disease models validates seven genes including HMOX1 and IGF2R in common with previously identified PD genes in DisGeNet. We further confirm that quercetin and rutin can partially alleviate cell death in this model and show that the expression of genes related to PD as well as those related to mitochondria and energy metabolism is back to basal levels in control cells upon quercetin or rutin pre-treatment. When RNA-seq analysis was performed on drug-pretreated LUHMES PD models, protein folding, misfolded protein binding, and unfolded protein response (UPR) pathways, as well as other genes including ETV5, ALS2CR13, PARK7, PINK1, LRRK2, and NGFR, were found to be affected. Finally, using a systems biology approach, we also identified novel target genes for potential drug repurposing. We believe these findings offer new avenues of innovative therapeutic strategies for PD.

Keywords:  LUHMES; Parkinson’s disease; Quercetin; RNA-seq; Rutin; Systems biology

DOI:  https://doi.org/10.1007/s12035-025-05505-0
Aging Dis. 2025 Dec 05.

Phosphorylated Ubiquitin as a Clinical Biomarker for Mitochondrial Damage in Neurodegenerative Diseases.

Fabienne C Fiesel, Jens O Watzlawik, Michael G Heckman, Sophia G Blumenfeld, Michael J Rigby, Mohammed Kehili, Katja Lohmann, Christine Klein, Derek P Narendra, Clemens R Scherzer, Nilufer Ertekin-Taner, Neill R Graff-Radford, Zbigniew K Wszolek, Owen A Ross, Wolfdieter Springer.

Phosphorylated ubiquitin (pS65-Ub) is generated by the kinase-ligase pair PINK1-Parkin to selectively label damaged mitochondria for degradation via the autophagy-lysosome system (mitophagy). Consistent with increasing mitochondrial and lysosomal dysfunctions, pS65-Ub accumulates with aging in human autopsy brain and in mice. pS65-Ub levels are strongly and independently elevated in brains from subjects with Alzheimer's or Parkinson's disease compared to age-matched, neurologically normal controls. Furthermore, pS65-Ub levels have been used to identify disease risk and potential resilience factors in cells and in human brain. However, it remains unknown whether pS65-Ub measured in biofluids may also be suitable as a clinical biomarker. Here, we used a validated sandwich ELISA based on the Mesoscale discovery platform to assess pS65-Ub levels in over 1500 plasma samples from different cohorts across a spectrum of mild cognitive impairment, Alzheimer's disease, or Parkinson's disease. We further analyzed almost 150 CSF samples from two independent case-control series with Parkinson's disease to determine whether pS65-Ub levels are associated with disease status and other clinical parameters. While pS65-Ub levels are significantly changed with disease compared to controls in certain samples, current measurements in plasma are not sufficiently discriminatory to serve as a robust diagnostic marker. However, in CSF, pS65-Ub levels were decreased in patients with Parkinson's disease compared to controls, and there was better discrimination between these groups. Our data indicate that pS65-Ub shows promise as a biomarker in CSF but will require further replication in larger cohorts and possibly in combination with additional other measures.

DOI: https://doi.org/10.14336/AD.2025.1220
Adv Healthc Mater. 2025 Dec 17. e03579

Immuno-Regulation of Brain Region-Specific Organoids Containing Isogenic Microglia-Like Cells.

Chang Liu, Justice Ene, Wenyan Lu, Falak Syed, Li Sun, Ana-Caroline Raulin, Yi Ren, Xueju Wang, Takahisa Kanekiyo, Yan Li.

  Most brain organoids derived from human induced pluripotent stem cells (iPSCs) lack microglia and thus immune function. Microglia-like cells (MGCs) can be differentiated from iPSCs, while the characteristics of isogenic MGC-containing brain organoids in modeling neurodegeneration and cell-cell communications have not been well investigated. In this study, iPSC-derived MGCs are co-cultured with isogenic forebrain cortical organoids (iFCo), which are stimulated with extracellular vesicles (EVs) of brain organoids differentiated from Alzheimer's disease (AD) patient-derived iPSCs (APOE ε4/ε4 and presenilin 1). The AD EV-stimulated co-culture organoids are treated with EVs from healthy MGCs or co-culture. Differential responses of the co-cultured organoids and the MGCs to AD EVs are demonstrated. The co-cultured organoids mitigated pro-inflammatory gene expressions. EVs from healthy MGCs or co-culture reduced the expression of IL-12β, iNOS, TREM2, and CASS4, which are associated with neural inflammation and degeneration, as well as showed regulation on genes involved in microglial activation and carbon metabolism. AD EV cargo analysis by proteomics and microRNA-sequencing revealed APOE and APP proteins and microRNAs regulated pathways such as mitophagy. This study paves the way for understanding the role of microglia and brain organoids in modeling neural degeneration and the development of EV-based cell-free therapeutics for AD treatment.

Keywords:  brain organoids; co‐culture; extracellular vesicles; human pluripotent stem cells; microglia‐like cells; multi‐omics; neural degeneration

DOI:  https://doi.org/10.1002/adhm.202503579
Biophys J. 2025 Dec 18. pii: S0006-3495(25)00767-2. [Epub ahead of print]

Mitochondrial position responds to glucose stimulation in a model of the pancreatic beta cell.

Luis Perez, Xue Wen Ng, Michael Mohs, David W Piston, Shankar Mukherji.

The compartmentalization of eukaryotic cells into membrane-bound organelles with specific subcellular positioning enables precise spatial and temporal control of cellular functions. Although functionally significant mitochondrial localization has been demonstrated in cells such as neurons, it remains unclear how general these cell principles are. Here, we examine the spatial organization of mitochondria within MIN6 pancreatic beta cells under variable glucose conditions. We observe glucose-dependent redistributions of mitochondria, favoring peripheral localization at elevated glucose levels. Our results, formalized into a stochastic model of mitochondrial trafficking, suggest that active mitochondrial transport along microtubules and PKA signaling activity, but not ATP synthesis, are critical regulators of this redistribution. These results suggest that environmentally responsive mitochondrial subcellular positioning may represent a general regulatory mechanism in even nonpolarized cell types.

DOI: https://doi.org/10.1016/j.bpj.2025.11.018
Neuromethods. 2024 ;210 39-59

Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).

Zhe Zhang, Shinghua Ding.

  Predictive disease models play significant roles in advancing our knowledge of the pathology of human disease. In this field, animal models have been extensively employed and have provided crucial insights into the pathophysiological mechanisms of human disease. However, they often fail to fully capture many human phenotypes due to significant species differences in genomic responses. Human induced pluripotent stem cells (hiPSCs) are genetically reprogrammed cells that exhibit qualities remarkably similar to those of embryonic stem cells (ESC) and have emerged as a promising source for cell therapy and fundamental research in pathology. The ability to reprogram human fibroblast cells (hFBs) to iPSCs provides an opportunity to model human diseases. However, even hiPSCs from different persons have different genetic background, thus generation of isogenic unaffected control hiPSCs is necessary to study model human disease. Here, we describe methods to generate isogenic hFBs using CRISPR/Cas9 gene editing method, and subsequently reprogram them into iPSCs using commercially available Sendai virus vectors. Specifically, using the CRISPR/Cas9 system and Sendai virus vector, isogenic iPSC lines can be generated. This protocol provides a structured approach for obtaining multiple isogeneic hiPSC lines, which facilitate the modeling of various human diseases.

Keywords:  CRISPR/Cas 9 gene editing; Reprogramming; Sendai vectors; human fibroblast; isogenic iPSCs

DOI:  https://doi.org/10.1007/978-1-0716-3999-3_4
J Pharm Anal. 2025 Nov;15(11): 101272

Mitochondrial membrane chromatography: Discovery of mitochondrial targeting modulators.

Wu Su, Yu Kong, Hua Li, Yongyao Wang, Lizhuo Wang, Le Shi, Huaizhen He, Shengli Han, Hui Guo, Jiankang Liu, Jiangang Long.

  Mitochondria are fundamental organelles that play a crucial role in cellular energy metabolism, substance metabolism, and various essential cellular signaling pathways. The dysfunction of mitochondria is significantly implicated in the onset and progression of aging, neurodegenerative diseases, metabolic disorders, and tumors, thereby rendering mitochondria-targeted regulation, a vital strategy for disease prevention and treatment. The recently developed mitochondrial membrane chromatography (MMC) technique, which immobilizes mitochondrial proteins as a chromatographic separation medium, has shown great potential for efficiently screening mitochondria-targeted modulators from complex compound library. In contrast to traditional screening methods, MMC has no need to purify mitochondrial proteins and can preserve its in situ and physiological conformation. Consequently, it presents broader application prospects for screening mitochondrial modulators as well as investigating receptor-ligand interactions involving any target protein associated with mitochondria. This review aims to elucidate the critical role of mitochondria in the development and progression of major chronic diseases, discuss recent advancements and applications of MMC, and propose future directions for MMC in the identification of novel mitochondrial modulators.

Keywords:  Major chronic diseases; Mitochondrial membrane chromatography; Mitochondrial modulators; Mitochondrial nutrients; Molecular screening

DOI:  https://doi.org/10.1016/j.jpha.2025.101272
Alzheimers Dement. 2025 Dec;21(12): e70977

Proteomic analysis links truncated tau to lysosome motility, autophagy, and endo-lysosomal dysfunction.

Despoina Goniotaki, Maximilian Hausherr, Steven Lynham, Ayushin Ale, George Chennell, Stefania Marcotti, Katrin Marcus, Wendy Noble, Diane P Hanger, Graham Fraser, Deepak P Srivastava.

   INTRODUCTION: Tauopathies involve progressive accumulation of abnormal tau species that disrupt the autophagy-lysosomal pathway (ALP), critical for degrading intracellular macromolecules and aggregates, leading to toxicity and cell death. This study examines how overexpression of the N-terminally truncated Tau35 protein affects proteolytic pathways, including autophagy and endo-lysosomal processes.
METHODS: Using the Tau35 mouse model and SH-SY5Y human neuroblastoma cells stably expressing Tau35 or full-length tau, we assessed protein degradation and lysosomal function via Western blotting, proteomics of lysosome-enriched brain fractions, cathepsin activity assays, endocytosis/proteolysis assays, and live-cell imaging using LysoTracker.
RESULTS: We identified early endo-lysosomal alterations associated with Tau35 expression, including increased endocytosis, disrupted autophagic flux, proteolytic impairment, and lysosomal motility defects.
DISCUSSION: These findings extend previous research by elucidating Tau35-induced dysfunction in intracellular degradation systems and offer mechanistic insight into tauopathy progression. This work provides a foundation for developing targeted therapies to restore acidification, proteostasis, and lysosomal function in tauopathies.
HIGHLIGHTS: Tau35, an N-terminally truncated tau fragment, disrupts proteolytic pathways: We show that Tau35 overexpression leads to significant alterations in autophagy and endo-lysosomal function. Endo-lysosomal dysfunction is an early pathological event: Our findings demonstrate early-stage increases in endocytosis, impaired proteolytic activity, altered autophagic flux, and disrupted lysosomal motility in Tau35-expressing models. In vivo and in vitro models confirm consistent pathogenic signatures: Parallel studies in a Tau35 mouse model and SH-SY5Y cells reveal converging cellular and molecular dysfunctions. Lysosome-enriched proteomics reveals novel pathway alterations: Proteomic profiling of lysosomal fractions identifies Tau35-specific protein dysregulation contributing to disease pathology. Mechanistic insights into tauopathy progression: These results provide a mechanistic understanding of how truncated tau species contribute to neuronal dysfunction, offering a rationale for targeting endo-lysosomal pathways in therapeutic development.

Keywords:  LysoTracker; SH‐SY5Y cells; Tau35; autophagy‐lysosomal pathway; endocytosis; live‐cell imaging; mice; proteolysis; proteomics; tauopathies

DOI:  https://doi.org/10.1002/alz.70977
Nat Commun. 2025 Dec 14. 16(1): 11260

RHOA regulates mitochondria-ER contact sites through modulation of the VAPB/PTPIP51 tether.

Zheng Yang, Shintaro Nakajima, Hsiuchen Chen, Yogaditya Chakrabarty, Huibao Feng, David C Chan.

The mitochondria-endoplasmic reticulum contact site (MERCS) is critical for calcium exchange, phospholipid transfer, and bioenergetics. Impairment of MERCS is implicated in numerous pathological conditions, including cancer and neurodegenerative diseases. Remodeling of MERCS can affect calcium signaling or metabolism, but the mechanisms involved in dynamic MERCS remodeling are unknown. Employing a genome-wide CRISPRi screen, we uncover the ability of the small GTPase RHOA to tune the cellular MERCS level. RHOA knockdown, or increasing its degradation by CUL3 overexpression, reduces the MERCS level; conversely, upregulation of RHOA increases the MERCS level. RHOA binds to the ER protein VAPB and regulates complex formation between VAPB and mitochondrial PTPIP51, which form a tethering complex at the interface between ER and mitochondria. Furthermore, this regulatory mechanism is perturbed by disease alleles of RHOA, CUL3, and VAPB involved in cancer, hyperkalemia, and neurodegeneration, suggesting that MERCS may be affected in a range of pathological conditions. This study identifies RHOA as a regulator of mitochondria-ER communication, providing mechanistic insights into the dynamic remodeling of MERCS and potential therapeutic strategies for diseases linked to MERCS dysfunction.

DOI: https://doi.org/10.1038/s41467-025-66138-4
J Cell Biol. 2026 Feb 02. pii: e202501135. [Epub ahead of print]225(2):

Maspardin/SPG21 controls lysosome motility and TFEB phosphorylation through RAB7 positioning.

Thomas Jacqmin, Florentine Gilis, Martine Albert, Jean-François Gaussin, Michel Jadot, Marielle Boonen.

Spastic paraplegia 21 is a neurodegenerative disease characterized by the degeneration of corticospinal axons. It is caused by mutations in the SPG21 gene, which encodes maspardin, a cytosolic protein of unknown function that associates with the late endosomal/lysosomal membrane. Intriguingly, we found that the phosphorylation level of the transcription factor EB (TFEB), a master regulator of the CLEAR gene network, is decreased in SPG21 knockout cells, leading to TFEB nuclear translocation. Our investigations revealed that the Rag-mediated presentation of TFEB to the mTOR kinase and its subsequent phosphorylation is disturbed by a delocalization of the RAB7 GTPase, a maspardin-binding partner, from retromer-positive late endosomes to lysosomes. This redistribution decreases the interaction between RAB7 and its GTPase-activating protein (GAP), TBC1D5. Consequently, RAB7 remains primarily GTP-bound, recruiting more FYCO1 to lysosomes and promoting the anterograde movement of these organelles along microtubules. These findings identify maspardin as a newly discovered RAB7 effector and shed light on several consequences of its deficiency.

DOI: https://doi.org/10.1083/jcb.202501135
Front Immunol. 2025 ;16 1712452

Dual role of complement in neuronal repair.

Agnieszka Lukomska, Peter Ciesielski, Mariusz Z Ratajczak, Magdalena Kucia.

  The complement system, long regarded as an arm of innate immunity, is now recognized as an important modulator of nervous system pathophysiology. Following acute injury or in chronic neurodegenerative diseases, promoting neuronal survival and axon regeneration remains a formidable clinical challenge. This review synthesizes the extensive, paradoxical evidence of complement's dual role in neurodegeneration and repair. We examine how complement activation is both detrimental-driving neuroinflammation, apoptosis, and pathological autophagy via receptors like C5aR1 and its interaction with the NLRP3 inflammasome-and beneficial, promoting C5a-mediated phagocyte recruitment for debris clearance and C3-dependent synaptic stripping for circuit remodeling. This review's unique contribution is its integration of these classic extracellular pathways with the recently discovered intracellular complement system, or 'complosome.' We explore how the complosome offers a novel mechanistic framework linking complement to fundamental cellular processes, including metabolism and survival, particularly through its intricate connection with the master regenerative mTOR pathway. This highlights complement not as a simple inflammatory switch, but as a sophisticated signaling network. Understanding this duality is essential for developing therapies that selectively suppress complement-driven damage while enhancing its regenerative functions.

Keywords:  C3aR; C5aR; NLRP3; axon regeneration; complosome; mTOR

DOI:  https://doi.org/10.3389/fimmu.2025.1712452
Sci Rep. 2025 Dec 13.

A human iPSC-based neural spheroid platform for modelling glioblastoma infiltration using high-content imaging.

Victoria S K Tsang, Federica Riccio, Aimee S Wilson, Hannah Nudds, Jason D Coombes, Heiko Wurdak, Harry J C J Bulstrode, Ivo Lieberam, Davide Danovi.

  Glioblastoma is the most aggressive adult brain tumour, characterised by resistance to therapy and high recurrence due to diffuse infiltration. We developed a physiologically relevant co-culture model, combining patient-derived glioblastoma cell lines with cortical-like neural spheroids differentiated from human induced pluripotent stem cells. Using high-content imaging, we demonstrate that GBM20 and GBM1 cell lines migrate directionally along axons toward neural spheroids in live imaging assays and infiltrate spheroids extensively in endpoint assays, unlike non-cancerous neural stem cells. A proof-of-principle drug screen identified PF 573228 (FAK inhibitor) and motixafortide (CXCR4 inhibitor) as potent suppressors of GBM20 and GBM1 infiltration, respectively. Bulk RNA sequencing revealed gene expression profiles correlating with invasive behaviour and drug sensitivity. This platform offers a valuable model for studying glioblastoma infiltration along axons and provides proof-of-principle that migration can serve as a measurable and actionable phenotype to screen therapeutic vulnerabilities in glioblastoma.

Keywords:  Cancer migration; Glioblastoma; High-content imaging; Induced-pluripotent stem cells; Neural spheroid; Stem cell modelling

DOI:  https://doi.org/10.1038/s41598-025-30914-5
Microbiol Mol Biol Rev. 2025 Dec 18. e0029724

Vesicle-driven endomembrane systems in fungi.

Rebekkah E Pope, Rolf A Prade.

  SUMMARYIn fungi, the endomembrane system is a pleiomorphic, dynamic network of organelles, driven by vesicle trafficking pathways, which maintain cellular homeostasis, hyphal polar growth, and the secretion of proteins and metabolites. In syncytial hyphae, spatial specialization of organelles and other cellular components of the endomembrane system is evident to support growth and adaptation. Young, apical regions of hyphae contain a Golgi-Spitzenkörper-exocyst triad for rapid polar expansion, whereas distal, older hyphal regions employ unconventional secretion via multivesicular bodies (MVBs), septal vesicle fusion, and extracellular vesicles (EVs) to enhance nutrient acquisition for the entirety of the mycelium. Vesicular trafficking integrates distinct endomembrane compartments into specialized pathways that involve vesicle biogenesis, transport, and fusion to sustain polarized growth and secretion. Actin and microtubules provide tracks for vesicle motility, while Rab GTPases regulate vesicle localization and fusion events. The ESCRT machinery governs MVB formation and scission, COPI/II regulate bidirectional endoplasmic reticulum-Golgi transport, SNARE proteins allow for vesicle and target membrane fusion, and the exocyst complex tethers vesicles to exocytic regions of the plasma membrane. Together, these components form dynamic endomembrane assembly lines that coordinate many cellular processes. The "distance hypothesis" predicts that extracellular vesicle-mediated secretion predominates in subapical regions as tip growth slows. This mechanism extends the secretory capabilities of hyphae and promotes broader distribution of secreted enzymes along hyphae. Having a better understanding of spatially regulated secretion pathways will advance our understanding of fungal cell biology and provide strategies to optimize fungi for industrial protein production.

Keywords:  Aspergillus nidulans; COPI/II; ESCRT; Rab GTPases; SNAREs; Spitzenkörper; actin filaments; endomembrane metabolism; exocyst; fungi; microtubules; multivesicular bodies; polar hyphal growth; protein secretion; secretomes; vesicles; yeast

DOI:  https://doi.org/10.1128/mmbr.00297-24