bims-axbals 2025-05-18 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–05–18
24 papers selected by
TJ Krzystek

Proteomics Analysis of the TDP-43 Interactome in Cellular Models of ALS Pathogenesis.
A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons.
Network analysis of the cerebrospinal fluid proteome reveals shared and unique differences between sporadic and familial forms of amyotrophic lateral sclerosis.
Autophagy- and oxidative stress-related protein deregulation mediated by extracellular vesicles of human MJD/SCA3 iPSC-derived neuroepithelial stem cells and differentiated neural cultures.
Astrocytes carrying LRRK2 G2019S exhibit increased levels of clusterin chaperone via miR-22-5p and reduced ability to take up α-synuclein fibrils.
Berberine can be a Potential Therapeutic Agent in Treatment of Huntington's Disease: A Proposed Mechanistic Insight.
Lysosomal Repair in Health and Disease.
Small-molecule dissolution of stress granules by redox modulation benefits ALS models.
Profiling human iPSC-derived sensory neurons for analgesic drug screening using a multi-electrode array.
Atg9 is a conserved regulator of lysosome repair.
Targeting autophagy in astrocytes: a potential for neurodegenerative disease intervention.
Discovery of Golgi Membrane-associated Degradation Pathway, GOMED: A Focus on 15 Years of Ultrastructural Analyses.
Induced mitochondrial deficit by NDUFS3 transient silencing reduces RAB7 expression and causes lysosomal dysfunction in pancreatic cancer cells.
Single-cell mitochondrial morphomics reveals cellular heterogeneity and predicts complex I, III, and ATP synthase Inhibition responses.
Human iPSC-Derived Neuron and Oligodendrocyte Co-culture as a Small-Molecule Screening Assay for Myelination.
Fat traffic control: S-acylation in axonal transport.
Design, synthesis and characterization of aryl bis-guanyl hydrazones as RNA binders of C9orf72 G4C2 extended repeats.
AI-guided laser purification of human iPSC-derived cardiomyocytes for next-generation cardiac cell manufacturing.
Snapshots of mitochondrial fission imaged by cryo-scanning transmission electron tomography.
Glymphatic flow reduced in Huntington disease.
Proteostasis and autophagy disruption by the aging-related VGVAPG hexapeptide - preliminary insights into a potential novel elastin-induced neurodegeneration pathway in an in vitro human cellular neuron model.
Molecular machineries shaping the mitochondrial inner membrane.
The wave nature of the action potential.
MEK1/2 inhibitors suppress pathological α-synuclein and neurotoxicity in cell models and a humanized mouse model of Parkinson's disease.

J Neurochem. 2025 May;169(5): e70079

Proteomics Analysis of the TDP-43 Interactome in Cellular Models of ALS Pathogenesis.

Flora Cheng, Tyler Chapman, Juliana Venturato, Jennilee M Davidson, Stella A Polido, Livia Rosa-Fernandes, Rebecca San Gil, Hannah J Suddull, Selina Zhang, Chiara Y Macaslam, Paulina Szwaja, Roger Chung, Adam K Walker, Stephanie L Rayner, Marco Morsch, Albert Lee.

  Cytoplasmic aggregation and nuclear depletion of TAR DNA-binding protein 43 (TDP-43) is a hallmark pathology of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). However, the protein interactome of TDP-43 remains incompletely defined. In this study, we aimed to identify putative TDP-43 protein partners within the nucleus and the cytoplasm and with different disease models of TDP-43 by comparing TDP-43 interaction partners in three different cell lines. We verified the levels of interaction of protein partners under stress conditions as well as after introducing TDP-43 variants containing ALS missense mutations (G294V and A315T). Overall, we identified 58 putative wild-type TDP-43 interactors, including novel binding partners responsible for RNA metabolism and splicing. Oxidative stress exposure broadly led to changes in TDP-43WT interactions with proteins involved in mRNA metabolism, suggesting a dysregulation of the transcriptional machinery early in disease. Conversely, although G294V and A315T mutations are both located in the C-terminal domain of TDP-43, both mutants presented different interactome profiles with most interaction partners involved in translational and transcriptional machinery. Overall, by correlating different cell lines and disease-simulating interventions, we provide a list of high-confidence TDP-43 interaction partners, including novel and previously reported proteins. Understanding pathological changes to TDP-43 and its specific interaction partners in different models of stress is critical to better understand TDP-43 proteinopathies and provide novel potential therapeutic targets and biomarkers.

Keywords:   ALS ; APEX ; MND ; TDP‐43; interactomics; proteomics

DOI:  https://doi.org/10.1111/jnc.70079
Cell Rep. 2025 May 10. pii: S2211-1247(25)00466-8. [Epub ahead of print]44(5): 115695

A multimodal screening platform for endogenous dipeptide repeat proteins in C9orf72 patient iPSC neurons.

Benedikt V Hölbling, Yashica Gupta, Paolo M Marchi, Magda L Atilano, Michael Flower, Enric Ureña, Rajkumar A Goulden, Hannah K Dobbs, Eszter Katona, Alla Mikheenko, Ashling Giblin, Ali Raza Awan, Chloe L Fisher-Ward, Niamh O'Brien, Deniz Vaizoglu, Liam Kempthorne, Katherine M Wilson, Lauren M Gittings, Mireia Carcolé, Marc-David Ruepp, Sarah Mizielinska, Linda Partridge, Pietro Fratta, Sarah J Tabrizi, Bhuvaneish T Selvaraj, Siddharthan Chandran, Emma Armstrong, Paul Whiting, Adrian M Isaacs.

  Repeat expansions in C9orf72 are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation generates neurotoxic dipeptide repeat proteins (DPRs). To study endogenous DPRs, we inserted the minimal HiBiT luciferase reporter downstream of sense repeat derived DPRs polyGA or polyGP in C9orf72 patient iPSCs. We show these "DPReporter" lines sensitively and rapidly report DPR levels in lysed and live cells and optimize screening in iPSC neurons. Small-molecule screening showed the ERK1/2 activator periplocin dose dependently increases DPR levels. Consistent with this, ERK1/2 inhibition reduced DPR levels and prolonged survival in C9orf72 repeat expansion flies. CRISPR knockout screening of all human helicases revealed telomere-associated helicases modulate DPR expression, suggesting common regulation of telomeric and C9orf72 repeats. These DPReporter lines allow investigation of DPRs in their endogenous context and provide a template for studying endogenous RAN-translated proteins, at scale, in other repeat expansion disorders.

Keywords:  C9orf72; CP: Neuroscience; RAN translation; amyotrophic lateral sclerosis; frontotemporal dementia; repeat expansion

DOI:  https://doi.org/10.1016/j.celrep.2025.115695
Mol Neurodegener. 2025 May 15. 20(1): 58

Network analysis of the cerebrospinal fluid proteome reveals shared and unique differences between sporadic and familial forms of amyotrophic lateral sclerosis.

Adam N Trautwig, Edward J Fox, Eric B Dammer, Anantharaman Shantaraman, Lingyan Ping, Duc M Duong, Caroline M Watson, Fang Wu, Seneshaw Asress, Qi Guo, Allan I Levey, James J Lah, Federico Verde, Alberto Doretti, Antonia Ratti, Nicola Ticozzi, Cindy V Ly, Timothy M Miller, Mark A Garret, James D Berry, Eleanor V Thomas, Christina N Fournier, Zachary T McEachin, Nicholas T Seyfried, Jonathan D Glass.

   BACKGROUND: Amyotrophic Lateral Sclerosis (ALS), a neurodegenerative disease involving loss of motor neurons, typically results in death within 3-5 years of disease onset. Although roughly 10% of cases can be linked to a specific inherited mutation (e.g., C9orf72 hexanucleotide repeat expansion or SOD1 mutation), the cause(s) of most cases are unknown. Consequently, there is a critical need for biomarkers that reflect disease onset and progression across ALS subgroups.
METHODS: We employed tandem mass tag mass spectrometry (TMT-MS) based proteomics on cerebrospinal fluid (CSF) to identify and quantify 2105 proteins from sporadic, C9orf72, and SOD1 ALS patients, asymptomatic C9orf72 expansion carriers, and controls (N = 101). To verify trends in our Emory University cohort we used data-independent acquisition (DIA-MS) on an expanded, four center cohort. This expanded cohort of 259 individuals included 50 sporadic ALS (sALS), 43 C9orf72 ALS, 22 SOD1 ALS, 72 asymptomatic gene carriers (59 C9orf72 and 13 SOD1) and 72 age-matched controls. We identified 2330 proteins and used differential protein abundance and network analyses to determine how protein profiles vary across disease subtypes in ALS CSF.
RESULTS: Differential abundance and co-expression network analysis identified proteomic differences between ALS and control, as well as differentially abundant proteins between sporadic, C9orf72 and SOD1 ALS. A panel of proteins differentiated forms of ALS that are indistinguishable in a clinical setting. An additional panel differentiated asymptomatic from symptomatic C9orf72 and SOD1 mutation carriers, marking a pre-symptomatic proteomic signature of genetic forms of ALS. Leveraging this large, multicenter cohort, we validated our ALS CSF network and identified ALS-specific proteins and network modules.
CONCLUSIONS: This study represents a comprehensive analysis of the CSF proteome across sporadic and genetic causes of ALS that resolves differences among these ALS subgroups and also identifies proteins that distinguish symptomatic from asymptomatic gene carriers. These new data point to varying pathogenic pathways that result in an otherwise clinically indistinguishable disease.

Keywords:  Amyotrophic Lateral Sclerosis ALS; C9orf72; Cerebrospinal Fluid (CSF); Differentially Abundant Proteins (DAP); SOD1; Weighted Gene Co-Expression Network Analysis (WGCNA)

DOI:  https://doi.org/10.1186/s13024-025-00838-9
Cell Death Dis. 2025 May 15. 16(1): 383

Autophagy- and oxidative stress-related protein deregulation mediated by extracellular vesicles of human MJD/SCA3 iPSC-derived neuroepithelial stem cells and differentiated neural cultures.

Liliana S Mendonça, Ricardo Moreira, Daniel Henriques, Mónica Zuzarte, Teresa M Ribeiro-Rodrigues, Henrique Girão, Luís Pereira de Almeida.

Extracellular vesicles (EVs) have been associated with the transport of molecules related to the pathological processes in neurodegenerative diseases. Machado-Joseph disease (MJD) is a neurodegenerative disorder triggered by mutant ataxin-3 protein that causes protein misfolding and aggregation resulting in neuronal death. To evaluate EVs' role in the potential spread of disease-associated factors in MJD, in this study, EVs were isolated from human Control (CNT) and MJD induced-pluripotent stem cell-derived neuroepithelial stem cells (iPSC-derived NESC) and their differentiated neural cultures (cell cultures composed of neurons and glia). EVs were characterized and investigated for their ability to interfere with cell mechanisms known to be impaired in MJD. The presence of mRNA and proteins related to autophagy, cell survival, and oxidative stress pathways, and the mutant ataxin-3, was evaluated in the EVs. SOD1, p62, and Beclin-1 were found present both in CNT and MJD EVs. Lower levels of the p62 autophagy-related protein and higher levels of the oxidative stress-related SOD1 protein were found in MJD EVs. The oxidative stress-related CYCS mRNA and autophagy-related SQSTM1, BECN1, UBC, ATG12, and LC3B mRNAs were detected in EVs and no significant differences in their levels were observed between CNT and MJD EVs. The internalization of EVs by human CNT neurons was demonstrated, and no effect of the EVs administration was observed on cell viability. Moreover, the incubation of MJD EVs (isolated from NESC or differentiated neural cultures) with human CNT differentiated neural cells resulted in the reduction of SOD1 and autophagy-related proteins ATG3, ATG7, Beclin-1, LC3B, and p62 levels. Finally, a tendency for accumulation of ataxin-3-positive aggregates in CNT differentiated neural cells co-cultured with MJD differentiated neural cells was observed. Overall, our data indicate that EVs carry autophagy- and oxidative stress-related proteins and mRNAs and provide evidence of MJD EVs-mediated interference with autophagy and oxidative stress pathways.

DOI: https://doi.org/10.1038/s41419-025-07659-0
Acta Neuropathol Commun. 2025 May 12. 13(1): 98

Astrocytes carrying LRRK2 G2019S exhibit increased levels of clusterin chaperone via miR-22-5p and reduced ability to take up α-synuclein fibrils.

Alice Filippini, Giulia Carini, Alessandro Barbon, Massimo Gennarelli, Isabella Russo.

  Accumulating evidence highlights that dysfunction of astrocyte biology might contribute to Parkinson's disease (PD) onset and progression. Leucine-rich repeat kinase 2 (LRRK2), a gene linked to genetic and familial PD, has been reported to affect astrocytic-related functions, including the ingestion of alpha-synuclein (α-syn) aggregates. In this context, we recently showed that the extracellular chaperone clusterin (Clu) binds to and limits the uptake of alpha-syn fibrils by astrocytes. Thus, starting from these premises, we explored whether LRRK2 G2019S affects aggregated α-syn ingestion through the Clu-related pathway and the underlying molecular mechanisms. We first validated in our LRRK2 G2019S knock-in (KI) mouse strain that primary astrocytes exhibited an impaired ability to ingest fibrillary α-syn. Then, we investigated whether LRRK2 G2019S affects this pathway through the modulation of Clu. In this regard, we collected several results showing that LRRK2 regulates Clu levels in astrocytes. Specifically, brain slices and primary astrocytes from KI mice with the LRRK2 G2019S pathological mutation exhibit increased levels of Clu protein compared to their respective wild-type (WT). Accordingly, we observed an opposite effect in brain slices and primary astrocytes from LRRK2 knock-out (KO) mice in comparison to their respective WT. To gain insights into the molecular mechanism underlying LRRK2-dependent Clu modulation, we found that LRRK2 controls Clu expression at the translation level through the action of miR-22-5p. In addition, we demonstrated that treatment with miR-22-5p mimic improves the ability of LRRK2 G2019S-KI astrocytes to take up α-syn pffs. Taken together, our findings indicate that the LRRK2-Clu pathway is involved in the ingestion of a-syn fibrils and that the impairment of α-syn uptake in LRRK2 G2019S-KI astrocytes is associated to Clu levels. Future studies will allow us to understand whether the modulation of astrocytic LRRK2 G2019S-Clu pathway might attenuate the neuronal spreading of α-syn pathology in PD.

Keywords:  Astrocytes; Clusterin; LRRK2; Parkinson’s disease; miRNA

DOI:  https://doi.org/10.1186/s40478-025-02015-x
Mol Neurobiol. 2025 May 16.

Berberine can be a Potential Therapeutic Agent in Treatment of Huntington's Disease: A Proposed Mechanistic Insight.

Seema Sharma, Inderpreet Kaur, Naina Dubey, Neelima Goswami, Sampat Singh Tanwar.

  Huntington's disease (HD) is a genetic neurodegenerative disorder caused by CAG repeat expansion in the HTT gene, producing mutant huntingtin (mHTT) protein. This leads to neuronal damage through protein aggregation, transcriptional dysregulation, excitotoxicity, and mitochondrial dysfunction. mHTT impairs protein clearance and alters gene expression, energy metabolism, and synaptic function. Therapeutic strategies include enhancing mHTT degradation, gene silencing via antisense oligonucleotides and RNAi, promoting neuroprotection through BDNF signaling, and modulating neurotransmitters like glutamate and dopamine. Berberine, a natural isoquinoline alkaloid, has emerged as a promising therapeutic option for HD due to its multifaceted neuroprotective properties. Research indicates that berberine can mitigate the progression of neurodegenerative diseases, including HD, by targeting various molecular pathways. It exhibits antioxidant, anti-inflammatory, and autophagy-enhancing effects, which are crucial in reducing neuronal damage and apoptosis associated with HD. These properties make berberine a potential candidate for therapeutic intervention in HD, as demonstrated in both cellular and animal models. Berberine activates the PI3K/Akt pathway, which is vital for cell survival and neuroprotection. It reduces oxidative stress and neuroinflammation, both of which are implicated in HD pathology. Berberine enhances autophagic processes, promoting the degradation of mutant huntingtin protein, a key pathological feature of HD. In transgenic HD mouse models, berberine administration has been shown to alleviate motor dysfunction and prolong survival. It effectively reduces the accumulation of mutant huntingtin in cultured cells, suggesting a direct impact on the disease's molecular underpinnings. Berberine's safety profile, established through its use in treating other conditions, supports its potential for clinical trials in HD patients. Its ability to modulate neurotransmitter levels and engage multiple signaling pathways further underscores its therapeutic promise. While berberine shows significant potential as a therapeutic agent for HD, further research is necessary to fully elucidate its mechanisms and optimize its clinical application. The current evidence in the review paper, primarily from preclinical studies, provides a strong foundation for future investigations into berberine's efficacy and safety in human HD patients.

Keywords:  Apoptosis; Berberine; Clinical trial; Gut microbiome-brain axis; Huntington's disease; Inflammation; Neurodegeneration; Neurotransmitter; Synaptic Plasticity

DOI:  https://doi.org/10.1007/s12035-025-05054-6
J Cell Physiol. 2025 May;240(5): e70044

Lysosomal Repair in Health and Disease.

Jinrui Xun, Jay Xiaojun Tan.

  Lysosomes are essential organelles degrading a wide range of substrates, maintaining cellular homeostasis, and regulating cell growth through nutrient and metabolic signaling. A key vulnerability of lysosomes is their membrane permeabilization (LMP), a process tightly linked to diseases including aging, neurodegeneration, lysosomal storage disorders, and cardiovascular disease. Research progress in the past few years has greatly improved our understanding of lysosomal repair mechanisms. Upon LMP, cells activate multiple membrane remodeling processes to restore lysosomal integrity, such as membrane invagination, tubulation, lipid patching, and membrane stabilization. These repair pathways are critical in preserving cellular stress tolerance and preventing deleterious inflammation and cell death triggered by lysosomal damage. This review focuses on the expanding mechanistic insights of lysosomal repair, highlighting its crucial role in maintaining cellular health and the implications for disease pathogenesis and therapeutic strategies.

Keywords:  Atg8ylation; CASM; ESCRT; Lysosomal repair; PITT; annexins; lysosomal membrane permeabilization; microlysophagy; stress granules

DOI:  https://doi.org/10.1002/jcp.70044
Nat Chem Biol. 2025 May 14.

Small-molecule dissolution of stress granules by redox modulation benefits ALS models.

Hiroyuki Uechi, Sindhuja Sridharan, Jik Nijssen, Jessica Bilstein, Juan M Iglesias-Artola, Satoshi Kishigami, Virginia Casablancas-Antras, Ina Poser, Eduardo J Martinez, Edgar Boczek, Michael Wagner, Nadine Tomschke, António M de Jesus Domingues, Arun Pal, Thom Doeleman, Sukhleen Kour, Eric Nathaniel Anderson, Frank Stein, Hyun O Lee, Xiaojie Zhang, Anatol W Fritsch, Marcus Jahnel, Julius Fürsch, Anastasia C Murthy, Simon Alberti, Marc Bickle, Nicolas L Fawzi, André Nadler, Della C David, Udai B Pandey, Andreas Hermann, Florian Stengel, Benjamin G Davis, Andrew J Baldwin, Mikhail M Savitski, Anthony A Hyman, Richard J Wheeler.

Neurodegenerative diseases, such as amyotrophic lateral sclerosis, are often associated with mutations in stress granule proteins. Aberrant stress granule condensate formation is associated with disease, making it a potential target for pharmacological intervention. Here, we identified lipoamide, a small molecule that specifically prevents cytoplasmic condensation of stress granule proteins. Thermal proteome profiling showed that lipoamide stabilizes intrinsically disordered domain-containing proteins, including SRSF1 and SFPQ, which are stress granule proteins necessary for lipoamide activity. SFPQ has redox-state-specific condensate dissolving behavior, which is modulated by the redox-active lipoamide dithiolane ring. In animals, lipoamide ameliorates aging-associated aggregation of a stress granule reporter protein, improves neuronal morphology and recovers motor defects caused by amyotrophic lateral sclerosis-associated FUS and TDP-43 mutants. Thus, lipoamide is a well-tolerated small-molecule modulator of stress granule condensation, and dissection of its molecular mechanism identified a cellular pathway for redox regulation of stress granule formation.

DOI: https://doi.org/10.1038/s41589-025-01893-5
Cell Rep Methods. 2025 May 06. pii: S2667-2375(25)00087-6. [Epub ahead of print] 101051

Profiling human iPSC-derived sensory neurons for analgesic drug screening using a multi-electrode array.

Christian Kuete Fofie, Rafael Granja-Vazquez, Vincent Truong, Patrick Walsh, Theodore Price, Swati Biswas, Gregory Dussor, Joseph Pancrazio, Benedict Kolber.

  Chronic pain is a global health issue, yet effective treatments remain limited due to poor preclinical-to-human translation. To address this, we developed a high-content screening (HCS) platform using hiPSC-derived nociceptors to identify analgesics targeting the peripheral nervous system. These cells, cultured on multi-well microelectrode arrays, achieved nearly 100% active electrodes by week 2, maintaining stable activity for at least 2 weeks. After 28 days, we assessed drug effects on neuronal activity, achieving strong assay performance (robust Z' > 0.5). Pharmacological tests confirmed responses to key analgesic targets, including ion channels (Nav, Cav, Kv, and TRPV1), neurotransmitter receptors (AMPAR and GABA-R), and kinase inhibitors (tyrosine and JAK1/2). Transcriptomic analysis validated target expression, though levels differed from primary human DRG cells. The platform was used to screen over 700 natural compounds, demonstrating its potential for analgesic discovery. This HCS platform facilitates the rapid discovery of uncharacterized analgesics, reducing preclinical-to-human translation failure.

Keywords:  CP: Stem cell; DRG; analgesic discovery; chronic pain; hiPSC; high-content screening; human induced pluripotent stem cell; nociceptor

DOI:  https://doi.org/10.1016/j.crmeth.2025.101051
J Cell Biol. 2025 Jul 07. pii: e202504129. [Epub ahead of print]224(7):

Atg9 is a conserved regulator of lysosome repair.

Ruoxi Wang, Eric H Baehrecke.

The ATG9 transmembrane protein scrambles lipids to regulate phagophore formation during autophagy. Two recent studies from Peng et al. (https://doi.org/10.1083/jcb.202411092) and De Tito et al. (https://doi.org/10.1101/2024.07.23.604321) identify ATG9 as a conserved regulator of lysosome repair in Caenorhabditis elegans and human cells, but differences in repair mechanisms exist between these taxa.

DOI: https://doi.org/10.1083/jcb.202504129
Front Cell Neurosci. 2025 ;19 1584767

Targeting autophagy in astrocytes: a potential for neurodegenerative disease intervention.

Maja Potokar, Jernej Jorgačevski.

  Autophagy contributes to cellular homeostasis by regulating the degradation and recycling of damaged organelles and misfolded proteins. In the central nervous system (CNS), impaired autophagy contributes to inflammation, disrupts cellular metabolism, and leads to the accumulation of toxic protein aggregates that accelerate the progression of neurodegenerative diseases. In addition to its role in protein and organelle turnover, autophagy facilitates the elimination of pathogenic bacteria and viruses, whose infections can also lead to neurological diseases and neuroinflammatory processes. Astrocytes, the most abundant glial cells in the CNS, play a crucial role in maintaining neuronal homeostasis by regulating neurotransmitter balance, ion exchange, and metabolic support. During neurodegeneration, they become reactive, actively participating in neuroinflammatory responses by releasing proinflammatory cytokines, activating microglia, and removing toxic aggregates. Cytokine-mediated responses and metabolic changes in astrocytes influence neuronal viability and neurotransmission. Autophagy in astrocytes plays an important role in tuning the astrocyte-dependent activity of neurons under physiological conditions and in pathological activation of astrocytes by disease, injury or pathogenic stimuli. In this review, we highlight the contribution of astrocytes to neurodegeneration from the perspective of changes in their cytoskeleton, the autophagy process in which the cytoskeleton plays a crucial role, and the metabolic support of neurons. The modulation of autophagy at different stages has the potential to serve as an additional therapeutic target in CNS diseases.

Keywords:  astrocyte; autophagy; cytoskeleton; mitophagy; neurodegeneration; neuroinflammation

DOI:  https://doi.org/10.3389/fncel.2025.1584767
Microscopy (Oxf). 2025 May 13. pii: dfaf023. [Epub ahead of print]

Discovery of Golgi Membrane-associated Degradation Pathway, GOMED: A Focus on 15 Years of Ultrastructural Analyses.

Satoko Arakawa, Hirofumi Yamaguchi, Shigeomi Shimizu.

  In this review, we focus on the ultrastructural characteristics of the Golgi membrane-associated degradation (GOMED) pathway, which have been clarified by electron microscopy and highlight recent advances in the elucidation of its molecular mechanism and physiological roles. The discovery of GOMED, an Atg5/Atg7-independent degradation pathway that differs from canonical autophagy in membrane origin, stimuli, and substrate specificity, has substantially expanded our understanding of intracellular degradation systems. In 2009, we identified GOMED as a novel, evolutionarily conserved autophagic pathway and demonstrated its role in intracellular degradation across eukaryotes, from yeast to mammals. We identified the conserved protein Hsv2/Wipi3 as an essential GOMED protein, which translocates to the trans-Golgi upon induction and remodels Golgi membranes into cup-shaped structures that engulf cytoplasmic components for lysosomal degradation. These processes contribute to organelle and secretory granule turnover, as well as mitochondrial clearance during erythroid differentiation. Moreover, neuronal-specific ablation of Wipi3 in mice causes severe cerebellar degeneration, implicating GOMED in tissue development and homeostasis. As these mechanisms are associated with diseases, such as neurodegenerative disorders and cancer, GOMED mechanisms should also be considered when establishing therapeutic strategies for these diseases.

Keywords:  Autophagy; GOMED; Golgi; Mitophagy; Neurodegeneration; Wipi3

DOI:  https://doi.org/10.1093/jmicro/dfaf023
Cell Commun Signal. 2025 May 14. 23(1): 224

Induced mitochondrial deficit by NDUFS3 transient silencing reduces RAB7 expression and causes lysosomal dysfunction in pancreatic cancer cells.

Giulia Girolimetti, Sinforosa Gagliardi, Paola Cordella, Grazia Bramato, Riccardo Di Corato, Roberta Romano, Flora Guerra, Cecilia Bucci.

   BACKGROUND: RAB7 is a small GTPase with multiple cellular roles, regulating late endocytic trafficking and lysosomal biogenesis, influencing mitochondria-lysosome crosstalk, and contributing to many mitochondrial processes. Mitochondrial dysfunctions are widely reported in cancer and the development of cancer therapeutic strategies targeting mitochondria gained momentum in recent years. Mitochondrial impairment can cause alterations of mitochondria-lysosome crosstalk and can influence lysosomal function. Here, we used cell models of pancreatic cancer, one of the deadliest cancers worldwide, to cause a transient mild mitochondrial deficit lowering NDUFS3 protein levels in order to investigate the consequences on RAB7 and on the late endocytic pathway and, thus, the contribution of the mitochondria-lysosomes communication alterations to cancer progression.
METHODS: NDUFS3 and RAB7 downregulation was obtained by RNA interference (RNAi). Seahorse assays, Western blot analysis, mitochondrial staining, and Transmission Electron Microscopy (TEM) were used to assess silencing effects on mitochondrial structure and functioning. Western blotting was used to investigate expression of late endocytic pathway proteins and of the invasion marker vimentin. Confocal microscopy was used to analyze the mitochondrial network and lysosomal assessment. Zymography was performed to evaluate the ability to digest the extracellular matrix linked to cancer migration. SRB and colony assays were performed to assess cancer viability and proliferation. Wound healing assay and FluoroBlok membranes were used to determine migration and invasiveness.
RESULTS: In pancreatic cancer cells, transient silencing of the NDUFS3 protein caused mitochondrial deficit, slower oxidative metabolism, and mitochondrial morphology alterations. In this context, we observed RAB7 downregulation and impairment of the late endocytic pathway. In addition, NDUFS3-silenced RAB7-downregulated cells showed less invasive tumorigenic potential revealed by reduced levels of vimentin and other Epithelial-to-Mesenchymal Transition proteins, decreased viability, migration and invasiveness. Moreover, we found that modulation of RAB7 expression may regulate vimentin levels and influence mitochondrial morphology and levels of mitochondrial proteins.
CONCLUSIONS: Overall, our data show that mitochondrial deficit determines alterations of the crosstalk with lysosomes, leading to dysfunctions, and that this process is regulated by RAB7 acting as an oncogene. This highlights the synergic role of RAB7 and mitochondrial dysfunction, focusing on a cellular mechanism that may boost the effect of mitochondrial dysfunction in the cells, leading to the reduction of the tumorigenic potential.

Keywords:  Endocytosis; Lysosomes; Mitochondria; Pancreatic cancer; RAB7

DOI:  https://doi.org/10.1186/s12964-025-02214-y
Sci Rep. 2025 May 14. 15(1): 16715

Single-cell mitochondrial morphomics reveals cellular heterogeneity and predicts complex I, III, and ATP synthase Inhibition responses.

Ratneswary Sutharsan, Maddi Biaut Hontaas, Yan Li, Hao Xiong, Hartwig Preckel, Carolyn M Sue, Gautam Wali.

Mitochondrial heterogeneity drives diverse cellular responses in neurodegenerative diseases, complicating the evaluation of mitochondrial dysfunction. In this study, we describe a high-throughput imaging and analysis approach to investigate cell-to-cell mitochondrial variability. We applied known mitochondrial function inhibitors - rotenone, antimycin, and oligomycin to inhibit complexes I, III, and V (ATP synthase) function in human induced pluripotent stem cell-derived cortical neurons, a model commonly used in neurodegenerative disease research. We captured a large number of cell images and extracted a diverse range of mitochondrial morphological features related to shape, size, texture, and spatial distribution, for an unbiased and comprehensive analysis of mitochondrial morphology. Group-level cell analysis, which examines the collective responses of cells exposed to the same mitochondrial inhibitor, showed that cells treated with rotenone, antimycin, or oligomycin clustered together based on their shared morphological changes. Rotenone and antimycin, both targeting different complexes of the electron transport chain, formed sub-clusters within a larger cluster. In contrast, oligomycin, which inhibits ATP synthase, resulted in a distinct cluster likely due to its differing effect on ATP production. Single-cell analysis using dimensionality reduction techniques revealed distinct subpopulations of cells with varying degrees of sensitivity to each mitochondrial inhibitor, identifying the most affected cells. Mitochondrial feature differential expression analysis showed that neurite-related mitochondrial features, such as intensity and size, were more severely impacted than cell body-related mitochondrial features, particularly with rotenone and antimycin, which target the electron transport chain. In contrast, oligomycin which affects ATP synthesis by directly inhibiting ATP synthase showed relatively less severe alterations in neurite-related mitochondrial features, highlighting a distinct effect of the mode of action between inhibitors. By incorporating the most affected cells into machine learning models, we significantly improved the prediction accuracy of mitochondrial dysfunction outcomes - 81.97% for antimycin, 75.12% for rotenone, and 94.42% for oligomycin. This enhancement underscores the value of targeting highly responsive cell subpopulations, offering a more precise method for evaluating mitochondrial modulators and therapeutic interventions in neurodegenerative diseases.

DOI: https://doi.org/10.1038/s41598-025-99972-z
Bio Protoc. 2025 May 05. 15(9): e5227

Human iPSC-Derived Neuron and Oligodendrocyte Co-culture as a Small-Molecule Screening Assay for Myelination.

Stefanie Elke Chie, Zsofia Szentpetery, Melanie Generali, Tanja Kuhlmann, Giancarlo Natalucci, Maria Consolata Miletta.

  Neurons and oligodendrocytes are the building blocks of the brain. Neurons form synaptic connections and transmit signals, while oligodendrocytes, including oligodendrocyte precursor cells (OPCs) and their derivatives, are vital for central nervous system maintenance and myelination. The demand for human-specific neuron-oligodendrocyte model systems to study these interactions has grown, yet co-culture protocols remain limited. Recent advancements in the field provide methods for deriving co-cultures of neurons and OPCs from human induced pluripotent stem cells (hiPSC), each with distinct benefits and challenges. This study presents a time-efficient, reproducible method to derive neurons and O4-expressing oligodendrocytes, followed by a straightforward co-culture system that minimizes astrocyte differentiation and ensures robust neuron and oligodendrocyte populations. Key features • Reliable, stable generation of neurons and O4-expressing oligodendrocytes within a practical timeframe. • Co-culture system utilizing hIPSC-derived neurons and O4-expressing oligodendrocytes. • Maturation of neurons and oligodendrocytes achieved within 10 days of co-culturing. Graphical overview Graphical overview. The diagram outlines the sequential steps involved in the preparation, differentiation, and analysis phases. Key stages include the differentiation of neural progenitor cells (NPCs) into O4-expressing oligodendrocytes and neurons separately and then combining them into a co-culture, which can then be used for further experiments.

Keywords:  Co-culture; Human NPCs; Myelin; Neurons; O4-expressing oligodendrocyte; Screening assay; hiPSC

DOI:  https://doi.org/10.21769/BioProtoc.5227
Mol Pharmacol. 2025 Apr 16. pii: S0026-895X(25)15299-1. [Epub ahead of print]107(6): 100039

Fat traffic control: S-acylation in axonal transport.

Amelia H Doerksen, Nisandi N Herath, Shaun S Sanders.

  Neuronal axons serve as a conduit for the coordinated transport of essential molecular cargo between structurally and functionally distinct subcellular compartments via axonal molecular machinery. Long-distance, efficient axonal transport of membrane-bound organelles enables signal transduction and neuronal homeostasis. Efficient axonal transport is conducted by dynein and kinesin ATPase motors that use a local ATP supply from metabolic enzymes tethered to transport vesicles. Molecular motor adaptor proteins promote the processive motility and cargo selectivity of fast axonal transport. Axonal transport impairments are directly causative or associated with many neurodegenerative diseases and neuropathologies. Cargo specificity, cargo-adaptor proteins, and posttranslational modifications of cargo, adaptor proteins, microtubules, or the motor protein subunits all contribute to the precise regulation of vesicular transit. One posttranslational lipid modification that is particularly important in neurons in regulating protein trafficking, protein-protein interactions, and protein association with lipid membranes is S-acylation. Interestingly, many fast axonal transport cargos, cytoskeletal-associated proteins, motor protein subunits, and adaptors are S-acylated to modulate axonal transport. Here, we review the established regulatory role of S-acylation in fast axonal transport and provide evidence for a broader role of S-acylation in regulating the motor-cargo complex machinery, adaptor proteins, and metabolic enzymes from low-throughput studies and S-acyl-proteomic data sets. We propose that S-acylation regulates fast axonal transport and vesicular motility through localization of the proteins required for the motile cargo-complex machinery and relate how perturbed S-acylation contributes to transport impairments in neurological disorders. SIGNIFICANCE STATEMENT: This review investigates the regulatory role of S-acylation in fast axonal transport and its connection to neurological diseases, with a focus on the emerging connections between S-acylation and the molecular motors, adaptor proteins, and metabolic enzymes that make up the trafficking machinery.

Keywords:  S-acylation; fast axonal transport; glycolysis; molecular motor proteins; neurological disorders; palmitoylation

DOI:  https://doi.org/10.1016/j.molpha.2025.100039
Eur J Med Chem. 2025 May 07. pii: S0223-5234(25)00501-X. [Epub ahead of print]293 117736

Design, synthesis and characterization of aryl bis-guanyl hydrazones as RNA binders of C9orf72 G4C2 extended repeats.

Alice Maiocchi, Martina Pedrini, Veronica Ferrari, Agata Sofia Assunçao Carreira, Vincenzo Maria D'Amore, Federica Santoro, Anna Di Porzio, Maddalena Bosetti, Riccardo Cristofani, Alessandra Silvani, Diego Brancaccio, Luciana Marinelli, Francesco Saverio Di Leva, Alessandro Provenzani, Angelo Poletti, Pierfausto Seneci.

  Expanded G4C2 repeats derived from mutations of the C9orf72 gene are causative factors in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients, leading to multiple pathological events. Bis thiophene para dinicotinimidamide 2a was reported to preferentially stabilize G-quadruplex G4C2 RNA structures at sub-micromolar concentrations. We replaced its amidine groups with BBB-compliant guanyl hydrazones, and carried out scaffold variations to improve water solubility. An eight-membered array was built around bis-thiophene- (4b-6a), bis-oxazole- (7b), diphenylurea diamide- (8b) and phenyldioxy ditriazolephenyl scaffolds (9a,b). Biological profiling of the array identified 4b as a promising, drug-like hit, active in cellular assays on ALS patient-derived cells.

Keywords:  ALS; C9orf72 gene; Expanded G(4)C(2) repeats; Guanyl hydrazones; NMR binding studies; RNA transcripts; RNA-Small molecule modeling

DOI:  https://doi.org/10.1016/j.ejmech.2025.117736
Commun Biol. 2025 May 13. 8(1): 745

AI-guided laser purification of human iPSC-derived cardiomyocytes for next-generation cardiac cell manufacturing.

Prakaimuk Saraithong, Peyton Krajcarski, Yukako Kusaka, Moe Yamada, Junichi Matsumoto, Hailey Cunningham, Sama Salih, Darby Jones, Devika Baddhan, Christian Hausner, Justus Anumonwo, Anthony Rosenzweig, Mary M Navarro, Luis Villa Diaz, Joseph Criscione, Deok-Ho Kim, Todd J Herron.

Current methods for producing cardiomyocytes from human induced pluripotent stem cells (hiPSCs) using 2D monolayer differentiation are often hampered by batch-to-batch variability and inefficient purification processes. Here, we introduce CM-AI, a novel artificial intelligence-guided laser cell processing platform designed for rapid, label-free purification of hiPSC-derived cardiomyocytes (hiPSC-CMs). This approach significantly reduces processing time without the need for chronic metabolic selection or antibody-based sorting. By integrating real-time cellular morphology analysis and targeted laser ablation, CM-AI selectively removes non-cardiomyocyte populations with high precision. This streamlined process preserves cardiomyocyte viability and function, offering a scalable and efficient solution for cardiac regenerative medicine, disease modeling, and drug discovery.

DOI: https://doi.org/10.1038/s42003-025-08162-0
J Cell Sci. 2025 May 01. pii: jcs263639. [Epub ahead of print]138(9):

Snapshots of mitochondrial fission imaged by cryo-scanning transmission electron tomography.

Peter Kirchweger, Sharon Grayer Wolf, Neta Varsano, Tali Dadosh, Guenter P Resch, Michael Elbaum.

  Mitochondria undergo constant remodeling via fission, fusion, extension and degradation. Fission, in particular, depends on the accumulation of mitochondrial fission factor (MFF) and subsequent recruitment of the dynamin-related protein DRP1 (also known as DNM1L). We used cryo-scanning transmission electron tomography (cryo-STET) to investigate mitochondrial morphologies in MFF mutant (MFF-/-) mouse embryonic fibroblast (MEF) cells in ATP-depleting conditions that normally induce fission. The capability of cryo-STET to image through the cytoplasmic volume to a depth of 1 µm facilitated visualization of intact mitochondria and their surroundings. We imaged changes in mitochondrial morphology and cristae structure, as well as contacts with the endoplasmic reticulum (ER), degradative organelles and the cytoskeleton at stalled fission sites. We found disruption of the outer mitochondrial membrane at contact sites with the ER and degradative organelles at sites of mitophagy. We identified fission sites where the inner mitochondrial membrane is already separated while the outer membrane is still continuous. Although MFF is a general fission factor, these observations demonstrate that mitochondrial fission can proceed to the final stage in its absence. The use of cryo-STET allays concerns about the loss of structures due to sample thinning required for tomography using cryo-transmission electron microscopy.

Keywords:  Cryo-ET; Cryo-FM; Cryo-STET; Mitochondrial dynamics; Mitochondrial fission; Mitochondrial fission factor

DOI:  https://doi.org/10.1242/jcs.263639
Nat Rev Neurol. 2025 May 15.

Glymphatic flow reduced in Huntington disease.

Ian Fyfe.

DOI: https://doi.org/10.1038/s41582-025-01103-9
Neurochem Int. 2025 May 08. pii: S0197-0186(25)00065-8. [Epub ahead of print] 105992

Proteostasis and autophagy disruption by the aging-related VGVAPG hexapeptide - preliminary insights into a potential novel elastin-induced neurodegeneration pathway in an in vitro human cellular neuron model.

Bartosz Skóra, Konrad A Szychowski.

  The hexapeptide Val-Gly-Val-Ala-Pro-Gly (VGVAPG) is the most readily released product of elastin degradation, a process closely associated with aging. Recent studies have demonstrated the ability of this peptide to upregulate Sirtuin 2 (SIRT2) mRNA and protein expression. The correlation between HRD1 ligase (Synoviolin 1) and the degradation of SIRT2 has been previously reported in the literature. This study aimed to explore the impact of VGVAPG-induced interaction between HRD1 and SIRT2 and its effects on autophagy in differentiated SH-SY5Y cells in vitro (a simplified model of neurons). The results revealed that VGVAPG decreases HRD1 mRNA and protein expression while correlating with SIRT2 overexpression. Further analysis showed reduced SEL1L protein levels and an increase in p97/VCP protein expression. Additionally, enhanced phosphorylation of IRE1α indicated induction of ER stress in the tested cell model without affecting mTOR. Decreased proteasome activity and accumulation of ubiquitin were also noted. This phenomenon triggered VGVAPG-induced autophagy, as evidenced by increased expression of autophagy-related proteins ATG16L1, ATG5, ATG18, and FIP200. However, autophagy was suppressed probably as a result of VGVAPG-induced phosphorylation of ERK1/2. These findings demonstrate that the aging-related hexapeptide VGVAPG downregulates the function of the SEL1L-HRD1 complex, leading to SIRT2 accumulation and subsequent ER stress due to ERAD and UPS. This cascade, in turn, activates autophagy as an alternative clearance pathway aimed at restoring proteostasis; however, the process becomes dysregulated, leading to persistent ER stress. This dual effect may have significant implications in neurobiology, given the well-established correlation between autophagy impairment and aging-related neurodegenerative disorders.

Keywords:  HRD1; Sirtuin 2; VGVAPG; autophagy; elastin-derived peptide; neurodegeneration

DOI:  https://doi.org/10.1016/j.neuint.2025.105992
Nat Rev Mol Cell Biol. 2025 May 14.

Molecular machineries shaping the mitochondrial inner membrane.

Oliver Daumke, Martin van der Laan.

Mitochondria display intricately shaped deep invaginations of the mitochondrial inner membrane (MIM) termed cristae. This peculiar membrane architecture is essential for diverse mitochondrial functions, such as oxidative phosphorylation or the biosynthesis of cellular building blocks. Conserved protein nano-machineries such as F1Fo-ATP synthase oligomers and the mitochondrial contact site and cristae organizing system (MICOS) act as adaptable protein-lipid scaffolds controlling MIM biogenesis and its dynamic remodelling. Signal-dependent rearrangements of cristae architecture and MIM fusion events are governed by the dynamin-like GTPase optic atrophy 1 (OPA1). Recent groundbreaking structural insights into these nano-machineries have considerably advanced our understanding of the functional architecture of mitochondria. In this Review, we discuss how the MIM-shaping machineries cooperate to control cristae and crista junction dynamics, including MIM fusion, in response to cellular signalling pathways. We also explore how mutations affecting MIM-shaping machineries compromise mitochondrial functions.

DOI: https://doi.org/10.1038/s41580-025-00854-z
Front Cell Neurosci. 2025 ;19 1467466

The wave nature of the action potential.

Vitaly L Galinsky, Lawrence R Frank.

  An alternative to the standard Hodgkin-Huxley model for the action potential in axons is presented. It is based on our recently developed theory of electric field wave propagation in anisotropic and inhomogeneous brain tissues, which has been shown to explain a broad range of observed coherent synchronous brain electrical processes. We demonstrate that this theory also explains the spiking behavior of single neurons, thereby bridging the gap between the fundamental element of brain electrical activity-the neuron-and large-scale coherent synchronous electrical activity. We demonstrate that our recently developed theory of electric field wave propagation in anisotropic and inhomogeneous brain tissues, which has been shown to explain a broad range of observed coherent synchronous brain electrical processes, also applies to the spiking behavior of single neurons, thus bridging the gap between the fundamental element of brain electrical activity (the neuron) and large-scale coherent synchronous electrical activity. Our analysis indicates that a non-linear system with several small parameters can mathematically describe the membrane interface of the axonal cellular system. This enables the rigorous derivation of an accurate yet simpler non-linear model through the formal small-parameter expansion. The resulting action potential model exhibits a smooth, continuous transition from the linear wave oscillatory regime to the non-linear spiking regime, as well as a critical transition to a non-oscillatory regime. These transitions occur with changes in the criticality parameter and include several different bifurcation types, representative of the various experimentally detected neuron types. This new theory addresses the limitations of the Hodgkin-Huxley model, including its inability to explain extracellular spiking, efficient brain synchronization, saltatory conduction along myelinated axons, and various other observed coherent macroscopic brain electrical phenomena. We also demonstrate that our approach recovers the standard cable axon theory, utilizing the relatively simple assumptions of piece-wise homogeneity and isotropy. However, the diffusion process described by the cable equation is not capable of supporting action potential propagation across a wide range of experimentally reported axon parameters.

Keywords:  action potential; brain physics; critical dynamics; neuron; wave dynamics

DOI:  https://doi.org/10.3389/fncel.2025.1467466
Sci Transl Med. 2025 May 14. 17(798): eadp4625

MEK1/2 inhibitors suppress pathological α-synuclein and neurotoxicity in cell models and a humanized mouse model of Parkinson's disease.

Huilan Wang, Qing Wang, Haoxiang Xu, Yuanzheng Wu, Siulam Cheung, Qianhui Xu, Chengfang Pan, Jingyu Cao, Zhiyuan Cao, Ruonan Yang, Yu Ding, Yiyan Fei, Yongfeng Chen, Jian Wang, Cong Liu, Boxun Lu.

The abnormal accumulation of misfolded proteins is a common hallmark of many neurodegenerative disorders. Among these proteins, α-synuclein (αsyn) is a well-characterized pathogenic protein in Parkinson's disease (PD) and other synucleinopathies. αsyn can be hyperphosphorylated and form pathological aggregates, leading to neurodegeneration. Thus, chemical modulators of pathological αsyn may suppress its downstream toxicity and provide entry points to therapeutic intervention. Here, we identified mitogen-activated protein kinase kinase 1/2 (MEK1/2) inhibitors as negative modulators of basal αsyn in wild-type cells and that pathological αsyn in αsyn preformed fibrils (αsyn-PFF) induced the neuroblastoma cell line SHSY-5Y, PC12 cells, and primary cultured neurons. We further demonstrated that these inhibitors suppressed Ser129 phosphorylated αsyn (p-αsyn) through the kinase PLK2 downstream of MEK1/2-ERK2 in PD cell models. We established a humanized PD mouse model by injecting human αsyn-PFF into mice with homozygous knock-in of human SNCA. Oral administration of blood-brain barrier-penetrable MEK1/2 inhibitors lowered pathological αsyn and rescued PD-relevant phenotypes with an acceptable safety profile in these mice. Collectively, these data highlight MEK1/2 inhibitors as a potential therapeutic strategy for PD.

DOI: https://doi.org/10.1126/scitranslmed.adp4625