bims-axbals 2025-11-02 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–11–02
thirty-two papers selected by
TJ Krzystek

TDP-43-dependent mis-splicing of KCNQ2 triggers intrinsic neuronal hyperexcitability in ALS/FTD.
Intrinsically accelerated cellular degradation is amplified by TDP-43 loss in ALS-vulnerable motor neurons in a zebrafish model.
Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo.
CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival.
A guide to selecting high-performing antibodies for VCP (UniProt ID: P55072) for use in western blot, immunoprecipitation, and immunofluorescence.
VPS35 mutation inhibits PINK1/parkin-mediated mitophagy via increased LRRK2 kinase activity.
Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks.
Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2.
Targeting STAU1 prevents p53 apoptotic signaling in neurodegeneration.
The Parkinson's disease-associated LRRK2-G2019S variant restricts serine metabolism, leading to microglial inflammation and dopaminergic neuron degeneration.
Potential Role of Membrane Contact Sites in the Dysregulation of the Crosstalk Between Mitochondria and Lysosomes in Alzheimer's Disease.
Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes.
Beyond Antioxidants: The Emerging Role of Nrf2 Activation in Amyotrophic Lateral Sclerosis (ALS).
Clonidine prevents radiation-induced cell death in human brain organoids.
C9orf72 Dipeptide Repeat Proteinopathy Is Linked to Increased Histone H3 Phosphorylation on Serine 10.
Advancements in differentiation of induced pluripotent stem cells into specialized neuronal subtypes.
Tubulin regulates stability and localization of STMN2 by binding preferentially to its soluble form.
C-terminus-dependent detection of lysosomal alpha-synuclein in nigral Parkinson's disease human brain neurons.
Axonal injury signaling is restrained by a spared synaptic branch.
The Rab7-Epg5 and Rab39-ema modules cooperatively position autophagosomes for efficient lysosomal fusions.
Extracellular Vesicles-Dependent Secretion Regulates Intracellular CYFIP2 Protein Homeostasis in Cortical Neurons.
Metabolic rewiring prevents neurodegeneration caused by chronic mitochondrial dysfunction.
Tackling ciliary specialization to understand phenotypic variability in human primary ciliopathies.
Close-up of vesicular ER exit sites by volume electron imaging using FIB-SEM.
Marf- and Opa1-Dependent Formation of Mitochondrial Network Structure Is Required for Cell Growth and Subsequent Meiosis in Drosophila Males.
Impairment of lysosomal quality control in Huntington disease.
The Other Side of the Same Coin: Beyond the Coding Region in Amyotrophic Lateral Sclerosis.
Autophagy-Lysosome Pathway in Multiple System Atrophy Pathogenesis: The Best Is Yet to Come.
Protocol for in vitro immunofluorescence staining in a Transwell co-culture system.
Evolution of Axonal Injury in the Closed Head Impact Model of Engineered Rotational Acceleration in Adult Ferrets.
AAV9-Mediated Intrastriatal Delivery of Mutant HTT With 82 CAG Repeats Induces Huntington's Disease-Like Pathology and Behavioral Deficits in Mice.
Brain organoid models of Huntington's disease shift the focus towards neurodevelopment.

Nat Neurosci. 2025 Oct 31.

TDP-43-dependent mis-splicing of KCNQ2 triggers intrinsic neuronal hyperexcitability in ALS/FTD.

Brian J Joseph, Kelly A Marshall, Peter Harley, Jacob R Mann, Francesco Alessandrini, Carlos G Vanoye, Wanhao Chi, Mercedes Prudencio, Dina Simkin, Tzu-Ting Kao, Reshma R Desai, Matthew J Keuss, Simone Barattucci, Matteo Zanovello, Puja R Mehta, Jean-Marc DeKeyser, Francesco Limone, Jonathan Lee, Anna-Leigh Brown, Marcel F Leyton-Jaimes, Leslie A Nash, Irune Guerra San Juan, Eleonora Aronica, Brian J Wainger, Mala Shah, Anand Goswami, Neil A Shneider, Dennis W Dickson, Juan Burrone, Chaolin Zhang, Hynek Wichterle, Leonard Petrucelli, Jonathan K Watts, Alfred L George, Pietro Fratta, Kevin Eggan, Evangelos Kiskinis.

Motor neuron hyperexcitability is a broadly observed yet poorly understood feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Nuclear depletion and cytoplasmic aggregation of the RNA splicing protein TAR DNA-binding protein 43 (TDP-43) are observed in most ALS and FTD patients. Here we show that TDP-43 dysfunction causes mis-splicing of KCNQ2, which encodes a voltage-gated potassium channel (Kv7.2) that regulates neuronal excitability. Using iPSC-derived neurons and postmortem ALS/FTD brain and spinal cord tissue we find widespread, disease-specific and TDP-43-specific skipping of an exon encoding the KCNQ2 pore domain. The mis-spliced mRNA escapes degradation and is translated into a nonfunctional protein with severely reduced ion conductance that aggregates in the endoplasmic reticulum and causes intrinsic hyperexcitability in ALS neuronal models. This event, which correlates with higher phosphorylated TDP-43 levels and earlier age of disease onset in patients, can be rescued by splice-modulating antisense oligonucleotides that dampen hyperexcitability in induced pluripotent stem cell cortical neurons and spinal motor neurons with TDP-43 depletion. Our work reveals that nuclear TDP-43 maintains the fidelity of KCNQ2 expression and function and provides a mechanistic link between established excitability disruption in ALS/FTD patients and TDP-43 dysfunction.

DOI: https://doi.org/10.1038/s41593-025-02096-w
Nat Commun. 2025 Oct 27. 16(1): 9213

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

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

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

DOI: https://doi.org/10.1038/s41467-025-65097-0
iScience. 2025 Oct 17. 28(10): 113460

Endolysosomal dysfunction impairs proteostasis and induces neurodegeneration in vivo.

Wei Shao, Ellen A Albagli, Karen Jansen-West, Lillian M Daughrity, Jimei Tong, Anxhela Hysi, Peizhou Jiang, Tiffany W Todd, Emma Lee, Desiree Zanetti Alepuz, Yari Carlomagno, Mei Yue, Judith A Dunmore, Monica Castanedes-Casey, Paula Castellanos Otero, Aishe Kurti, Mercedes Prudencio, Casey N Cook, Dennis W Dickson, Tania F Gendron, Leonard Petrucelli, Yong-Jie Zhang.

  Transactive response (TAR) DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of the frontotemporal dementia (FTD)-amyotrophic lateral sclerosis (ALS) spectrum. Dysfunction of the endolysosomal system, which plays a crucial role in protein trafficking and maintaining proteostasis, has been implicated in FTD-ALS pathogenesis. While the impact of endolysosomal dysfunction on TDP-43 pathology remains unclear, we demonstrated that disrupting the endolysosomal pathway by expressing the constitutively active endosomal protein, Rab5Q79L, induces TDP-43 aggregation in cultured cells. Here, we generated a mouse model expressing GFP-tagged Rab5Q79L, demonstrating that GFP-Rab5Q79L mice exhibit early motor deficits and endolysosomal dysfunction, including enlarged endosomes, abnormal lysosome morphology, and p62- or ubiquitin-positive inclusions. These mice also developed significant neuronal loss, neuroinflammation, phosphorylated TDP-43 (pTDP-43) inclusions, and nuclear envelope and nuclear pore structural defects reminiscent of FTD-ALS. Accordingly, GFP-Rab5Q79L mice will prove useful in expanding our understanding of endolysosomal dysfunction in proteostasis and pTDP-43 pathology.

Keywords:  developmental neuroscience; molecular neuroscience; technical aspects of cell biology

DOI:  https://doi.org/10.1016/j.isci.2025.113460
Cell Death Dis. 2025 Oct 31. 16(1): 772

CLN7 protein functions at the interface between endolysosomes and stress granules to promote cell survival.

Aseel Sharaireh, Marta Guevara-Ferrer, Anna M Ludlaim, Jonathan D Humphries, Alexander M Phillips, Andrew W Dowsey, Zehan Zhang, John R Counsell, Richard D Unwin, Sara E Mole, Ahad A Rahim, Tristan R McKay.

Inherited biallelic mutations in the CLN7 gene result in the variant late infantile onset neuronal ceroid lipofuscinosis, a subtype of Batten disease (BD), a severe and fatal childhood neurodegenerative disease. Intriguingly, CLN7 genetic variants have also been associated with retinopathies, amyotrophic lateral sclerosis, and frontotemporal dementia. CLN7 encodes a transmembrane protein localizing to endolysosomal membranes with outward-facing chloride channel activity. Loss of CLN7 function results in cortical neurons accumulating swollen lipofuscin-containing lysosomes, leading to neuroinflammation and neurodegeneration. The molecular mechanisms underlying CLN7 BD neuropathology are not completely understood. We have generated iPSC lines from two CLN7 BD patients and age-matched unaffected controls to interrogate intracellular molecular phenotypes in iPSC-derived neural progenitor cells (iNPC). Taking a multi-omics approach we have identified disease-modified activities in endolysosomal transport in iNPCBD that lead to lysosomal dysfunction and decreased mitophagy, resulting in the accumulation of metabolically defective mitochondria. We further observe a breakdown in nuclear functions that centre on RNA processing and nuclear export, linking to CLN7 protein interactions at the stress granule. We have identified dual and distinct functions for CLN7, promoting cell survival during the cellular stress response. CLN7 loss of function in BD results in neuronal apoptosis.

DOI: https://doi.org/10.1038/s41419-025-08063-4
F1000Res. 2025 ;14 891

A guide to selecting high-performing antibodies for VCP (UniProt ID: P55072) for use in western blot, immunoprecipitation, and immunofluorescence.

Vera Ruíz Moleón, Maryam Fotouhi, Joel Ryan, Donovan Worrall, Riham Ayoubi, Vincent Francis, Peter S McPherson, Carl Laflamme.

  Valosin-containing protein (VCP) is a highly conserved and essential ATPase involved in many cellular processes like neuronal function, protein degradation, organelle maintenance, and stress response regulation. Understanding the specific mechanisms by which VCP plays in health and disease can provide novel insides in therapeutic targets, a process that would be facilitated by the availability of high-quality antibodies. Here we have characterized sixteen VCP commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  P55072; Transitional endoplasmic reticulum ATPase; VCP; Valosin-containing protein; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.169502.1
Brain. 2025 Oct 30. pii: awaf414. [Epub ahead of print]

VPS35 mutation inhibits PINK1/parkin-mediated mitophagy via increased LRRK2 kinase activity.

Liselot Manders, Thibaut Heyninck, Dorien Imberechts, Bjørn Holst, Rejko Krüger, Wim Vandenberghe.

  The p.D620N mutation in VPS35 causes an autosomal dominant form of Parkinson's disease via mechanisms that are poorly understood. PINK1 and parkin, two proteins whose loss of function underlies autosomal recessive Parkinson's disease, cooperate to mediate mitophagy, a quality control pathway for selective elimination of damaged mitochondria. PINK1/parkin-mediated mitophagy is disrupted by LRRK2 mutations, which are the most prevalent cause of autosomal dominant Parkinson's disease. Here, we investigated whether the p.D620N VPS35 mutation has an effect on PINK1/parkin-mediated mitophagy. We identified a novel family with autosomal dominant Parkinson's disease caused by a p.D620N VPS35 mutation. We cultured skin fibroblasts and iPSC-derived dopaminergic neurons from the proband and from a second, unrelated Parkinson's disease patient with the p.D620N VPS35 mutation, and compared them with isogenic and non-isogenic control cells. PINK1/parkin-mediated mitophagy was severely impaired in VPS35 mutant fibroblasts and neurons, while non-selective, starvation-induced autophagy and lysosomal degradative capacity were preserved. siRNA-mediated VPS35 knockdown rescued the mitophagy defect in VPS35 mutant cells, whereas overexpression of wild-type VPS35 did not, suggesting a gain-of-function mechanism of the mutation. The VPS35 mutation did not interfere with activation of PINK1 or parkin after mitochondrial depolarization, but impaired mitochondrial recruitment of the autophagy receptor optineurin. LRRK2 kinase activity was increased in the VPS35 mutant cells, as shown by enhanced levels of the T73-phosphorylated form of the LRRK2 substrate RAB10. The enhanced level of phosphorylated RAB10 in VPS35 mutant cells was decreased by treatment with LRRK2 kinase inhibitors and by VPS35 knockdown. Importantly, the mitophagy defect of VPS35 mutant fibroblasts and neurons was fully rescued by LRRK2 kinase inhibitors as well as by overexpression of PPM1H, a phosphatase that dephosphorylates multiple RAB substrates of LRRK2. Finally, in situ proximity ligation experiments revealed that endogenous VPS35 and LRRK2 are proximity partners in human dopaminergic neurons and that this proximity relationship is enhanced by the VPS35 mutation. In conclusion, the VPS35 mutation impairs PINK1/parkin-mediated mitophagy via a gain-of-function mechanism that involves stimulation of LRRK2 kinase activity. Thus, a VPS35/LRRK2 axis linked to dominant Parkinson's disease intersects with a pathway mediated by proteins encoded by the recessive Parkinson's disease genes.

Keywords:  Parkinson’s disease; RAB; autophagy; induced pluripotent stem cell; lysosome; mitochondrion

DOI:  https://doi.org/10.1093/brain/awaf414
Cell Rep. 2025 Oct 29. pii: S2211-1247(25)01265-3. [Epub ahead of print]44(11): 116494

Phospho-proteome profiling in human neurons reveals targets of TBK1 in ALS/FTD-associated autophagy networks.

Julie Smeyers, Juan A Oses-Prieto, Lin Yadanar, Man Wang, Marci Iadarola, Serena Lu, Karen S Wang, Taylor H Watanabe, Jayanta Debnath, Alma L Burlingame, Daniel A Mordes.

  Loss-of-function variants in TBK1, encoding a protein kinase, are strongly associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how haploinsufficiency for TBK1 leads to age-related neurodegeneration remains unresolved. Here, we utilize sets of isogenic induced pluripotent stem cells (iPSCs) with loss of TBK1 or loss of optineurin (OPTN) for quantitative global proteomics and phospho-proteomics in both stem cells and excitatory neurons. We found that TBK1 sustains the abundance and phosphorylation of its interacting adapter proteins, AZI2/NAP1, TANK, and TBKBP1/SINTBAD. Moreover, TBK1 regulates the phosphorylation of endo-lysosomal proteins, such as GABARAPL2, the late-endosome GTPase RAB7A, and selective autophagy cargo receptor proteins-including novel phospho-sites in p62/SQSTM1-in neurons. Finally, we provide a census of the phospho-proteome in nascent human neurons for further studies. Overall, TBK1 serves as a point of convergence in ALS/FTD-linked endo-lysosomal networks that act in a cell-autonomous manner to maintain protein homeostasis in neurons.

Keywords:  ALS; CP: Molecular biology; CP: Neuroscience; OPTN; TBK1; autophagy; dementia; iPSC disease modeling; neurodegeneration; phospho-proteomics; proteomics

DOI:  https://doi.org/10.1016/j.celrep.2025.116494
Elife. 2025 Oct 31. pii: RP100799. [Epub ahead of print]13

Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2.

Siyu Chen, Tamar Basiashvili, Joshua Hutchings, Marta Sanz Murillo, Amalia Villagran Suarez, Erica Xiong, Jaime Alegrio Louro, Andres E Leschziner, Elizabeth Villa.

  Parkinson's disease (PD) is the second most common neurodegenerative disorder. Mutations in human leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy and in situ cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2's kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length human LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.

Keywords:  LRRK2; human; microtubules; molecular biophysics; neuroscience; parkinson's disease; structural biology

DOI:  https://doi.org/10.7554/eLife.100799
Cell Death Dis. 2025 Oct 27. 16(1): 761

Targeting STAU1 prevents p53 apoptotic signaling in neurodegeneration.

Mandi Gandelman, Sharan Paul, Karla P Figueroa, Justine Sundrud, Warunee Dansithong, Daniel R Scoles, Stefan M Pulst.

Stress responses and neuronal death mediated by the p53 pathway play a central role in the progression of neurodegenerative disease, constituting a common target to extend neuronal function and survival. Interaction of p53 and its signaling network with RNA-binding proteins (RBPs) helps fine-tune its activation and the resulting cell fates. Preclinical therapeutics based on depletion of the RBP STAUFEN-1 (STAU1) protein successfully prevent neurodegeneration, however, the specific mechanisms are not fully understood. STAU1 is pathologically overabundant in multiple neurological disorders and contributes to neurodegeneration by exacerbating autophagy dysfunction, endoplasmic reticulum stress, and RNA-protein condensate accumulation. We previously showed that lowering STAU1 levels mitigates these disease-related features and prevents neuronal death in animal models of amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) and spinocerebellar ataxia type 2 (SCA2). Here, we show by combined transcriptomic and functional analyses that STAU1 reduction results in the inhibition of apoptosis through the p53 pathway. In both proliferating and post-mitotic cell types-human iPSC-derived neurons, mouse cortical neurons, SH-SY5Y cells, and fibroblasts-STAU1 reduction effectively prevented p53-mediated apoptosis and DNA damage induced by Nutlin-3 and etoposide. Further examination in C9orf72-expanded patient-derived fibroblasts and a C9orf72 mouse model of ALS/FTD, which exhibit baseline overabundance of STAU1 and activation of the p53 pathway, confirmed that STAU1 reduction also prevented p53-driven pro-apoptotic signaling. These findings establish STAU1 as a novel modulator of DNA damage and p53-dependent apoptosis, suggesting that targeting STAU1 could be a promising approach to prevent neurodegeneration in ALS/FTD.

DOI: https://doi.org/10.1038/s41419-025-08067-0
J Neuroinflammation. 2025 Oct 27. 22(1): 244

The Parkinson's disease-associated LRRK2-G2019S variant restricts serine metabolism, leading to microglial inflammation and dopaminergic neuron degeneration.

Henry Kurniawan, Sarah L Nickels, Alise Zagare, Elisa Zuccoli, Isabel Rosety, Gemma Gomez-Giro, Enrico Glaab, Jens C Schwamborn.

  A growing body of evidence implicates inflammation as a key hallmark in the pathophysiology of Parkinson's disease (PD), with microglia playing a central role in mediating neuroinflammatory signaling in the brain. However, the molecular mechanisms linking microglial activation to dopaminergic neuron degeneration remain poorly understood. In this study, we investigated the contribution of the PD-associated LRRK2-G2019S mutation to microglial neurotoxicity using patient-derived induced pluripotent stem cell (iPSC) models. We found that LRRK2-G2019S mutant microglia exhibited elevated activation markers, enhanced phagocytic capacity, and increased secretion of pro-inflammatory cytokines such as TNF-α. These changes were associated with metabolic dysregulation, including upregulated glycolysis and impaired serine biosynthesis. In 3D midbrain organoids, these overactivated microglia resulted in dopaminergic neuron degeneration. Notably, treating LRRK2-G2019S microglia with oxamic acid, a glycolysis inhibitor, attenuated microglial inflammation and reduced neuronal loss. Our findings underscore the link between metabolic targeting in microglia and dopaminergic neuronal loss in LRRK2-G2019S mutation, and highlight a potential strategy that warrants further preclinical evaluation.

Keywords:  Glycolysis; IPSC; LRRK2-G2019S; Metabolism; Microglia; Organoids; Parkinson’s disease; Serine

DOI:  https://doi.org/10.1186/s12974-025-03577-2
Int J Mol Sci. 2025 Oct 10. pii: 9858. [Epub ahead of print]26(20):

Potential Role of Membrane Contact Sites in the Dysregulation of the Crosstalk Between Mitochondria and Lysosomes in Alzheimer's Disease.

Giulia Girolimetti, Matteo Calcagnile, Cecilia Bucci.

  Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a gradual decline in cognitive abilities and a progressive loss of the neuronal system resulting from neuronal damage and death. The maintenance of neuronal homeostasis is intricately connected to the crosstalk and balance among organelles. Indeed, intracellular organelles are not just isolated compartments in the cell; instead, they are interdependent structures that can communicate through membrane contact sites (MCSs), forming physical connection points represented by proteinaceous tethers. Mitochondria and lysosomes have fundamental physiological functions within neurons, and accumulating evidence highlights their dysfunctions as AD features, strongly associated with the neurodegenerative process underlying the development and progression of AD. This review explores mitochondria-lysosome communication through MCSs, the tethering proteins and their functions in the cell, discussing the methodological challenges in measuring the structure and dynamics of contacts, and the potential role of altered mitochondria-lysosome communication in the context of organelle dysfunction related to neuron impairment in AD pathogenesis. The different abundance of the tethering proteins was considered in healthy physiological and in AD-related conditions to assess the possible organelle communication dysregulation and the subsequent cellular function alterations, and to evaluate the role of mitochondria-lysosome MCSs in the pathogenesis of this disorder.

Keywords:  Alzheimer’s disease; lysosomes; membrane contact site; mitochondria

DOI:  https://doi.org/10.3390/ijms26209858
J Vis Exp. 2025 Oct 10.

Modeling Mitochondrial Disease Using Brain Organoids: A Focus on Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like Episodes.

Shihori Kawano, Chika Saegusa, Yusuke Masano, Franziska Becker, Mari Nakamura, Seiji Shiozawa, Junji Fujikura, Takafumi Toyohara, Takaaki Abe, Hideyuki Okano, Masato Fujioka.

Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS) are mitochondrial disorders most commonly caused by a m.3243A>G variant in mitochondrial tRNALeu. To investigate the pathophysiology of MELAS, we generated brain organoids from multiple induced pluripotent stem cell (iPSC) lines derived from a patient with MELAS carrying the m.3243A>G variant. These lines share an identical nuclear genetic background but differ in their heteroplasmy levels involving the m.3243A>G variant. We observed significant differences in organoid size, morphology, and neural induction efficiency, which correlated with the degree of heteroplasmy. Dissociated neurons from the organoids were transferred into a 2D-culture system, which is convenient and suitable for high-throughput drug screening. The organoids also exhibited significant differences in the formation of neural networks, depending on heteroplasmy levels. Our results suggest that patient-derived iPSC-based organoid models represent a useful platform for studying MELAS mechanisms and for drug screening. This video presents comprehensive and user-friendly methods, including protocols for generating organoids and evaluating phenotypes.

DOI: https://doi.org/10.3791/69303
Int J Mol Sci. 2025 Oct 10. pii: 9872. [Epub ahead of print]26(20):

Beyond Antioxidants: The Emerging Role of Nrf2 Activation in Amyotrophic Lateral Sclerosis (ALS).

Minoo Sharbafshaaer, Roberta Pepe, Rosaria Notariale, Fabrizio Canale, Gioacchino Tedeschi, Alessandro Tessitore, Paolo Bergamo, Francesca Trojsi.

  Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder involving the progressive degeneration of upper and lower motor neurons. While oxidative stress, RNA-binding protein (RBP) pathology, mitochondrial dysfunction, and glial-neuronal dysregulation is involved in ALS pathogenesis, current therapies provide limited benefit, underscoring the need for multi-target disease-modifying strategies. Nuclear factor erythroid 2-related factor 2 (Nrf2), classically regarded as a master regulator of redox homeostasis, has recently emerged as a central integrator of cellular stress responses relevant to ALS. Beyond its canonical antioxidant function, Nrf2 regulates critical pathways involved in mitochondrial quality control, proteostasis, nucleocytoplasmic transport, RNA surveillance, and glial reactivity. Experimental models demonstrate that astrocyte-specific Nrf2 activation enhances glutathione metabolism, suppresses neuroinflammation, promotes stress granule disassembly, and reduces RBP aggregation. In C9orf72-linked ALS, Nrf2 activation mitigates dipeptide repeat protein toxicity and restores RNA processing fidelity via modulation of nonsense-mediated decay and R-loop resolution. Recent advances in Nrf2-targeted interventions including Keap1-Nrf2 protein-protein interaction inhibitors, dual Nrf2/HSF1 activators, and cell-type-selective Adeno-associated virus 9 (AAV9) vectors show promise in preclinical ALS models. These multimodal approaches highlight Nrf2's therapeutic versatility and potential to address the upstream convergence points of ALS pathogenesis. Taken together, positioning Nrf2 as a systems-level regulator offers a novel framework for developing precision-based therapies in ALS. Integrating Nrf2 activation with RNA- and glia-directed strategies may enable comprehensive modulation of disease progression at its molecular roots.

Keywords:  Keap1 inhibitors; Nrf2; RNA-binding proteins; amyotrophic lateral sclerosis; glial cells; mitochondrial dysfunction; neuroinflammation; nucleocytoplasmic transport; oxidative stress; stress granules

DOI:  https://doi.org/10.3390/ijms26209872
Sci Rep. 2025 Oct 31. 15(1): 38113

Clonidine prevents radiation-induced cell death in human brain organoids.

Martin Lundberg, Samudyata, Marja Koskuvi, Asimenia Gkogka, Mahnaz Nikpour, Melis Çelik, Erik Smedler, Bo Stenerlöw, Per-Arne Lönnqvist, Martin Schalling, Carl M Sellgren.

  Radiotherapy is a standard treatment of pediatric brain tumors. Though the survival rate has improved for many tumor types, most patients suffer long-term cognitive decline and there is currently no way of preventing radiation-induced damage to healthy brain tissue. Here, we used a human forebrain organoid model to investigate if the α2-adrenoceptor and I1-imidazoline receptor agonist clonidine could prevent radiotoxicity. We found that treatment of organoids with clonidine significantly reduced radiation-induced loss of neural progenitor cells, neurons, astrocytes, and oligodendrocyte lineage cells. Moreover, clonidine reduced overall DNA damage and signs of reactive gliosis in organoids. Our findings demonstrate that pharmacological rescue of radiation neurotoxicity is possible in a human brain organoid model and provides a rationale for future drug repurposing studies aiming to prevent radiation-induced brain injury in children treated with radiotherapy.

Keywords:  Brain organoids; Cell death; Clonidine; Induced pluripotent stem cells; Neuroprotection; Radiotherapy

DOI:  https://doi.org/10.1038/s41598-025-26170-2
ACS Omega. 2025 Oct 21. 10(41): 48395-48411

C9orf72 Dipeptide Repeat Proteinopathy Is Linked to Increased Histone H3 Phosphorylation on Serine 10.

Samantha N Cobos, Raven M A Fisher, Seth A Bennett, Chaim Janani, David K Dansu, Matthew M Cleere, Arefa Yeasmin, Gabriel Cruz, Sidra Qureshi, William Villasi, Rania Frederic, Kyle Chen, Mila Mirzakandova, George Angelakakis, Elizaveta Son, Andrew Elgendy, Mariana P Torrente.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal illnesses forming a neurodegenerative disease continuum. While most ALS/FTD cases are sporadic, a small proportion of cases are linked to mutations in many genes. Among these, hexanucleotide repeat expansions in the C9orf72 gene are the most common and lead to the formation of dipeptide repeat proteins (DPRs), including a proline-arginine dipeptide (PR), which aggregate in the cytoplasm of decaying neurons. As genetics alone fails to explain the etiology of ALS/FTD, it is possible that epigenetic mechanisms - such as histone post-translational modifications (PTMs) - are involved in disease processes. A Saccharomyces cerevisiae (PR)50 overexpression model displays overt growth suppression and aggregation. Here, we exploit this model as a discovery platform to comprehensively characterize changes in the levels of PTMs on Histones H3 and H4. We find that overexpression of (PR)50 is associated with increased levels of phosphorylation on Histone H3 at Serine 10 (H3S10ph). Furthermore, (PR)50 overexpression revealed modest increases in the levels of other marks associated with increased gene expression. Remarkably, decreased abundance of Ipl1, the kinase responsible for phosphorylating H3S10 in yeast, leads to amelioration of the growth suppression phenotype and restores H3S10ph levels even in the context of (PR)50 overexpression. Recapitulating our results in yeast, several c9orf72 ALS patient-derived fibroblasts and induced pluripotent stem cell (iPSCs) lines display similar increases in H3S10ph levels. Altogether, these findings reveal a previously undiscovered connection between H3S10ph and c9 ALS/FTD proteinopathy that could reveal novel targets for the treatment of this disease.

DOI: https://doi.org/10.1021/acsomega.5c05836
Neural Regen Res. 2025 Oct 30.

Advancements in differentiation of induced pluripotent stem cells into specialized neuronal subtypes.

Selena Setsu, Hideyuki Okano, Satoru Morimoto.

   ABSTRACT: The ability to generate specialized human neurons from induced pluripotent stem cells has revolutionized neuroscience, regenerative medicine, and drug discovery. Since their discovery, induced pluripotent stem cells have emerged as an ethically favorable and versatile platform to model human neurological diseases, offering new insights beyond traditional animal models. In the past decade, rapid advances have enabled the efficient differentiation of induced pluripotent stem cells into diverse neuronal subtypes, including glutamatergic neurons, GABAergic neurons, dopaminergic neurons, serotonergic neurons, motor neurons, sensory neurons, Purkinje cells, sympathetic neurons, parasympathetic neurons, and noradrenergic neurons. Tailored combinations of developmental signaling molecules, transcription factor programming, and small molecule modulation have dramatically improved the reproducibility, scalability, and functional maturity of these differentiated neurons. These advancements are particularly timely as they underpin the next generation of disease modelling platforms, high-throughput drug screening systems, and emerging cell-based therapies for conditions such as Parkinson's disease, amyotrophic lateral sclerosis, epilepsy, and Alzheimer's disease. Moreover, the field is moving toward standardized, chemically defined protocols and improved validation pipelines, including electrophysiological assays and molecular profiling, to ensure the authenticity and maturity of induced pluripotent stem cell-derived neurons. Notably, recent breakthroughs in sympathetic and parasympathetic neuron derivation are expanding the scope of induced pluripotent stem cell technology into autonomic nervous system research and cardiac neuromodulation studies. However, challenges remain, including variability across induced pluripotent stem cell lines, incomplete neuronal maturation, and scalability constraints for clinical-grade applications. Addressing these hurdles through optimization of patterning cues, co-culture systems, and advanced bioprocessing strategies will be crucial to realizing the full translational potential of induced pluripotent stem cell-derived neurons. Collectively, the methodologies and developments summarized here mark a major step toward achieving faithful, efficient, and scalable generation of human neurons in vitro, laying the foundation for personalized neurology and regenerative medicine.

Keywords:  Purkinje cells; cell therapy; disease modelling; dopaminergic neurons; drug screening; glutamatergic neurons; induced pluripotent stem cells; motor neurons; neuronal differentiation; regenerative medicine; sensory neurons

DOI:  https://doi.org/10.4103/NRR.NRR-D-25-00630
J Cell Biol. 2025 Dec 01. pii: e202502192. [Epub ahead of print]224(12):

Tubulin regulates stability and localization of STMN2 by binding preferentially to its soluble form.

Xiang Deng, Gary A Bradshaw, Marian Kalocsay, Timothy Mitchison.

The small, tubulin-binding protein STMN2 is highly expressed in neurons and is implicated in amyotrophic lateral sclerosis. STMN2 degrades rapidly and accumulates at axotomy sites, suggesting fast turnover is crucial for its neuroprotective function. We show that STMN2 was primarily degraded by the ubiquitin-proteasome system. Its membrane-targeting N-terminal domain promoted fast turnover, whereas its tubulin-binding domain promoted stabilization. Proximity labeling and imaging showed that tubulin binding reduced STMN2 targeting to trans-Golgi network membranes. Pull-down assays showed that tubulin binds preferentially to soluble over membrane-bound STMN2. Our observations suggest that STMN2 interconverts between a soluble, tubulin-bound form and a membrane-bound, tubulin-free form, and is rapidly degraded when released from both membranes and tubulin. We propose that tubulin binding sequesters and stabilizes STMN2, while its neuroprotective function involves an unknown membrane activity.

DOI: https://doi.org/10.1083/jcb.202502192
Mol Neurodegener. 2025 Oct 31. 20(1): 116

C-terminus-dependent detection of lysosomal alpha-synuclein in nigral Parkinson's disease human brain neurons.

Martino L Morella, Bana Al Khayrat, Tim E Moors, Lisanne In't Veld, Irene Frigerio, Vinod Udayar, Bram L van der Gaag, Wilma D J van de Berg.

  The abnormal accumulation of alpha-Synuclein (αSyn) within neurons is a hallmark of synucleinopathies, such as Parkinson's disease (PD), and could stem from impaired protein degradation. Genetic, in vitro, and post-mortem studies have suggested that lysosomal dysfunction and impaired proteolytic activity play important roles in the pathogenesis of PD. Lysosomes have been proposed as key sites for αSyn degradation, but direct evidence of the lysosomal localization of endogenous αSyn in the human brain is limited. This study aimed to investigate the localization of αSyn proteoforms, including different post-translational modifications (PTMs), within lysosomes of post-mortem human nigral neurons. We analyzed formalin-fixed, paraffin-embedded brain tissue from donors diagnosed with PD, PD with Dementia (PDD) or incidental Lewy body disease (iLBD). Substantia nigra sections were assessed using an extensive panel of αSyn-specific antibodies, including PTM-specific antibodies, and selected lysosomal markers via multiplex immunofluorescence, confocal and stimulated emission depletion (STED) microscopy. Here, we demonstrate widespread accumulation of αSyn within lysosomes in nigral dopaminergic neuron somas of donors with PD/PDD and iLBD. This lysosomal αSyn appeared morphologically distinct from cytosolic inclusions such as Lewy bodies (LBs) and related macro-aggregates, and was present both in cells with and without these larger αSyn deposits. When present, macro-aggregates were consistently accompanied by ring-shaped lysosomal structures. Compared to other neuronal morphologies, lysosomal αSyn was the most frequent morphology at early Braak stages (1-4), with a decline at later stages (5-6). Interestingly, lysosomal αSyn was detected solely by targeting the N-terminus or the NAC domain of αSyn, and not with antibodies targeting Serine 129-phosphorylated αSyn or other epitopes at the C-terminus (CT), suggesting that lysosome-associated αSyn lacks the CT. Our findings reveal two co-existing pools of neuronal somatic αSyn: a CT-negative lysosome-associated form, and a primarily non-lysosomal CT-positive form. Overall, we provide direct evidence of lysosomal involvement in cellular αSyn metabolism in post-mortem human PD brain.

Keywords:  Alpha-synuclein; C-terminus; Lysosome; Morphology; Parkinson’s; Truncation

DOI:  https://doi.org/10.1186/s13024-025-00884-3
Elife. 2025 Oct 29. pii: RP104896. [Epub ahead of print]13

Axonal injury signaling is restrained by a spared synaptic branch.

Laura J Smithson, Juliana L Zang, Lucas Junginger, Thomas J Waller, Lauren Reilly-Jankowiak, Sophia A Khan, Ye Li, Dawen Cai, Catherine A Collins.

  The intrinsic ability of injured neurons to degenerate and regenerate their axons facilitates nervous system repair; however, this ability is not engaged in all neurons and injury locations. Here, we investigate the regulation of a conserved axonal injury response pathway with respect to the location of damage in branched motoneuron (MN) axons in Drosophila larvae. The dileucine zipper kinase (DLK; also known as MAP3K12 in mammals and Wallenda (Wnd) in Drosophila) is a key regulator of diverse responses to axonal injury. In three different populations of MNs, we observed the same striking result that Wnd/DLK signaling becomes activated only in response to injuries that remove all synaptic terminals. Injuries that spared even a small part of a synaptic terminal were insufficient to activate Wnd/DLK signaling, despite the presence of extensive axonal degeneration. The regulation of injury-induced Wnd/DLK signaling occurs independently of its previously known regulator, the Hiw/PHR ubiquitin ligase. We propose that Wnd/DLK signaling regulation is linked to the trafficking of a synapse-to-nucleus axonal cargo and that this mechanism enables neurons to respond to impairments in synaptic connectivity.

Keywords:  D. melanogaster; axonal degeneration; axonal injury signaling; axonal regeneration; neuroscience; spared synapse; structural plasticity

DOI:  https://doi.org/10.7554/eLife.104896
Elife. 2025 Oct 28. pii: RP102663. [Epub ahead of print]13

The Rab7-Epg5 and Rab39-ema modules cooperatively position autophagosomes for efficient lysosomal fusions.

Attila Boda, Villő Balázs, Anikó Nagy, Dávid Hargitai, Mónika Lippai, Zsófia Simon-Vecsei, Márton Molnár, Fanni Fürstenhoffer, Gábor Juhász, Péter Lőrincz.

  Macroautophagy, a major self-degradation pathway in eukaryotic cells, utilizes autophagosomes to transport self-material to lysosomes for degradation. While microtubular transport is crucial for the proper function of autophagy, the exact roles of factors responsible for positioning autophagosomes remain incompletely understood. In this study, we performed a loss-of-function genetic screen targeting genes potentially involved in microtubular motility. A genetic background that blocks autophagosome-lysosome fusions was used to accurately analyze autophagosome positioning. We discovered that pre-fusion autophagosomes move towards the non-centrosomal microtubule organizing center (ncMTOC) in Drosophila fat cells, which requires a dynein-dynactin complex. This process is regulated by the small GTPases Rab7 and Rab39 together with their adaptors: Epg5 and ema, respectively. The dynein-dependent movement of vesicles toward the nucleus/ncMTOC is essential for efficient autophagosomal fusions with lysosomes and subsequent degradation. Remarkably, altering the balance of kinesin and dynein motors changes the direction of autophagosome movement, indicating a competitive relationship where normally dynein-mediated transport prevails. Since pre-fusion lysosomes were positioned similarly to autophagosomes, it indicates that pre-fusion autophagosomes and lysosomes converge at the ncMTOC, which increases the efficiency of vesicle fusions.

Keywords:  D. melanogaster; autophagosome; cell biology; dynein; fusion; lysosome; microtubular transport; ncMTOC

DOI:  https://doi.org/10.7554/eLife.102663
Biomedicines. 2025 Oct 15. pii: 2518. [Epub ahead of print]13(10):

Extracellular Vesicles-Dependent Secretion Regulates Intracellular CYFIP2 Protein Homeostasis in Cortical Neurons.

Michael J Culp, Breandan J Rosolia, Cameron Keyser, Jingqi Yan.

  Background: Fragile X Syndrome (FXS) is the most common monogenic cause of autism spectrum disorders, and is characterized by the excessive immature excitatory synapses in cortical neurons, leading to excitatory/inhibitory imbalance and core autistic behaviors. This synaptic pathology has been attributed to dysregulated levels of synaptic proteins, including CYFIP2: a key regulator of synaptic structure and plasticity. However, the mechanism underlying the increased CYFIP2 protein level in FXS neurons remains unclear. Neurons abundantly secrete extracellular vesicles (EVs) enriched with bioactive cargos (proteins and miRNAs). Objectives: the goal of this research is to identify whether EV-dependent secretion plays important roles in regulating the intracellular CYFIP2 protein level in WT and FXS neurons. Methods and Results: our proteomic analysis reveals that CYFIP2 protein is packaged in EVs released by mouse cortical neurons. Pharmacological and genetic blockades of neuronal EV release significantly elevated intracellular CYFIP2 levels by 78 ± 14% and 168 ± 39%, respectively. Glutamate-evoked EV release significantly reduced the CYFIP2 level by 24 ± 2%. Neurons from Fmr1 KO mice, an FXS model, secreted significantly less EVs (46 ± 5%) than the wild type, and showed significantly elevated CYFIP2 (by 155 ± 31%). Evoking EV release in FXS neurons significantly lowered the intracellular CYFIP2 (by 53 ± 6%). Conclusions: these findings identify an EV-secretion-dependent mechanism that controls neuronal CYFIP2 level, implicating EV-mediated export in the regulation of synaptic protein homeostasis, synaptic remodeling, and FXS-associated synaptic deficits.

Keywords:  CYFIP2; Fragile X Syndrome; extracellular vesicles; neurons; synapse

DOI:  https://doi.org/10.3390/biomedicines13102518
Curr Biol. 2025 Oct 27. pii: S0960-9822(25)01263-1. [Epub ahead of print]

Metabolic rewiring prevents neurodegeneration caused by chronic mitochondrial dysfunction.

Shlesha Richhariya, Daniel Shin, Matthias Schlichting, Michael Rosbash.

  The mitochondrial fission-fusion cycle is often disrupted in neurodegenerative diseases, but this important, dynamic process is not well characterized in healthy long-lived neurons of animals. We used an efficient cell-type-specific CRISPR strategy to knock out key fission and fusion genes in specific Drosophila neurons. Neither process is essential for neuronal survival and function, but the fusion knockouts had a larger impact than that of fission, especially in older animals. Mutations in the human mitochondrial inner membrane fusion gene Opa1 often cause the disease optic atrophy. Importantly, knockout of Opa1 in neurons causes a dramatic age-dependent transcriptomic response. This response resembles those of cancer cells and includes the upregulation of glycolytic genes, including Lactate dehydrogenase (Ldh). A novel double knockout strategy indicates that Ldh enhances the reduced ATP levels of the fusion mutants and is essential to prevent age-dependent neurodegeneration. This neuroprotective upregulation of Ldh is largely mediated by the transcription factor ATF4. The identified relationship-dysfunctional mitochondrial fusion alters metabolism-is reminiscent of Warburg's original cancer hypothesis, albeit in neurons. These data underscore the similarity of the two molecular programs, which promote growth in cancer and viability in the case of neurodegeneration.

Keywords:  ATF4; CRISPR; Drosophila; Warburg effect; mitochondrial dynamics; neurodegeneration

DOI:  https://doi.org/10.1016/j.cub.2025.09.063
J Cell Sci. 2025 Oct 15. pii: jcs264177. [Epub ahead of print]138(20):

Tackling ciliary specialization to understand phenotypic variability in human primary ciliopathies.

Ruxandra Bachmann-Gagescu, John A Sayer.

  Primary cilia are crucial cellular organelles with vital roles in signal transduction and cellular function. Disruptions in primary ciliary structure or function underlie a group of genetic disorders known as primary ciliopathies. These disorders present as a diverse range of clinical features with prominent phenotypic variability, often complicating their diagnosis and the basic understanding of their underlying molecular mechanisms. To grasp this complexity, the ciliopathy field is moving from a static view of primary cilia towards a more comprehensive understanding of their dynamic and specialized cell-type-specific roles. By building on the large amount of knowledge gathered over the past decades and by employing recently developed tools, including multi-omics and human cell-based in vitro models, we can now interrogate ciliary specialization to understand the role of cilia in each tissue and the consequences of ciliary gene dysfunction on human health. This Perspective explores the current challenges and opportunities associated with these modern tools and databases, highlighting important action points to advance our understanding of this fascinating organelle and its role in human health and disease.

Keywords:  Cell-type-specific ciliary specialization; Fibro-cystic kidney disease; Multi-omics; Neurodevelopmental ciliopathies; Organoids; Phenotypic heterogeneity; Primary cilia; Retinal dystrophies; iPSC-derived models

DOI:  https://doi.org/10.1242/jcs.264177
J Cell Biol. 2026 Jan 05. pii: e202504178. [Epub ahead of print]225(1):

Close-up of vesicular ER exit sites by volume electron imaging using FIB-SEM.

Athul Nair, Akhil Nair, Patrick Stock, Arkash Jain, Elliott Somerville, Anwesha Sanyal, Jose Inacio Costa-Filho, Tom Kirchhausen.

The architecture of ER exit sites (ERES), the first sites of membrane remodeling in protein secretion, remains unclear, with descriptions ranging from vesicular clusters to extended tubular structures. We addressed this divergence by visualizing ERES in cells not overexpressing secretory cargo using large-scale volume-focused ion beam scanning EM (FIB-SEM) after high-pressure freeze substitution. Automated segmentation in EM (ASEM), our 3D U-Net pipeline trained with sparsely labeled 50-70-nm COPI vesicles near the Golgi, accurately detected them in HeLa, SVG-A, and iPSC-derived neurons. Using the same model, we identified abundant clusters of ∼5-40 larger vesicles (∼65-85 nm) confined within ∼250 nm3 regions adjacent to flattened ER domains, consistent with vesicular ERES. Similar assemblies also appeared alongside tubular networks and varicosities extending from enlarged ER domains, previously described as the sole ERES in HeLa cells. These findings reveal that vesicular ERES are widespread and morphologically diverse, resolving longstanding contradictions in early secretory pathway organization.

DOI: https://doi.org/10.1083/jcb.202504178
Int J Mol Sci. 2025 Oct 14. pii: 9991. [Epub ahead of print]26(20):

Marf- and Opa1-Dependent Formation of Mitochondrial Network Structure Is Required for Cell Growth and Subsequent Meiosis in Drosophila Males.

Tatsuru Matsuo, Mitsuki Yamanaka, Yoshihiro H Inoue.

  Mitochondria are dynamic organelles that undergo repeated fusion and fission. We studied how the distribution and shape of mitochondria change during Drosophila spermatogenesis and whether factors that regulate their dynamics are necessary for these changes. Unlike the shortened mitochondria seen in mitosis, an interconnected network of elongated mitochondria forms before meiosis and is maintained during meiotic divisions. Mitochondria are evenly divided into daughter cells, relying on microtubules and F-actin. To explore the role of mitochondrial network structure in cell growth and meiosis, we depleted the mitochondrial fusion factors Opa1 and Marf and the morphology proteins Letm1 and EndoB in spermatocytes. This knockdown led to inhibited cell growth and failed meiosis. As a result, the spermatocytes differentiated into spermatids without completing meiosis. The knockdown also inhibited the cytoplasmic and nuclear accumulation of Cyclin B before meiosis, and Cdk1 was not fully activated at the onset of meiosis. Notably, ectopic overexpression of Cyclin B partially rescued the failure of meiosis. Many spermatids from the spermatocytes subjected to the knockdowns contained multiple smaller nuclei and abnormally shaped Nebenkerns. These findings suggest that mitochondrial network structure, maintained by fusion and morphology factors, is essential for meiosis progression and Nebenkern formation in Drosophila spermatogenesis.

Keywords:  Cyclin B; Drosophila; meiosis; mitochondrial dynamics; nebenkern; spermatogenesis

DOI:  https://doi.org/10.3390/ijms26209991
Cell Death Dis. 2025 Oct 27. 16(1): 762

Impairment of lysosomal quality control in Huntington disease.

Paola Rusmini, Francesco Mina, Marta Valenza, Martina Vitali, Veronica Ferrari, Barbara Tedesco, Elena Casarotto, Marta Cozzi, Marta Chierichetti, Ali Mohamed, Paola Pramaggiore, Laura Cornaggia, Carmelo Milioto, Maria Brodnanova, Rocio Magdalena, Prashant Koshal, Margherita Piccolella, Riccardo Cristofani, Mariarita Galbiati, Valeria Crippa, Angelo Poletti.

Huntington disease (HD) is a neurodegenerative disease caused by a polyglutamine expansion (polyQ) in the Huntingtin protein (muHTT), which makes it prone to misfolding and aggregation. muHTT aggregates sequester a wide variety of proteins essential for cell homeostasis, including chaperones and transcription factors, and their depletion may contribute to HD pathogenesis. Lysosomes are the main hubs for degradative and signaling activities in cells, and their functionality is crucial for cell homeostasis, especially for neurons. Different forms of cellular stresses, including proteotoxic stresses, can alter lysosome integrity and induce lysosomal membrane permeabilization (LMP). Damaged lysosomes are recognized by galectins, in particular galectin-3 (LGALS3) with activation of the lysosome quality control (LQC) system responsible for repairing, degrading, or replacing leaky lysosomes. The system is transcriptionally regulated by the transcription factors EB and E3 (TFEB and TFE3, respectively). Using HD mouse and cell models, we demonstrated that TFEB and TFE3 are sequestered in muHTT aggregates, and muHTT proteins associates with LMP triggering the translocation of LGALS3 to the lumen of lysosomes, with a close relation between polyQ size and severity of these events. Moreover, we demonstrated that TFEB and TFE3 silencing or overexpression modulate muHTT aggregation. TFEB and TFE3 knockdown worsens muHTT aggregation, while their overexpression reduces muHTT inclusions and concurrently reduces LGALS3 accumulation via lysophagy and lysosome replacement. Our findings suggest that both TFEB and TFE3 are implicated in HD, and their sequestration in muHTT inclusions increase the vulnerability of neurons to lysosome injury, altering LQC and contributing to disease pathogenesis. In physiologial conditions, lysosome membrane permeabilization occurs and activates TFEB and TFE3 triggering a response to induce lysophagy and lysosome biogenesis. In HD, muHTT sequesters TFEB and TFE3 into inclusions and the reduced TFEB/TFE3 bioavailability prevents the activation of lysophagy and leading to the accumulation of damaged lysosomes. Created in BioRender.

DOI: https://doi.org/10.1038/s41419-025-08103-z
Pharmaceuticals (Basel). 2025 Oct 18. pii: 1573. [Epub ahead of print]18(10):

The Other Side of the Same Coin: Beyond the Coding Region in Amyotrophic Lateral Sclerosis.

Paola Ruffo, Benedetta Perrone, Francesco Perrone, Francesca De Amicis, Rodolfo Iuliano, Cecilia Bucci, Angela Messina, Francesca Luisa Conforti.

  Transposable elements (TEs), once regarded as genomic "junk," are now recognized as powerful regulators of gene expression, genome stability, and innate immunity. In the context of neurodegeneration, particularly Amyotrophic Lateral Sclerosis (ALS), accumulating evidence implicates TEs as active contributors to disease pathogenesis. ALS is a fatal motor neuron disease with both sporadic and familial forms, linked to genetic, epigenetic, and environmental factors. While coding mutations explain a subset of cases, advances in long-read sequencing and epigenomic profiling have unveiled the profound influence of non-coding regions-especially retrotransposons such as LINE-1, Alu, and SVA-on ALS onset and progression. TEs may act through multiple mechanisms: generating somatic mutations, disrupting chromatin architecture, modulating transcriptional networks, and triggering sterile inflammation via innate immune pathways like cGAS-STING. Their activity is normally repressed by epigenetic regulators, including DNA methylation, histone modifications, and RNA interference pathways; however, these controls are compromised in ALS. Taken together, these insights underscore the translational potential of targeting transposable elements in ALS, both as a source of novel biomarkers for patient stratification and disease monitoring, and as therapeutic targets whose modulation may slow neurodegeneration and inflammation. This review synthesizes the current knowledge of TE biology in ALS; integrates findings across molecular, cellular, and systems levels; and explores the therapeutic potential of targeting TEs as modulators of neurodegeneration.

Keywords:  amyotrophic lateral sclerosis; epigenetic regulation; retrotransposons; transposable elements

DOI:  https://doi.org/10.3390/ph18101573
Int J Mol Sci. 2025 Oct 20. pii: 10204. [Epub ahead of print]26(20):

Autophagy-Lysosome Pathway in Multiple System Atrophy Pathogenesis: The Best Is Yet to Come.

Panagiota Mavroeidi, Maria Xilouri.

  Multiple lines of evidence extracted from human post-mortem brain material and cellular and animal models of concomitant proteinopathies cumulatively suggest that the neuronal protein α-Synuclein exerts a strong influence on the pathogenesis of neurodegenerative comorbidities, collectively termed α-Synucleinopathies. Accumulation of α-Synuclein-positive inclusions in neurons or oligodendrocytes is the main histopathological hallmark of Parkinson's disease (PD) or multiple system atrophy (MSA), respectively. In addition, various pieces of data indicate that components of the autophagy-lysosomal pathway are altered in the context of α-Synucleinopathies. α-Synuclein itself is degraded by autophagy; however, aberrant protein conformations may impair lysosomal function. Genetic PD often involves components of the lysosome, including common genetic mutations in GBA1, which encodes for the lysosomal enzyme β-glucocerebrosidase. Alterations in lysosomal components that correlate with a commensurate increase in α-Synuclein deposition have been widely observed in PD brains. However, corresponding data in the context of MSA are emerging but remain less extensive than PD. In the current review, we focus on the pathological features as well as the impairments in the autophagy-lysosome pathway (ALP) that are associated with MSA and discuss the current challenges and future directions of therapeutic strategies targeting autophagy in experimental MSA-like models.

Keywords:  autophagy; lysosomes; multiple system atrophy (MSA); neurodegeneration; oligodendrocytes; therapeutics; α-Synuclein

DOI:  https://doi.org/10.3390/ijms262010204
STAR Protoc. 2025 Oct 23. pii: S2666-1667(25)00568-4. [Epub ahead of print]6(4): 104162

Protocol for in vitro immunofluorescence staining in a Transwell co-culture system.

Jenny Y Y Ooi, Yung Shing Tsang, Lawrence P McMahon, Limy Wong.

  Immunofluorescence (IF) is a key technique for localizing target molecules. Here, we present a protocol for in vitro IF staining optimized for skeletal muscle myotube formation cultured on Transwell inserts. We describe steps for primary cell culture, fixing, permeabilization, and antibody incubation. We then detail procedures for quantifying myotube differentiation by IF microscopy to calculate fusion index. This protocol will enable direct IF staining on cell monolayer seeded on permeable support in co-culture or other Transwell studies.

Keywords:  Molecular Biology; cell Biology; microscopy

DOI:  https://doi.org/10.1016/j.xpro.2025.104162
J Neurosci Res. 2025 Nov;103(11): e70090

Evolution of Axonal Injury in the Closed Head Impact Model of Engineered Rotational Acceleration in Adult Ferrets.

Justin L Krieg, Kosta Antolis, Carl Hooper, Hasini Kapuwelle, Rebecca George, William T O'Brien, Stuart J McDonald, Anna V Leonard, Renée J Turner, Frances Corrigan.

  Concussion-related symptoms, such as impaired balance, slower processing speed, attention deficits, memory dysfunction, and irritability, are thought to result from diffuse axonal injury (DAI), characterized by selective damage to white matter axons. Axons subjected to this mechanical stretch injury exhibit diverse pathological changes, including disruption of axonal transport, neurofilament compaction and degradation, myelin sheath disruption, and loss of sodium channels required for action potential generation and propagation. These distinct forms of axonal pathology may evolve differentially over time and preferentially localize to specific white matter tracts. In this study, we employed the clinically relevant ferret model of concussion using the closed head impact model of engineered rotational acceleration (CHIMERA). 55 male ferrets were randomly allocated to sham or injury groups and then to either 24 h, 72 h, or 14d survival time points. We confirmed that axonal transport disruption and neurofilament pathology represent independent processes, with minimal colocalization but a shared peak of around 72 h following injury. Furthermore, we observed a persistent loss of ankyrin-G, a critical anchoring protein for sodium channels at the node of Ranvier, up to 14d postinjury, suggesting that the resultant impairment in axonal transmission may underlie many concussion symptoms. Indeed, injured ferrets displayed significant deficits in balance, working memory, spatial memory, and recognition memory. These findings demonstrate that the CHIMERA model in ferrets recapitulates key axonal pathologies and their associated clinical manifestations following concussion. This model offers a valuable platform for investigating the temporal evolution of axonal injury and developing targeted therapeutic interventions to mitigate concussion-related deficits.

Keywords:  axonal injury; cognition; diffuse traumatic brain injury; ferret; motor; myelin; oligodendrocytes

DOI:  https://doi.org/10.1002/jnr.70090
Clin Genet. 2025 Oct 26.

AAV9-Mediated Intrastriatal Delivery of Mutant HTT With 82 CAG Repeats Induces Huntington's Disease-Like Pathology and Behavioral Deficits in Mice.

Lin Huang, Yazhou Tang, Yuan Wang, Yali Tan, Wei Lei, Xinyu Deng, Yongjie Luo.

  Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by CAG repeat expansion in the HTT gene. Existing toxin-induced and genetic models provide important insights, but none fully replicate the progressive pathology of HD. An AAV9-mediated striatal mouse model expressing mutant HTT with 82 CAG repeats was established to reproduce hallmark neuropathological changes and behavioral deficits. Male C57BL/6 mice received bilateral intrastriatal injections of AAV9-HTT-82Q or control AAV9-GFP. Behavioral performance was assessed by rotarod, balance beam, open field, and Y-maze tests. Neuropathology was examined with HE/Nissl staining, TUNEL assay, and immunofluorescence for mHTT, DARPP-32, GFAP, and Iba1. AAV9-82Q mice exhibited progressive motor coordination deficits on the rotarod from Week 4 and impaired beam traversal from Week 18. Open field testing revealed persistent hyperactivity from Week 8, while anxiety-like and cognitive measures showed only mild, non-significant trends. Histological analysis demonstrated extensive mHTT aggregation in the striatum, accompanied by neuronal pyknosis, vacuolization, and significant loss of Nissl-positive neurons. TUNEL staining confirmed increased apoptosis. Immunofluorescence further revealed selective reduction of DARPP-32+ medium spiny neurons, along with marked astrogliosis and microgliosis, indicating robust neurodegeneration and inflammatory responses. The AAV9-82Q model induces adult-onset, progressive HD-like pathology with early motor impairments, neuronal loss, and glial activation. It complements existing models and provides a reproducible platform for mechanistic studies and preclinical therapeutic evaluation.

Keywords:  Huntington disease; adeno‐associated viruses; mice model; neurodegeneration; striatum

DOI:  https://doi.org/10.1111/cge.70076
Dis Model Mech. 2025 Oct 01. pii: dmm052510. [Epub ahead of print]18(10):

Brain organoid models of Huntington's disease shift the focus towards neurodevelopment.

Wenqing Xu, Alessandro Prigione.

Huntington's disease (HD) is traditionally viewed as an age-related disorder. Emerging evidence suggests that mutant huntingtin (mHTT) disrupts early neurodevelopment, although the contribution of developmental alterations to the late disease onset remains to be clarified. Leveraging human pluripotent stem cell-derived brain organoids, we and others are exploring how mHTT affects the developing human brain. These models reveal impaired neural progenitor organization and function, accompanied by a mitochondrial stress response, indicating reduced capacity to manage cellular stress. Enhancing mitochondrial health and promoting neural cell resilience may thus represent potential strategies for improving the brain's compensatory mechanisms, thereby prolonging a healthy state. These insights highlight a potential window of opportunity for therapeutic interventions. Targeting mitochondrial fitness and neurodevelopmental pathways at early stages - long before clinical symptoms emerge - could help prevent or delay disease onset and progression in affected individuals.

DOI: https://doi.org/10.1242/dmm.052510