bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2025–06–29
forty-one papers selected by
TJ Krzystek
  1. Role of LRRK2 in axonal transport and Parkinson's disease.
  2. Nemo-like kinase disrupts nuclear import and drives TDP43 mislocalization in ALS.
  3. Ankyrin-G and Its Binding Partners in Neurons: Orchestrating the Molecular Structure of the Axon Initial Segment.
  4. [Autophagy Impairment in Parkinson's Disease: Approaches to Therapy].
  5. An organ-chip model of sporadic ALS using iPSC-derived spinal cord motor neurons and an integrated blood-brain-like barrier.
  6. Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line.
  7. The Role of Non-Coding RNAs in ALS.
  8. Phospholipid biogenesis maintains neuronal integrity during aging and axon regeneration.
  9. Perspectives in Amyotrophic Lateral Sclerosis: Biomarkers, Omics, and Gene Therapy Informing Disease and Treatment.
  10. Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis.
  11. Loss-of-function coding variants in the Ras of complex proteins/GTPase domain of leucine rich repeat kinase 2.
  12. From Gene to Pathways: Understanding Novel Vps51 Variant and Its Cellular Consequences.
  13. A novel frameshift mutation in Phosphoinositide 3-kinase regulatory subunit 1 ( PIK3R1 ) causes immunodeficiency and Amyotrophic Lateral Sclerosis (ALS).
  14. Restoration of myogenesis in ALS-myocytes through miR-26a-5p-mediated Smad4 inhibition and its impact on motor neuron development.
  15. Bulbar-Onset Amyotrophic Lateral Sclerosis Unmasked by General Anesthesia: A Case Report.
  16. Increased Activity-Dependent Bulk Endocytosis in Huntington's Disease Results From Huntingtin Haploinsufficiency.
  17. Polyalanine Expansion in PABPN1 Alters the Structure and Dynamics of Its Nuclear Aggregates in Differentiated Muscle Cells.
  18. AAV-based delivery of RNAi targeting ataxin-2 improves survival and pathology in TDP-43 mice.
  19. ALS mutations shift the isoelectric point of the KIF5A C-terminal inducing protein aggregation and TDP-43 mislocalization.
  20. Skeletal muscle, neuromuscular organoids and assembloids: a scoping review.
  21. Metal-Induced Genotoxic Events: Possible Distinction Between Sporadic and Familial ALS.
  22. Metabolic analysis of sarcopenic muscle identifies positive modulators of longevity and healthspan in C. elegans.
  23. Cholinergic basal forebrain neurons regulate vascular dynamics and cerebrospinal fluid flux.
  24. Human Pluripotent Stem Cell-Based Therapies for Parkinson's Disease: Challenges and Potential Solutions.
  25. Neuronal Deletion of Tumor Susceptibility Gene 101 (Tsg101) Causes Rapid Apoptotic Loss of Hippocampal CA3 Neurons.
  26. Secondary accumulation of lyso-platelet activating factors in lysosomal storage diseases.
  27. Key challenges and recommendations for defining organelle membrane contact sites.
  28. Cortical Actin Depolymerisation in 3D Cell Culture Enhances Extracellular Vesicle Secretion and Therapeutic Effects.
  29. The mechanisms underlying TDP-43-associated neurodegeneration in Alzheimer's disease and related dementias.
  30. Microaggrephagy: an ESCRT-I-PTPN23-dependent pathway for MAPT/tau aggregate clearance.
  31. Abnormal α-synuclein binds to synaptotagmin 13, impairing extracellular vesicle release in synucleinopathies.
  32. Myostatin Modulation in Spinal Muscular Atrophy: A Systematic Review of Preclinical and Clinical Evidence.
  33. Scalable production of human cortical organoids using a biocompatible polymer.
  34. Extension of replicative lifespan by synthetic engineered telomerase RNA in patient induced pluripotent stem cells.
  35. Mislocalization of nucleic acids is a convergent and targetable mechanism in Alzheimer's disease and frontotemporal dementia.
  36. The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy.
  37. Mitochondrial protein nmd regulates lipophagy and general autophagy during development.
  38. A lysosomal surveillance response to stress extends healthspan.
  39. Endogenous SNAP-Tagging of Munc13‑1 for Monitoring Synapse Nanoarchitecture.
  40. TNIP1 and Autophagy Receptors regulate STING Signaling.
  41. Nuclear Roles for Canonically Lysosomal Proteases.
  1. Biochem J. 2025 Jun 25. pii: BCJ20253133. [Epub ahead of print]482(13):
    Role of LRRK2 in axonal transport and Parkinson's disease.
    Björn Twellsieck, C Alexander Boecker.
      Axonal transport is crucial for neuronal health and function, facilitating the delivery of newly synthesized material from the soma via anterograde transport and the removal of aged proteins and damaged organelles for degradation via retrograde transport. Emerging evidence links Parkinson's disease (PD)-causing mutations in the leucine-rich repeat kinase 2 (LRRK2) gene to dysfunctional axonal transport. Pathogenic LRRK2 mutations induce increased LRRK2 kinase activity, leading to the hyperphosphorylation of RAB proteins, which are key regulators of intracellular trafficking and transport. Here, we review the current literature on how LRRK2 affects the axonal transport of different cargoes, focusing on synaptic vesicle precursors, mitochondria, and autophagosomes. We further discuss how LRRK2 influences cytoskeletal dynamics and how it affects vesicle trafficking at the Golgi, which may indirectly contribute to its effect on axonal transport. This review summarizes our current understanding of how pathogenic LRRK2 hyperactivation disrupts axonal transport and how this may be linked to the neurodegeneration of PD.
    Keywords:  LRRK2; Parkinson’s disease; RAB GTPases; axonal transport
    DOI:  https://doi.org/10.1042/BCJ20253133
  2. J Clin Invest. 2025 06 24. pii: e188138. [Epub ahead of print]
    Nemo-like kinase disrupts nuclear import and drives TDP43 mislocalization in ALS.
    Michael E Bekier, Emile S Pinarbasi, Gopinath Krishnan, Jack J Mesojedec, Madelaine Hurley, Harisankar Harikumar Sheela, Catherine A Collins, Layla T Ghaffari, Martina de Majo, Erik M Ullian, Mark Koontz, Sarah Coleman, Xingli Li, Elizabeth Mh Tank, Jacob Waksmacki, Fen-Biao Gao, Sami J Barmada.
      Cytoplasmic TDP43 mislocalization and aggregation are pathological hallmarks of amyotrophic lateral sclerosis (ALS). However, the initial cellular insults that lead to TDP43 mislocalization remain unclear. In this study, we demonstrate that Nemo-like kinase (NLK) - a proline-directed serine/threonine kinase - promotes the mislocalization of TDP43 and other RNA-binding proteins by disrupting nuclear import. NLK levels are selectively elevated in neurons exhibiting TDP43 mislocalization in ALS patient tissues, while genetic reduction of NLK reduces toxicity in human neuron models of ALS. Our findings suggest that NLK is a promising therapeutic target for neurodegenerative diseases.
    Keywords:  ALS; Cell biology; Molecular pathology; Neuroscience; Transport
    DOI:  https://doi.org/10.1172/JCI188138
  3. Biomolecules. 2025 Jun 19. pii: 901. [Epub ahead of print]15(6):
    Ankyrin-G and Its Binding Partners in Neurons: Orchestrating the Molecular Structure of the Axon Initial Segment.
    Xiaowei Zhu, Yanyan Yu, Zhuqian Jiang, Yoshinori Otani, Masashi Fujitani.
      The axon initial segment (AIS) is a specialized subcellular domain that plays an essential role in action potential initiation and the diffusion barrier. A key organizer of the AIS is Ankyrin-G, a scaffolding protein responsible for clustering voltage-gated ion channels, cell adhesion molecules (CAMs), and cytoskeletal components at this critical neuronal domain. Recent proteomic analyses have revealed a complex network of proteins in the AIS, emphasizing Ankyrin-G's central role in its molecular architecture. This review discusses new findings in the study of AIS-associated proteins. It explains how Ankyrin-G and its binding partners (such as ion channels, CAMs, spectrins, actin, and microtubule-associated proteins including end-binding protein 3, tripartite motif-containing protein 46, and calmodulin-regulated spectrin-associated protein 2) organize their structure. Understanding the dynamic regulation and molecular interactions within the AIS offers insights into neuronal excitability and reveals potential therapeutic targets for axonal dysfunction-related diseases. Through these dynamic interactions, Ankyrin-G ensures the proper alignment and dense clustering of key channel complexes, thereby maintaining the AIS's distinctive molecular and functional identity. By further unraveling the complexity of Ankyrin-G's interactome, our understanding of AIS formation, maintenance, and plasticity will be considerably enhanced, contributing to the elucidation of the pathogenesis of neurological and neuropsychiatric disorders.
    Keywords:  Ankyrin-G; axon initial segment; binding partners; proteome
    DOI:  https://doi.org/10.3390/biom15060901
  4. Mol Biol (Mosk). 2025 Jan-Feb;59(1):59(1): 60-79
    [Autophagy Impairment in Parkinson's Disease: Approaches to Therapy].
    T S Usenko.
      Parkinson's disease (PD) is one of the most common neurodegenerative disorders and is characterized by progressive motor impairment due to the death of dopaminergic neurons in the substantia nigra (SN) of the brain. PD affects more than 1% of the population over 60 years of age worldwide. Despite significant progress in understanding the pathogenesis of PD, including genetic and biochemical aspects, current therapy is limited to symptomatic treatments. Recent evidence suggests that impaired autophagy leads to the accumulation of abnormal proteins and, particularly, α-synuclein, aggregated forms of which are neurotoxic to dopaminergic neurons in the SN. Notably, PD is predominantly sporadic. However, monogenic PD forms have also been described. PD forms associated with mutations of the GBA1 or LRRK2 gene are among the most common PD forms with known etiology. Leucine-rich repeat kinase 2 (LRRK2), which is encoded by LRRK2, and the lysosomal enzyme glucocerebrosidase (GCase), which is encoded by GBA1, are involved in the same endolysosomal pathway. LRRK2 and GCase dysfunction reported in PD, especially in cases with mutations of the respective genes, can impair the endolysosomal pathway, the lysosomal function, and possibly autophagy. The review highlights the molecular mechanisms of autophagy and the prospects for targeted therapy of PD via induction of autophagy by influencing the key players in the process.
    Keywords:  GBA1; LRRK2; Parkinson's disease; autophagy; inducers; mTOR; targeted therapy
    DOI:  https://doi.org/10.31857/S0026898425010053, EDN: HDEMSA
  5. Cell Stem Cell. 2025 Jun 17. pii: S1934-5909(25)00222-X. [Epub ahead of print]
    An organ-chip model of sporadic ALS using iPSC-derived spinal cord motor neurons and an integrated blood-brain-like barrier.
    Deepti Lall, Michael J Workman, Samuel Sances, Briana N Ondatje, Shaughn Bell, George Lawless, Amanda Woodbury, Dylan West, Amanda Meyer, Andrea Matlock, Vineet Vaibhav, Jennifer E Van Eyk, Clive N Svendsen.
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder in which motor neurons (MNs) of the brain and spinal cord degenerate, leading to paralysis. Generating MNs from patient-specific induced pluripotent stem cells (iPSCs) may help elucidate early stages of disease. Here, we combined MNs from patients with early-onset disease with brain microvascular endothelial-like cells in a microfluidic device we termed spinal cord chips (SC-chips) and added media flow, which enhanced neuronal maturation and improved cellular health. Bulk transcriptomic and proteomic analyses of SC-chips revealed differences between control and ALS samples, including increased levels of neurofilaments. Single-nuclei RNA sequencing revealed the presence of two MN subpopulations and an ALS-specific dysregulation of glutamatergic and synaptic signaling. This ALS SC-chip model generates a diversity of mature MNs to better understand ALS pathology in a model that has an active blood-brain barrier-like system for future drug screening.
    Keywords:  amyotrophic lateral sclerosis; glutamatergic synapse; microfluidic organ-chips; motor neuron identity; neuronal maturation; synaptic signaling
    DOI:  https://doi.org/10.1016/j.stem.2025.05.015
  6. bioRxiv. 2025 Mar 26. pii: 2025.03.25.645331. [Epub ahead of print]
    Temporal dynamics of proteome and phosphorproteome during neuronal differentiation in the reference KOLF2.1J iPSC line.
    Ying Hao, Ziyi Li, Erika Lara, Daniel M Ramos, Marianist Santiana, Benjamin Jin, Jacob Epstein, Jasmin Camacho, Nicole Carmiol, Isabelle Kowal, Paige Jarreau, Cory A Weller, Sydney Klaisner, Laurel A Screven, Caroline B Pantazis, Mike A Nalls, Priyanka Narayan, Luigi Ferrucci, Andrew B Singleton, Michael E Ward, Mark R Cookson, Yue Andy Qi.
      Induced pluripotent stem cell (iPSC)-derived neurons have emerged as a powerful model to investigate both neuronal development and neurodegenerative diseases. Although transcriptomics and imaging have been applied to characterize neuronal development signatures, comprehensive datasets of protein and post-translational modifications (PTMs) are not readily available. Here, we applied quantitative proteomics and phosphoproteomics to profile the differentiation of the KOLF2.1J iPSC line, the first reference line of the iPSC Neurodegenerative Disease Initiative (iNDI) project. We developed an automated workflow enabling high-coverage enrichment of proteins and phosphoproteins. Our results revealed molecular signatures across proteomic and phosphoproteomic landscapes during differentiation of iPSC-derived neurons. Proteomic data highlighted distinct changes in mitochondrial pathways throughout the course of differentiation, while phosphoproteomics revealed specific regulatory dynamics in GTPase signaling pathways and microtubule proteins. Additionally, phosphosite dynamics exhibited discordant trends compared to protein expression, particularly in processes related to axon functions and RNA transport. Furthermore, we mapped the kinase dynamic changes that are critical for neuronal development and maturation. We developed an interactive Web app ( https://niacard.shinyapps.io/Phosphoproteome/ ) to visualize temporal landscape dynamics of protein and phosphosite expression. By establishing baselines of proteomic and phosphoproteomic profiles for neuronal differentiation, this dataset offers a valuable resource for future research into neuronal development and neurodegenerative diseases using this reference iPSC line.
    Highlights: Temporal dynamics of proteome and phosphoproteome profiles in KOLF2.1J iPSC derived neurons.Phosphoproteomics highlights GTPase signaling and microtubule regulation in neuronal differentiation.Kinome mapping reveals a shift in kinase activity patterns from early to late differentiation.Shinyapp for visualizing the trajectory of protein and phosphosite expression during neuronal differentiation.
    DOI:  https://doi.org/10.1101/2025.03.25.645331
  7. Genes (Basel). 2025 May 23. pii: 623. [Epub ahead of print]16(6):
    The Role of Non-Coding RNAs in ALS.
    Alessandra Falduti, Adele Giovinazzo, Elisa Lo Feudo, Valentina Rocca, Filippo Brighina, Angela Messina, Francesca Luisa Conforti, Rodolfo Iuliano.
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons, leading to muscle weakness, paralysis, and eventually death. The pathogenesis of ALS is influenced by genetic factors, environmental factors, and age-related dysfunctions. These factors, taken together, are responsible for sporadic cases of ALS, which account for approximately 85-90% of ALS cases, while familial ALS accounts for the remaining 10-15% of cases, usually with dominant traits. Despite advances in understanding and studying the disease, the cause of the onset of ALS remains unknown. Emerging evidence suggests that non-coding RNAs, including microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), play crucial roles in the pathogenesis of the disease. An abnormal expression of these molecules is implicated in various ALS-related processes, including motor neuron survival, protein aggregation, and inflammation. Here, we describe the dysregulation of non-coding RNAs in the pathogenic mechanism of ALS, highlighting the potential roles of miRNAs, lncRNAs, and circRNAs as biomarkers or therapeutic targets to examine the progression of the disease.
    Keywords:  amyotrophic lateral sclerosis (ALS); microRNAs; neurodegenerative disease; non-coding RNAs
    DOI:  https://doi.org/10.3390/genes16060623
  8. Genetics. 2025 Jun 25. pii: iyaf122. [Epub ahead of print]
    Phospholipid biogenesis maintains neuronal integrity during aging and axon regeneration.
    Seungmee Park, Yishi Jin, Andrew D Chisholm.
      Neurons maintain their morphology over prolonged periods of adult life with limited regenerative capacity. Among the various factors that shape neuronal morphology, lipids function as membrane components, signaling molecules, and regulators of synaptic plasticity. Here, we tested genes involved in phospholipid biosynthesis and identified their roles in axon regrowth and maintenance. CEPT-2 and EPT-1 are enzymes catalyzing the final steps in the de novo phospholipid synthesis (Kennedy) pathway. Loss of function mutants of cept-2 or ept-1 show reduced axon regrowth and failure to maintain axon morphology. We demonstrate that CEPT-2 is required cell-autonomously to prevent age-related axonal morphology defects. We further investigated genetic interactions of cept-2 or ept-1 with dip-2, a conserved regulator of lipid metabolism that affects axon morphology maintenance and regrowth after injury. Loss of function in dip-2 led to suppression of axon regrowth defects observed in either cept-2 or ept-2 mutants, suggesting that DIP-2 acts to counterbalance phospholipid synthesis. Our findings reveal the genetic regulation of lipid metabolism as critical for axon maintenance following injury and during aging.
    Keywords:  Kennedy pathway; aging; axon degeneration; axon regeneration; phosphatidylcholine; phosphatidylethanolamine
    DOI:  https://doi.org/10.1093/genetics/iyaf122
  9. Int J Mol Sci. 2025 Jun 13. pii: 5671. [Epub ahead of print]26(12):
    Perspectives in Amyotrophic Lateral Sclerosis: Biomarkers, Omics, and Gene Therapy Informing Disease and Treatment.
    Nina Bono, Flaminia Fruzzetti, Giorgia Farinazzo, Gabriele Candiani, Stefania Marcuzzo.
      Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of upper and lower motor neurons, leading to muscle weakness, paralysis, and ultimately respiratory failure. Despite advances in understanding its genetic basis, particularly mutations in Chromosome 9 Open Reading Frame 72 (C9orf72), superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP), and Fused in Sarcoma (FUS) gene, current diagnostic methods result in delayed intervention, and available treatments offer only modest benefits. This review examines innovative approaches transforming ALS research and clinical management. We explore emerging biomarkers, including the fluid-based markers such as neurofilament light chain, exosomes, and microRNAs in biological fluids, alongside the non-fluid-based biomarkers, including neuroimaging and electrophysiological markers, for early diagnosis and patient stratification. The integration of multi-omics data reveals complex molecular mechanisms underlying ALS heterogeneity, potentially identifying novel therapeutic targets. We highlight current gene therapy strategies, including antisense oligonucleotides (ASOs), RNA interference (RNAi), and CRISPR/Cas9 gene editing systems, alongside advanced delivery methods for crossing the blood-brain barrier. By bridging molecular neuroscience with bioengineering, these technologies promise to revolutionize ALS diagnosis and treatment, advancing toward truly disease-modifying interventions for this previously intractable condition.
    Keywords:  advanced diagnosis; amyotrophic lateral sclerosis; bioengineering; nanotechnology; systems biology approach; therapeutic strategies
    DOI:  https://doi.org/10.3390/ijms26125671
  10. Int J Mol Sci. 2025 Jun 07. pii: 5481. [Epub ahead of print]26(12):
    Flow Cytometric Quantification of Mitochondrial Properties: A High-Throughput Approach for Single Organelle Analysis.
    Andrew J Piasecki, Hannah C Sheehan, Jonathan L Tilly, Dori C Woods.
      Recent advances in flow cytometry facilitate the detection of subcellular components, such as organelles and vesicles. Fluorescence-activated mitochondria sorting (FAMS) is a flow cytometry-based technique that allows for quantitative analysis and sorting of mitochondria as individual organelles from various tissues and in vitro cell culture. This manuscript details three novel applications of this technique to study mitochondrial function on an organelle-specific level, which is not possible with other approaches. Specifically, we detail the further development and versatility of this nanoscaled flow cytometry approach, including assays to quantitatively assess mitochondrial subpopulations, mitochondrial protein translocation, and both free-floating and EV-encapsulated secreted mitochondria. We demonstrate a multi-parameter quantitative assay for the analysis of mitochondrial autophagy using antibodies targeting the proteins PINK1 and Parkin corresponding to ΔΨM and further show how these can be assessed for mtDNA content on a single organelle level. Further, we establish parameters for the size and surface marker-based analysis of EVs, many of which contain identifiable and respiring mitochondria, as well as free-floating respiratory-competent mitochondria. These results display the versatility of nanoscaled flow cytometry in terms of both sample input and target organelle and provide an important methodological means for the quantitative assessment of mitochondrial features.
    Keywords:  extracellular vesicle sorting; flow cytometry; fluorescence-activated mitochondria sorting; mitochondria; organelle sorting
    DOI:  https://doi.org/10.3390/ijms26125481
  11. Protein Sci. 2025 Jul;34(7): e70190
    Loss-of-function coding variants in the Ras of complex proteins/GTPase domain of leucine rich repeat kinase 2.
    Sarah Butterfield, Susanne Herbst, Patrick Alfryn Lewis.
      The LRRK2 gene is a key contributor to the genetic risk of Parkinson's disease, and a priority drug target for the disorder. Leucine Rich Repeat Kinase 2, the protein product of LRRK2, is a multidomain enzyme implicated in a range of cellular processes-including endolysosomal trafficking and damage response. Based on the report that truncation and structural variants resulting in loss of LRRK2 protein are observed in human populations, genomic sequence repositories were queried for coding variants affecting key catalytic residues in LRRK2-resulting in the identification of three variants (K1347E, K1347R, and T1348P) predicted to ablate the capacity of LRRK2 to bind GTP. Biochemical and cellular characterization of these variants confirmed loss of GTP binding, as well as reduced or loss of kinase activity. These data demonstrate the presence of rare coding enzymatic loss-of-function variants in humans, with implications for our understanding of LRRK2 as a driver of disease and as a drug target.
    Keywords:  GTPase; LRRK2; Parkinson's; leucine rich repeat kinase 2; lysosomes
    DOI:  https://doi.org/10.1002/pro.70190
  12. Int J Mol Sci. 2025 Jun 14. pii: 5709. [Epub ahead of print]26(12):
    From Gene to Pathways: Understanding Novel Vps51 Variant and Its Cellular Consequences.
    Damla Aygun, Didem Yücel Yılmaz.
      Disorders of vesicular trafficking and genetic defects in autophagy play a critical role in the development of metabolic and neurometabolic diseases. These processes govern intracellular transport and lysosomal degradation, thereby maintaining cellular homeostasis. In this article, we present two siblings with a novel homozygous variant in VPS51 (Vacuolar protein sorting 51) gene (c.1511C>T; p.Thr504Met), exhibiting developmental delay, a thin corpus callosum, severe intellectual disability, epilepsy, microcephaly, hearing loss, and dysphagia. This study aimed to investigate the effects of the novel VPS51 gene variation at the RNA and protein level in fibroblasts derived from patients. A comparative proteomic analysis, which has not been previously elucidated, was performed to identify uncharacterized proteins associated with vesicular trafficking. Furthermore, the impact of disrupted pathways on mitochondria-lysosome contact sites was assessed, offering a thorough pathophysiological evaluation of GARP/EARP (Golgi Associated Retrograde Protein / Endosome Associated Retrograde Protein) complex dysfunction. An analysis of mRNA expression indicated decreased levels of the VPS51 gene, alongside modifications in the expression of autophagy-related genes (LC3B, p62, RAB7A, TBC1D15). Western blotting demonstrated a reduction in VPS51 and autophagy-related protein levels. Proteomic profiling revealed 585 differentially expressed proteins, indicating disruptions in vesicular trafficking, lysosomal function, and mitochondrial metabolism. Proteins involved in mitochondrial β-oxidation and oxidative phosphorylation exhibited downregulation, whereas pathways related to glycolysis and lipid synthesis showed upregulation. Live-cell confocal microscopy revealed a notable increase in mitochondria-lysosome contact sites in patient fibroblasts, suggesting that VPS51 protein dysfunction contributes to impaired organelle communication. The findings indicate that the novel VPS51 gene variation influences intracellular transport, autophagy, and metabolic pathways, offering new insights into its involvement in neurometabolic disorders.
    Keywords:  EARP; GARP; autophagy; mitochondria–lysosome contact; proteomics; vesicular traffic
    DOI:  https://doi.org/10.3390/ijms26125709
  13. bioRxiv. 2025 May 28. pii: 2025.05.23.655625. [Epub ahead of print]
    A novel frameshift mutation in Phosphoinositide 3-kinase regulatory subunit 1 ( PIK3R1 ) causes immunodeficiency and Amyotrophic Lateral Sclerosis (ALS).
    Brice Calco, Colin L Sweeney, Joseph Steiner, Tongguang Wang, Tovah E Markowitz, Subrata Paul, Bianca Palicha, Sara Dinges, Kelsey Yoo, Lisa Henderson, Valerie McDonald, Suk See De Ravin, Benjamin Greenberg, Christa S Zerbe, Luigi D Notarangelo, Steven M Holland, Avindra Nath, Farinaz Safavi.
      Mutations in PIK3R1 , a regulatory subunit of Class I PI3K, are implicated in immune disorders and neurological conditions. We identified a novel heterozygous pathogenic frameshift mutation (c.1710dup) in PIK3R1 in a patient with common variable immunodeficiency who developed slowly progressive Amyotrophic Lateral Sclerosis. Induced pluripotent stem cells (iPSCs) and iPSC-derived motor neurons (iMNs) demonstrated that this mutation resulted in PIK3R1 haploinsufficiency, with downstream activation of AKT, disruption of neuronal electrical function and increased apoptosis in iPSC-derived motor neurons. Single-cell RNA sequencing (scRNA-seq) and pathway analysis of differentially expressed genes showed apoptosis pathways were upregulated in neuronal clusters from iMNs harboring the PIK3R1 c.1710 dup mutation. Mutated iPSC-derived brain organoids were smaller than matched controls. scRNA-seq of brain organoids showed more active apoptosis in neuronal clusters of patient-derived brain organoids. These findings identify a critical and novel role for PIK3R1 haploinsufficiency in neuronal function and survival.
    DOI:  https://doi.org/10.1101/2025.05.23.655625
  14. Mol Ther Nucleic Acids. 2025 Sep 09. 36(3): 102581
    Restoration of myogenesis in ALS-myocytes through miR-26a-5p-mediated Smad4 inhibition and its impact on motor neuron development.
    Caterina Peggion, Raphael Severino Bonadio, Roberto Stella, Silvia Scalabrin, Laura Pasetto, Caterina Millino, Laura Camporeale, Beniamina Pacchioni, Valentina Bonetto, Alessandro Bertoli, Stefano Cagnin, Maria Lina Massimino.
      Amyotrophic lateral sclerosis (ALS) is the most common adult-onset paralytic disorder, characterized primarily by a progressive loss of motor neurons (MNs) in which degeneration skeletal muscle involvement has been demonstrated. Skeletal muscle is a plastic tissue that responds to insults through proliferation and differentiation of satellite cells. Skeletal muscle degeneration and regeneration are finely regulated by signals that regulate satellite cell proliferation and differentiation. It is known that satellite cell differentiation is impaired in ALS, but little is known about the involvement of microRNAs (miRNAs) and their role in intercellular communication in ALS. Here we demonstrated impaired differentiation of satellite cells derived from ALS mice related to the impairment of myogenic p38MAPK and protein kinase A (PKA)/pCREB signaling pathways that can be regulated by miR-882 and -134-5p. These miRNAs participate in autocrine signaling in association with miR-26a-5p that, secreted from wild-type (WT) and captured by ALS myoblasts, enhances ALS-related myoblast differentiation by repressing Smad4-related signals. Moreover, miR-26a-5p and -431-5p work in a paracrine way ameliorating motoneuron differentiation. These findings emphasize the need to better understand intercellular communication and its role in ALS pathogenesis and progression. They also suggest that miRNAs could be targeted or used as therapeutic agents for myofiber and MN regeneration.
    Keywords:  MT: non-coding RNAs; amyotrophic lateral sclerosis; cell communication; miRNA; myogenesis; neurogenesis; neuromuscular disorders; primary stem cells; skeletal muscle
    DOI:  https://doi.org/10.1016/j.omtn.2025.102581
  15. Cureus. 2025 May;17(5): e84823
    Bulbar-Onset Amyotrophic Lateral Sclerosis Unmasked by General Anesthesia: A Case Report.
    Amine Bouabdallaoui, Salma Taouihar, Naji Yahya, Nawal Adali, Hicham Nassik.
      Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects the motor neurons in the brain and spinal cord, resulting in severe muscular weakness, atrophy, and loss of motor control. Patients with ALS are often highly sensitive to anesthetic drugs, specifically neuromuscular blocking agents, which can exacerbate muscle weakness and contribute to prolonged postoperative recovery times. In this article, we report a case of bulbar ALS diagnosed after postoperative extubation failure in a 51-year-old patient. Practitioners should consider this disease in cases of difficult postoperative ventilatory weaning and should be aware of the impact of surgery and anaesthesia on disease progression.
    Keywords:  amyotrophic lateral sclerosis; anesthesia; case reports; general surgery; neurology
    DOI:  https://doi.org/10.7759/cureus.84823
  16. J Neurochem. 2025 Jun;169(6): e70134
    Increased Activity-Dependent Bulk Endocytosis in Huntington's Disease Results From Huntingtin Haploinsufficiency.
    Han C G Tan, Robyn L McAdam, Andrew Morton, Michael A Cousin, Karen J Smillie.
      Huntington's disease (HD) is a life-limiting, progressive monogenic neurodegenerative disorder characterised by chorea, hypokinesis and psychosocial symptoms. HD is characterised by a variable CAG expansion in exon 1 of the HTT gene, which encodes the huntingtin (htt) protein. This expansion results in an extended polyglutamine tract, which is widely thought to confer a toxic gain of function on the protein that is responsible for disease progression. Most individuals with HD are heterozygous for this mutation, meaning that loss of wild-type htt function may also contribute to disease pathology. We previously identified that the recycling of synaptic vesicle proteins at the presynapse was specifically disrupted in striatal neurons from a preclinical model of HD, the HttQ140/Q140 knockin mouse. This defect was only revealed during high activity and, notably, was due to loss of wild-type htt function. The dominant endocytosis mode at the presynapse during high activity is activity-dependent bulk endocytosis (ADBE). Therefore, we determined whether dysfunction in this pathway was linked to this recycling defect. We revealed that three independent neuronal subtypes derived from HttQ140/Q140 mice displayed enhanced recruitment, but no change in the extent of ADBE via the evoked uptake of fluid phase markers. Importantly, this phenotype was due to a loss of wild-type htt function, since depletion of htt in Htt+/+ neurons mimicked the defect, and removal of mutant htt from HttQ140/Q140 neurons did not correct this dysfunction. Neurons from HttQ140/+ mice, which mimic the human condition, also displayed increased activity-dependent triggering of ADBE, suggesting that htt haploinsufficiency may be responsible. This was confirmed by the inability of zinc finger proteins that selectively target mutant htt to correct this defect in HttQ140/+ neurons. Therefore, htt haploinsufficiency drives dysfunction in a key endocytosis mode that is dominant during high neuronal activity, providing a potential mechanism for circuit dysfunction that results in neurodegeneration in later life in HD.
    Keywords:  Huntington's disease; endocytosis; haploinsufficiency; mouse; presynapse; synaptic vesicle
    DOI:  https://doi.org/10.1111/jnc.70134
  17. FASEB J. 2025 Jun 30. 39(12): e70748
    Polyalanine Expansion in PABPN1 Alters the Structure and Dynamics of Its Nuclear Aggregates in Differentiated Muscle Cells.
    Sander D Mallon, Erik Bos, Vahid Sheikhhassani, Milad Shademan, Lenard M Voortman, Alireza Mashaghi, Thomas H Sharp, Vered Raz.
      Intracellular protein aggregation is a hallmark of aging and contributes to pathology in some age-associated diseases. In hereditary adult-onset neuromuscular diseases (NMDs), protein aggregates play a key role in disease onset and progression. The wild-type Poly(A) binding protein nuclear 1 (PABPN1) forms benign nuclear aggregates, whereas a short trinucleotide expansion leads to the formation of pathogenic aggregates, a hallmark of Oculopharyngeal Muscular Dystrophy (OPMD). In OPMD, the mutant PABPN1 causes skeletal muscle weakness. So far, the structural differences between benign and pathogenic protein aggregates and their effects on muscle cell biology remain poorly understood. We employed an array of advanced imaging modalities to explore the morphological differences between nuclear aggregates formed by non-pathogenic and pathogenic PABPN1 variants. Through analyses spanning micro- to nanoscale, we identified distinct structural features of aggregates formed by wild-type and expanded PABPN1. We demonstrate that these differences were more pronounced in differentiated muscle cells compared to proliferating cells. We further linked the structural features of PABPN1 aggregates to muscle cell biology, namely alterations in mitochondrial function and proteasomal activity. Our findings provide new insights into the structural distinctions between pathogenic and non-pathogenic aggregates and their implications for cellular dysfunction in NMDs.
    Keywords:  OPMD; aggregates structure; imaging; muscle; protein aggregates
    DOI:  https://doi.org/10.1096/fj.202501097R
  18. Nat Commun. 2025 Jun 25. 16(1): 5334
    AAV-based delivery of RNAi targeting ataxin-2 improves survival and pathology in TDP-43 mice.
    Defne A Amado, Ashley B Robbins, Katherine R Whiteman, Alicia R Smith, Guillem Chillon, Yonghong Chen, Joshua A Fuller, Nicholas A Patty, Aleksandar Izda, Congsheng Cheng, Shareen Nelson, Abigail I Dichter, Esteban O Mazzoni, Alex Mas Monteys, Beverly L Davidson.
      Amyotrophic lateral sclerosis (ALS) involves motor neuron death due to mislocalized TDP-43. Pathologic TDP-43 associates with stress granules (SGs), and lowering the SG-associated protein ataxin-2 (ATXN2) using Atxn2-targeting antisense oligonucleotides prolongs survival in TAR4/4 sporadic ALS mice but failed in clinical trials likely due to poor target engagement. Here we show that an AAV with potent motor neuron transduction delivering Atxn2-targeting miRNAs reduces Atxn2 throughout the central nervous system at doses 40x lower than published work. In TAR4/4 mice, miAtxn2 increased survival (50%) and strength, and reduced motor neuron death, inflammation, and phosphorylated TDP-43. TAR4/4 transcriptomic dysregulation recapitulated ALS gene signatures that were rescued by miAtxn2, identifying potential therapeutic mechanisms and biomarkers. In slow progressing hemizygous mice, miAtxn2 slowed disease progression, and in ALS patient-derived lower motor neurons, our AAV vector transduced >95% of cells and potently reduced ATXN2 at MOI 4 logs lower than previously reported. These data support AAV-RNAi targeting ATXN2 as a translatable therapy for sporadic ALS.
    DOI:  https://doi.org/10.1038/s41467-025-60497-8
  19. J Neurosci. 2025 Jun 24. pii: e1658242025. [Epub ahead of print]
    ALS mutations shift the isoelectric point of the KIF5A C-terminal inducing protein aggregation and TDP-43 mislocalization.
    Pietro Zanella, Isabel Loss, Rosanna Parlato, Jochen H Weishaupt, Carlo Sala, Chiara Verpelli, Tobias M Boeckers, Alberto Catanese.
      Amyotrophic Lateral Sclerosis (ALS) is a devastating neurodegenerative disease characterized by death of lower and upper motor neurons. Although the mechanism behind the selective neuron loss is still unclear, several heterogenous genes have been causally linked to ALS. KIF5A encodes for a neuronally enriched kinesin involved in protein transport and mutations within this gene have been causally linked to different motor neuron diseases. The mutations identified in ALS patients are mostly predicted to alter its mRNA splicing, leading to a frameshift mutation and an aberrant 39 amino acid-long sequence in the C-terminal domain of KIF5A.Here we found that ALS-related KIF5A mutations induce the accumulation of the mutant form of the protein in human motoneurons, which are also characterized by the cytosolic mislocalization of TDP-43. This ALS hallmark was even exacerbated upon overexpression of the ALS-KIF5A protein in cells differentiated from healthy controls and primary neurons, suggesting a pathological connection between the cellular load of the mutant protein and TDP-43 pathology. While the terminal domain of the WT isoform is characterized by an acid isoelectric point (pI), the ALS variant presents a basic pI due to the altered aminoacidic composition of this sequence. We thus generated a KIF5A ALS isoform that retained part of the aberrant sequence but with lower pI. The overexpression of this mutated variant led to significantly lower protein aggregation and TDP-43 mislocalization than the ALS mutant. Our data show that re-establishing the correct pI rescues KIFA aggregation and significantly reduces the cytoplasmic mislocalization of TDP-43.Significance Statement Amyotrophic Lateral Sclerosis is a lethal neurodegenerative disease to which no cure is still known. Heterogenous genes have been causally linked to ALS, yet, the exact pathomechanism responsible for neuronal death remains unclear. One such gene is KIF5A which encodes for a neuronally enriched kinesin. Identified mutations cause incorrect mRNA splicing resulting in an aberrant C-terminal aminoacidic sequence. Here, we identified TDP-43 cytosolic enrichment, a hallmark common to many ALS models, in two distinct hiPSC-derived motoneuron lines harboring the ALS mutation KIF5Ac2993-1 G>A Moreover, we generated a KIF5A isoform that retained most of the aberrant sequence but did not promote protein aggregation nor TDP-43 mislocalization upon overexpression. These results shed further light on the pathobiochemistry of the ALS-KIF5A cases.
    DOI:  https://doi.org/10.1523/JNEUROSCI.1658-24.2025
  20. EBioMedicine. 2025 Jun 26. pii: S2352-3964(25)00269-5. [Epub ahead of print]118 105825
    Skeletal muscle, neuromuscular organoids and assembloids: a scoping review.
    Jiang-Li Yang, Shi-Yan Qian, Min-Li Cheng, Li-Xiang Wang, Yu Wang, Jing-Jing Liu, Chen-Shi Xi, Yu-Xin Yang, Yue Li, Chong Gao, Guo-Qing Zheng.
      Skeletal muscle organoids (SKMOs), neuromuscular organoids (NMOs), and assembloids have emerged as powerful in vitro models that simulate the intricate cellular interactions between muscle and nerve, offering a promising approach to study function, development, and disease at the neuromuscular junction (NMJ). Given the relevance of NMJ dysfunction in diseases such as amyotrophic lateral sclerosis (ALS), these models provide insights into disease modelling. Scoping reviews are particularly valuable when exploring broad or emerging areas, as they help identify key concepts and evolving methodologies. Here, we conducted a scoping review by searching five databases, ultimately including 17 studies focussing on the development and application of SKMOs, NMOs, and assembloids in muscle function modelling. We highlight recent advancements and summarise various differentiation protocols, primarily utilising the Wnt signalling pathway agonist CHIR99021 and basic fibroblast growth factor (bFGF) to induce pluripotent stem cells into 2D neuromesodermal progenitors, further differentiated into SKMOs, NMOs, and assembloids. We also reviewed their cellular compositions, including motor neurons, neural stem cells, terminal Schwann cells, and astrocytes, alongside related research outcomes. Additionally, we discuss key challenges such as iPSC donor selection, standardisation, vascularisation, and 3D organoid imaging. This scoping review provides a foundation for future research on muscle function modelling.
    Keywords:  Application; Assembloids; Neuromuscular; Organoid; Scoping review; Skeletal muscle
    DOI:  https://doi.org/10.1016/j.ebiom.2025.105825
  21. Toxics. 2025 Jun 12. pii: 493. [Epub ahead of print]13(6):
    Metal-Induced Genotoxic Events: Possible Distinction Between Sporadic and Familial ALS.
    William Wu Kim, Gregory Zarus, Breanna Alman, Patricia Ruiz, Moon Han, Paul Mehta, Chao Ji, Hoormat Qureshi, James Antonini, Mohammad Shoeb.
      Metal exposure is a potential risk factor for amyotrophic lateral sclerosis (ALS). Increasing evidence suggests that elevated levels of DNA damage are present in both familial (fALS) and sporadic (sALS) forms of ALS, characterized by the selective loss of motor neurons in the brain, brainstem, and spinal cord. However, identifying and differentiating initial biomarkers of DNA damage response (DDR) in both forms of ALS remains unclear. The toxicological profiles from the Agency for Toxic Substances and Disease Registry (ATSDR) and our previous studies have demonstrated the influence of metal exposure-induced genotoxicity and neurodegeneration. A comprehensive overview of the ATSDR's toxicological profiles and the available literature identified 15 metals (aluminum (Al), arsenic (As), cadmium (Cd), chromium (Cr), cobalt (Co), copper (Cu), iron (Fe), lead (Pb), manganese (Mn), mercury (Hg), nickel (Ni), selenium (Se), uranium (U), vanadium (V), and zinc (Zn)) showing exposure-induced genotoxicity indicators associated with ALS pathogenesis. Genetic factors including mutations seen in ALS types and with concomitant metal exposure were distinguished, showing that heavy metal exposure can exacerbate the downstream effect of existing genetic mutations in fALS and may contribute to motor neuron degeneration in sALS. Substantial evidence associates heavy metal exposure to genotoxic endpoints in both forms of ALS; however, a data gap has been observed for several of these endpoints. This review aims to (1) provide a comprehensive overview of metal exposure-induced genotoxicity in ALS patients and experimental models, and its potential role in disease risk, (2) summarize the evidence for DNA damage and associated biomarkers in ALS pathogenesis, (3) discuss possible mechanisms for metal exposure-induced genotoxic contributions to ALS pathogenesis, and (4) explore the potential distinction of genotoxic biomarkers in both forms of ALS. Our findings support the association between metal exposure and ALS, highlighting under or unexplored genotoxic endpoints, signaling key data gaps. Given the high prevalence of sALS and studies showing associations with environmental exposures, understanding the mechanisms and identifying early biomarkers is vital for developing preventative therapies and early interventions. Limitations include variability in exposure assessment and the complexity of gene-environment interactions. Studies focusing on longitudinal exposure assessments, mechanistic studies, and biomarker identification to inform preventative and therapeutic strategies for ALS is warranted.
    Keywords:  ALS; DNA damage; biomarkers; metals; neurotoxicity; telomere
    DOI:  https://doi.org/10.3390/toxics13060493
  22. Redox Biol. 2025 Jun 14. pii: S2213-2317(25)00245-9. [Epub ahead of print]85 103732
    Metabolic analysis of sarcopenic muscle identifies positive modulators of longevity and healthspan in C. elegans.
    Steffi M Jonk, Alan Nicol, Vicki Chrysostomou, Emma Lardner, Shu-Che Yu, Gustav Stålhammar, Jonathan G Crowston, James R Tribble, Peter Swoboda, Pete A Williams.
      Sarcopenia is the age-related degeneration of skeletal muscle, resulting in loss of skeletal muscle tone, mass, and quality. Skeletal muscle is a source of systemic metabolites and macromolecules important for neuronal health, function, and healthy neuronal aging. Age-related loss of skeletal muscle might result in decreased metabolite and macromolecule availability, resulting in reduced neuronal function or increased susceptibility to unhealthy aging and neurodegenerative diseases. We aimed to identify muscle metabolite candidates that regulate healthy aging. C57BL/6J mice were aged to young adult (4 months) and old age (25 months) and skeletal muscle was collected. Age-related muscle loss was confirmed by reduced muscle mass, muscle fiber degeneration, reduced myosin intensity, in addition to a metabolic shift and increased DNA damage in skeletal muscle. Using a low molecular weight enriched metabolomics protocol, we assessed the metabolic profile of skeletal muscle from young adult and old age mice and identified 20 metabolites that were significantly changed in aged muscle. These metabolite candidates were tested in C. elegans assays of lifespan, healthspan, muscle, and mitochondrial morphology under normal and stressed conditions. We identified four metabolite candidates (beta-alanine, 4-guanidinobutanoic acid, 4-hydroxyproline, pantothenic acid) that, when supplemented in C. elegans provided robust gero- and mitochondrial protection. These candidates also affected life-, and health- span in C. elegans models of amyotrophic lateral sclerosis (ALS) and Duchenne muscular dystrophy (DMD). Our findings support that aging muscle can be used to identify novel metabolite modulators of lifespan and health and may show promise for future treatments of neurodegenerative and neuromuscular disorders.
    Keywords:  Aging; C. elegans; Metabolomics; Mitochondria; Sarcopenia
    DOI:  https://doi.org/10.1016/j.redox.2025.103732
  23. Nat Commun. 2025 Jun 23. 16(1): 5343
    Cholinergic basal forebrain neurons regulate vascular dynamics and cerebrospinal fluid flux.
    Kai-Hsiang Chuang, Xiaoqing Alice Zhou, Ying Xia, Zengmin Li, Lei Qian, Eamonn Eeles, Grace Ngiam, Jurgen Fripp, Elizabeth J Coulson.
      Brain waste is cleared via a cerebrospinal fluid (CSF) pathway, the glymphatic system, whose dysfunction may underlie many brain conditions. Previous studies show coherent vascular oscillation, measured by blood oxygenation level-dependent (BOLD) fMRI, couples with CSF inflow to drive fluid flux. Yet, how this coupling is regulated, whether it mediates waste clearance, and why it is impaired remain unclear. Here we demonstrate that cholinergic neurons modulate BOLD-CSF coupling and glymphatic function. We find BOLD-CSF coupling correlates cortical cholinergic activity in aged humans. Lesioning basal forebrain cholinergic neurons in female mice impairs glymphatic efflux and associated changes in BOLD-CSF coupling, arterial pulsation and glymphatic influx. An acetylcholinesterase inhibitor alters these dynamics, primarily through peripheral mechanisms. Our results suggest cholinergic loss impairs glymphatic function by a neurovascular mechanism, potentially contributing to pathological waste accumulation. This may provide a basis for developing diagnostics and treatments for glymphatic dysfunction.
    DOI:  https://doi.org/10.1038/s41467-025-60812-3
  24. Yonsei Med J. 2025 Jul;66(7): 395-404
    Human Pluripotent Stem Cell-Based Therapies for Parkinson's Disease: Challenges and Potential Solutions.
    Mi-Yoon Chang, Sang-Hun Lee.
      Over 20 years of research on human pluripotent stem cell (hPSC)-based therapies for Parkinson's disease (PD) has recently culminated in clinical trials. The first clinical report on autologous transplantation of patient-derived hPSCs showed significant motor symptom improvement, validating the therapeutic promise of this approach. However, critical challenges remain, notably the limited engraftment and survival of donor cells. Cellular stress incurred during in vitro differentiation of hPSCs into midbrain dopaminergic progenitors and neurons contributes to the reduced survival of grafted mDA neurons. Additionally, the host brain environment at the injection sites becomes hostile to transplanted cells due to needle trauma, immune rejection, and alpha-synuclein pathology present in the brains of PD patients. This review discusses potential strategies to address both intrinsic donor cell stress and hostile host brain environment, aiming to enhance the long-term efficacy and engraftment of hPSC-based cell therapies for PD.
    Keywords:  Human pluripotent stem cell; Parkinson's disease; alpha-synuclein; cell therapy; midbrain-type dopamine
    DOI:  https://doi.org/10.3349/ymj.2024.0447
  25. Biomolecules. 2025 May 28. pii: 786. [Epub ahead of print]15(6):
    Neuronal Deletion of Tumor Susceptibility Gene 101 (Tsg101) Causes Rapid Apoptotic Loss of Hippocampal CA3 Neurons.
    Will P Walker, Megan Lea Ratz-Mitchem, Kay-Uwe Wagner, Teresa M Gunn.
      Endosomal dysfunction is one of the earliest cellular signs in Alzheimer's disease. Tumor susceptibility gene 101 protein (TSG101) is a component of the endosomal sorting complex required for transport (ESCRT)-I, which plays a key role in sorting ubiquitinated cell surface proteins and lipids onto intraluminal vesicles of multivesicular bodies for trafficking to lysosomes or autophagosomes for degradation, or to the plasma membrane for exosomal secretion. TSG101-dependent trafficking has been implicated in the propagation and spread of misfolded proteins associated with neurodegenerative diseases. We used transgenesis mice to study the in vivo consequences of disrupting TSG101-dependent trafficking in adult neurons. Mice lacking Tsg101 in forebrain neurons (Tsg101ck2-null) showed rapid loss of hippocampal neurons and progressive forebrain atrophy. Astrogliosis was apparent in the dentate gyrus within 1 week of deleting Tsg101, followed by apoptosis of hippocampal CA3 neurons and accumulation of the autophagy adapter P62/SQSTM1 and ubiquitinated proteins. Failure to detect lipidated LC3 indicated autophagy was impaired rather than upregulated. Endosomal markers (RAB5 and RAB7) and amyloid protein also accumulated in hippocampal neurons of Tsg101ck2-null mice. Our data establish a critical role for TSG101 in neuronal survival and demonstrate the importance of the in vivo assessment of gene and protein functions.
    Keywords:  Tumor Susceptibility Gene 101 (Tsg101/TSG101); amyloid beta; endosomal trafficking; neurodegeneration
    DOI:  https://doi.org/10.3390/biom15060786
  26. Mol Genet Metab. 2025 Jun 17. pii: S1096-7192(25)00171-4. [Epub ahead of print]145(4): 109180
    Secondary accumulation of lyso-platelet activating factors in lysosomal storage diseases.
    Pamela Kell, Sonali Mishra, Heather L Gray-Edwards, Douglas R Martin, Angel Gaudioso Guirado, María Dolores Ledesma, Ernesto R Bongarzone, Mark S Sands, Ellen Sidransky, Daniel S Ory, Xuntian Jiang.
      Lysosomal storage diseases (LSDs) are a group of inherited disorders caused by defects in genes that encode lysosomal enzymes, transmembrane proteins, or transport proteins. These defects typically lead to the accumulation of undegraded substrates or obstructed substances in lysosomes, serving as primary storage materials. However, in certain LSDs, secondary storage products-such as glycosphingolipids, phospholipids, and cholesterol-can also accumulate in tissues, independent of the primary enzyme or protein defect. In our recent studies, we identified lyso-platelet activating factors (lyso-PAFs) as secondary storage compounds in multiple LSDs, including Niemann-Pick disease type C1 (NPC1), GM2 activator deficiency, and GM1 gangliosidosis (GM1). Our ongoing work suggests that lyso-PAFs are also prevalent secondary storage products in Niemann-Pick disease type A (NPA), Sandhoff disease (SD), Tay-Sachs disease (TSD), and Krabbe disease (KD). We observed that elevated lyso-PAF levels were significantly correlated with the accumulation of primary storage substances in these disorders, indicating their potential as biomarkers for disease progression in these LSDs. Moreover, treatment with adeno-associated virus (AAV)-based gene therapies led to a reduction in lyso-PAF levels in the central nervous systems of TSD sheep and GM1 cats, further supporting their potential as biomarkers for therapeutic efficacy. While it remains unclear whether changes in lyso-PAFs contribute directly to disease pathology or simply reflect disease progression, further research into the enzymes involved in their synthesis and degradation is essential for uncovering their functional role in the cellular physiology and pathology of LSDs. Thus, further exploration of lyso-PAF in biofluids as prognostic and pharmacodynamic biomarkers is warranted.
    Keywords:  Krabbe disease; Lyso-platelet activating factors; Niemann-Pick disease type A; Sandhoff disease; Tay-Sachs disease
    DOI:  https://doi.org/10.1016/j.ymgme.2025.109180
  27. Nat Rev Mol Cell Biol. 2025 Jun 23.
    Key challenges and recommendations for defining organelle membrane contact sites.
    Tito Calì, Emmanuelle M Bayer, Emily R Eden, György Hajnóczky, Benoit Kornmann, Laura Lackner, Jen Liou, Karin Reinisch, Hyun-Woo Rhee, Rosario Rizzuto, Luca Scorrano, Marisa Brini.
      Intracellular membrane contact sites (MCSs) between organelles have crucial roles in cellular signalling and homeostasis. These sites, which are often disrupted in pathological conditions, enable the exchange of ions, lipids and metabolites between membrane-bound compartments, helping cells adapt to varying physiological conditions. Specific tether proteins and complexes stabilize these interactions and mediate responses to different intracellular or extracellular stimuli. The study of MCSs has progressed in recent years, owing to the development of new methods such as genetically encoded reporter constructs, advanced imaging techniques, including super-resolution microscopy and electron tomography, and proteomic approaches based on mass spectrometry. These tools have enabled unprecedented visualization and quantification of organelle interactions, as well as identification of the molecular players involved. This Expert Recommendation aims to define and map the 'organelle contactome', describing key proteins involved in contact site formation and the roles of MCSs in cellular function. We also explore contact site dynamics and detail advantages and disadvantages of the methodologies for studying them. Importantly, we consolidate open questions in contact site research and discuss challenges and limitations of the current experimental approaches.
    DOI:  https://doi.org/10.1038/s41580-025-00864-x
  28. J Extracell Vesicles. 2025 Jun;14(6): e70109
    Cortical Actin Depolymerisation in 3D Cell Culture Enhances Extracellular Vesicle Secretion and Therapeutic Effects.
    Zhen Yang, Xiaoke Li, Qiyuan Lin, Fanfan Zhou, Kaini Liang, Jiao Jiao Li, Yudi Niu, Qingchen Meng, Tianyuan Zhao, Hao Li, Du Wang, Jianjing Lin, Hui Li, Bin Wang, Wei Liu, Yanan Du, Jianhao Lin, Dan Xing.
      Three-dimensional (3D) culture systems have been shown to enhance cellular secretion of small extracellular vesicles (sEVs) compared to two-dimensional (2D) culture. However, the molecular mechanisms driving sEV secretion and influencing their potential for disease treatment have not been elucidated. In this study, we discovered the depolymerisation of cortical actin as a new mechanism that leads to increased sEV release, and that in 3D cultured mesenchymal stem cells (MSCs), this process was modulated by the downregulation of integrin-α1 (ITGA1) and subsequent inhibition of the RhoA/cofilin signalling pathway. Interestingly, the knockdown of Rab27A and Rab27B significantly reduced sEV secretion by MSCs to 0.5- and 0.1-fold, respectively. However, there was no difference in expression levels of Rab27A/B between MSCs cultured in 2D and 3D environments. In addition, sEVs derived from 3D cultured MSCs demonstrated enhanced therapeutic function both in vitro and in rat models of osteoarthritis (OA) and wound healing. Collectively, this study illustrates a new mechanism for enhanced secretion of sEVs, involving RhoA/cofilin pathway-dependent cortical actin depolymerisation, which is independent of Rab27A/B. These findings provide novel insights for optimising the yield of stem cell-derived sEVs, as well as their therapeutic efficacy for treating chronic diseases.
    Keywords:  3D culture; Rab27A/B; actin depolymerisation; cortical actin; small extracellular vesicles
    DOI:  https://doi.org/10.1002/jev2.70109
  29. Mol Psychiatry. 2025 Jun 25.
    The mechanisms underlying TDP-43-associated neurodegeneration in Alzheimer's disease and related dementias.
    Ju Gao, Mao Ding, Siyue Qin, Devanshi Shukla, Lauren Vicuna, Jingjing Liang, Xinglong Wang.
      Alzheimer's disease (AD) and Alzheimer's disease-related dementias (ADRDs) are among the most prevalent neurodegenerative diseases, characterized by progressive cognitive decline driven by complex and overlapping pathological mechanisms. While amyloid plaques, neurofibrillary tangles, and Lewy bodies are well-established hallmarks, TAR DNA-binding protein 43 (TDP-43) pathology has emerged as a critical contributor to disease progression, particularly in cases exhibiting hippocampal sclerosis and severe brain atrophy. TDP-43 pathology is defined by its cytoplasmic mislocalization, aberrant aggregation, and nuclear depletion, leading to disruptions in RNA metabolism, stress granule dynamics, and mitochondrial function. Increasing evidence suggests that TDP-43 pathology not only exacerbates neuronal degeneration but also interacts with Aβ plaques, tau tangles, and α-synuclein aggregates, compounding neurodegenerative processes and accelerating cognitive decline. Despite its growing recognition, TDP-43 pathology remains underexplored compared to other proteinopathies in AD and ADRDs, highlighting the need for further mechanistic studies and targeted therapeutic development. In this review, we summarize the current understanding of TDP-43 pathology in AD and ADRDs, with a focus on its role in disease progression. We further discuss the molecular mechanisms underlying TDP-43-associated neurodegeneration in AD and ADRDs, emphasizing RNA dysregulation, mitochondrial dysfunction, disrupted protein homeostasis, stress response alternations, and nuclear-cytoplasmic transport impairments. Lastly, given the significant impact on disease pathology, we review ongoing efforts to treat TDP-43-associated neurodegeneration, including antisense oligonucleotides, small-molecule inhibitors, and peptide-based interventions aimed at restoring TDP-43 function or preventing its neurotoxicity and pathological aggregation.
    DOI:  https://doi.org/10.1038/s41380-025-03089-8
  30. Autophagy. 2025 Jun 26.
    Microaggrephagy: an ESCRT-I-PTPN23-dependent pathway for MAPT/tau aggregate clearance.
    Shoshiro Hirayama, Shigeo Murata.
      The clearance mechanisms for ubiquitinated protein aggregates, such as MAPT/tau in neurodegenerative diseases, remain incompletely understood, particularly regarding the role of microautophagy. To identify mediators of this process, we performed an unbiased genome-wide CRISPR knockout screen using cells propagating MAPT/tau repeat domain (MAPT/tauRD) aggregates. This screen identified the ESCRT-I complex and the accessory protein PTPN23 as essential for the clearance of ubiquitinated MAPT/tauRD aggregates via a microautophagy-dependent pathway, operating independently of macroautophagy and chaperone-mediated autophagy. We designate this pathway "microaggrephagy". Mechanistically, microaggrephagy involves the recognition of polyubiquitinated aggregates by the ESCRT-I subunit TSG101, with PTPN23 acting as an adaptor bridging ESCRT-I and ESCRT-III to facilitate microautophagic engulfment. Furthermore, a disease-associated mutation in the ESCRT-I component UBAP1 disrupts its interaction with PTPN23 and impairs MAPT/tau clearance, implicating dysfunction of this pathway in neurodegenerative pathogenesis. These findings establish microaggrephagy as a distinct cellular mechanism for degrading pathological protein aggregates, provide a molecular basis for its function, and suggest potential therapeutic targets for proteinopathies.
    Keywords:  ESCRT-I complex; MAPT/tau repeat domain (mapt/taurd); PTPN23; microaggrephagy; microautophagy; protein aggregates
    DOI:  https://doi.org/10.1080/15548627.2025.2525866
  31. Transl Neurodegener. 2025 Jun 23. 14(1): 32
    Abnormal α-synuclein binds to synaptotagmin 13, impairing extracellular vesicle release in synucleinopathies.
    Yasuo Miki, Shuji Shimoyama, Makoto T Tanaka, Hanae Kushibiki, Asa Nakahara, Xiaopeng Wen, Masanori Hijioka, Tomoya Kon, Megha Murthy, Tomonori Furukawa, Conceição Bettencourt, Fumiaki Mori, Hiroki Mizukami, Shirushi Takahashi, Mari Tada, Yoshihisa Kitamura, Akiyoshi Kakita, Thomas T Warner, Koichi Wakabayashi.
       BACKGROUND: Despite increasing in vitro research, direct evidence of how abnormal α-synuclein (α-Syn) dysregulates vesicular transport and synaptic function in the human brain is lacking.
    METHODS: We performed a transcriptome analysis using brain tissues from a multiple system atrophy (MSA) mouse model, which develops human α-Syn-positive glial cytoplasmic inclusion-like structures and neuronal cytoplasmic inclusion-like structures after tamoxifen injection. We then performed histologic and biochemical analyses using brain samples from 71 human cases (Parkinson's disease, n = 10; dementia with Lewy bodies [DLB], n = 19; MSA, n = 15; control: n = 27), a human blood sample (control: n = 1), and cultured cells.
    RESULTS: Based on the transcriptome of the MSA mouse model, we identified 10 vesicular transport proteins, including synaptotagmin 13 (SYT13), that might interact with α-Syn. Immunohistochemistry using human brain samples demonstrated that of the 10 vesicular transport proteins identified in the transcriptome analysis, only SYT13 was incorporated into both Lewy bodies and glial cytoplasmic inclusions. Proximity ligation assays revealed that SYT13 exhibited a higher degree of interactions with phosphorylated α-Syn than with endogenous α-Syn. Immunoprecipitation confirmed that SYT13 bound predominantly to phosphorylated α-Syn, SYT1, and the soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) complexes. Filter trap assays revealed interactions between SYT13 and soluble toxic β-sheet-rich α-Syn oligomers. Furthermore, fraction analysis showed a significant increase of SYT13 protein levels at the synapses in DLB and MSA. Notably, a correlation was observed between the levels of SYT13 and aggregated α-Syn at the synapses. SYT13 was observed to regulate extracellular vesicle release in association with SYT1 and the SNARE complexes in SH-SY5Y cells. SYT13 overexpression in SH-SY5Y cells impaired extracellular vesicle release. Consistently, the numbers of extracellular vesicles were significantly reduced in the brain homogenates of DLB and MSA cases compared with those in controls.
    CONCLUSIONS: Abnormal α-Syn impairs extracellular vesicle release through interactions with SYT13 in synucleinopathies. Our findings provide insights into therapeutic strategies for alleviating dysregulations of vesicular transport and synaptic function in patients with synucleinopathies.
    Keywords:  Extracellular vesicles; SNARE complex; SYT13; Synucleinopathy; α-Synuclein
    DOI:  https://doi.org/10.1186/s40035-025-00493-6
  32. Int J Mol Sci. 2025 Jun 18. pii: 5858. [Epub ahead of print]26(12):
    Myostatin Modulation in Spinal Muscular Atrophy: A Systematic Review of Preclinical and Clinical Evidence.
    Martina Gnazzo, Giulia Pisanò, Valentina Baldini, Giovanna Giacomelli, Silvia Scullin, Benedetta Piccolo, Emanuela Claudia Turco, Susanna Esposito, Maria Carmela Pera.
      Spinal Muscular Atrophy (SMA) is a genetic disorder characterized by the progressive loss of motor neurons and consequent muscle atrophy. Although SMN-targeted therapies have significantly improved survival and motor outcomes, residual muscle weakness remains a major clinical challenge, particularly in patients treated later in the disease course. Myostatin, a potent negative regulator of skeletal muscle mass, has emerged as a promising therapeutic target to address this gap. This review summarizes the preclinical and clinical evidence supporting the modulation of the myostatin pathway in SMA. Preclinical studies have demonstrated that inhibiting myostatin, especially when combined with SMN-enhancing agents, can increase muscle mass, improve motor function, and enhance neuromuscular connectivity in SMA mouse models. These findings provide a strong rationale for translating myostatin inhibition into clinical practice as an adjunctive strategy. Early clinical trials investigating myostatin inhibitors have shown favorable safety profiles and preliminary signs of target engagement. However, large-scale trials have yet to demonstrate widespread, robust efficacy across diverse patient populations. Despite this, myostatin pathway inhibition remains a compelling approach, particularly when integrated into broader treatment paradigms aimed at enhancing motor unit stability and function in individuals with SMA. Further clinical research is essential to validate efficacy, determine optimal timing, and define the patient subgroups most likely to benefit from myostatin-targeted therapies.
    Keywords:  SMN-targeted therapy; adjunctive treatment strategy; apitegromab; motor function restoration; muscle atrophy; myostatin inhibition; spinal muscular atrophy
    DOI:  https://doi.org/10.3390/ijms26125858
  33. Nat Biomed Eng. 2025 Jun 27.
    Scalable production of human cortical organoids using a biocompatible polymer.
    Genta Narazaki, Yuki Miura, Sergey D Pavlov, Mayuri Vijay Thete, Julien G Roth, Merve Avar, Sungchul Shin, Ji-Il Kim, Zuzana Hudacova, Sarah C Heilshorn, Sergiu P Pașca.
      The generation of neural organoids from human pluripotent stem cells holds great promise in modelling disease and screening drugs, but current approaches are difficult to scale due to undesired organoid fusion. Here we develop a scalable cerebral cortical organoid platform by screening biocompatible polymers that prevent the fusion of organoids cultured in suspension. We identify a cost-effective polysaccharide that increases the viscosity of the culture medium, significantly enhancing the yield of cortical organoids while preserving key features such as regional patterning, neuronal morphology and functional activity. We further demonstrate that this platform enables straightforward screening of 298 FDA-approved drugs and teratogens for growth defects using over 2,400 cortical organoids, uncovering agents that disrupt organoid growth and development. We anticipate this approach to provide a robust and scalable system for modelling human cortical development, and facilitate efficient compound screening for neuropsychiatric disorders-associated phenotypes.
    DOI:  https://doi.org/10.1038/s41551-025-01427-3
  34. Nat Biomed Eng. 2025 Jun 27.
    Extension of replicative lifespan by synthetic engineered telomerase RNA in patient induced pluripotent stem cells.
    Neha Nagpal, Suneet Agarwal.
      RNA engineering has yielded a new class of medicines but faces limitations depending on RNA size and function. Here we demonstrate the synthesis and enzymatic stabilization of telomerase RNA component (TERC), a therapeutically relevant long non-coding RNA (lncRNA) that extends telomere length and replicative lifespan in human stem cells. Compared with therapeutic mRNAs, engineered TERC RNA (eTERC) depends on avoiding nucleoside base modifications and incorporates a distinct trimethylguanosine 5' cap during in vitro transcription. We show that the non-canonical polymerase TENT4B can be repurposed to enzymatically stabilize synthetic RNAs of any size by catalysing self-limited 2'-O-methyladenosine tailing, which is critical for optimal eTERC function in cells. A single transient exposure to eTERC forestalls telomere-induced senescence in telomerase-deficient human cell lines and lengthens telomeres in induced pluripotent stem cells from nine patients carrying different mutations in telomere-maintenance genes, as well as primary CD34+ blood stem/progenitor cells. Our results provide methods and proof of functional reconstitution for a stabilized, synthetic human lncRNA. eTERC may have therapeutic potential to safely extend replicative capacity in human stem cells.
    DOI:  https://doi.org/10.1038/s41551-025-01429-1
  35. Cell Rep. 2025 Jun 24. pii: S2211-1247(25)00638-2. [Epub ahead of print]44(7): 115867
    Mislocalization of nucleic acids is a convergent and targetable mechanism in Alzheimer's disease and frontotemporal dementia.
    Christy Hung, Miriam Llorian, Koustav Pal, Frederick J Livesey, Jyoti Choudhary, Theodoros I Roumeliotis, Rickie Patani.
      Nucleocytoplasmic transport defects are observed in Alzheimer's disease (AD) and frontotemporal dementia (FTD). Here, we assess mRNA nucleocytoplasmic localization by performing transcriptome-wide profiling on nuclear and cytoplasmic fractions of human iPSC-derived cortical neurons from healthy individuals compared to those with familial AD or FTD. We find that AD- and FTD-causing mutations induce significant changes in mRNA nucleocytoplasmic distribution. We additionally observe the redistribution of mitochondria-related transcripts across AD and FTD neurons. The significantly increased mitochondrial RNA (mtRNA) in the cytosol of AD and FTD mutant neurons raised the possibility of leakage, which motivated us to investigate mtDNA leakage. We reveal abnormal cytoplasmic accumulation of mtDNA in AD and FTD cortical neurons together with evidence of mitochondrial aberrance. Importantly, mislocalisation of nucleic acids, mitochondrial dysfunction and cGAS-STING activation can be ameliorated through VCP D2 ATPase inhibition.
    Keywords:  Alzheimer's disease; CP: Molecular biology; CP: Neuroscience; VCP inhibition; cGAS-STING; frontotemporal dementia; mRNA; mitochondrial DNA; mitochondrial RNA; mitochondrial dysfunction; neurodegeneration; nucleocytoplasmic redistribution
    DOI:  https://doi.org/10.1016/j.celrep.2025.115867
  36. Elife. 2025 06 24. pii: RP101531. [Epub ahead of print]13
    The triad interaction of ULK1, ATG13, and FIP200 is required for ULK complex formation and autophagy.
    Yutaro Hama, Yuko Fujioka, Hayashi Yamamoto, Noboru Mizushima, Nobuo N Noda.
      In mammals, autophagosome formation, a central event in autophagy, is initiated by the ULK complex comprising ULK1/2, FIP200, ATG13, and ATG101. However, the structural basis and mechanism underlying the ULK complex assembly have yet to be fully clarified. Here, we predicted the core interactions organizing the ULK complex using AlphaFold, which proposed that the intrinsically disordered region of ATG13 engages the bases of the two UBL domains in the FIP200 dimer via two phenylalanines and also binds the tandem microtubule-interacting and transport domain of ULK1, thereby yielding the 1:1:2 stoichiometry of the ULK1-ATG13-FIP200 complex. We validated the predicted interactions by point mutations and demonstrated direct triad interactions among ULK1, ATG13, and FIP200 in vitro and in cells, wherein each interaction was additively important for autophagic flux. These results indicate that the ULK1-ATG13-FIP200 triadic interaction is crucial for autophagosome formation and provides a structural basis and insights into the regulation mechanism of autophagy initiation in mammals.
    Keywords:  ATG13; AlphaFold; FIP200; ULK complex; autophagy; cell biology; human
    DOI:  https://doi.org/10.7554/eLife.101531
  37. Autophagy. 2025 Jun 26.
    Mitochondrial protein nmd regulates lipophagy and general autophagy during development.
    Wei Wang, Xufeng Wang, Xiaoqi Zhou, Lu Jiang, Weina Shang, Liquan Wang, Chao Tong.
      Lipophagy engulfs lipid droplets and delivers them to lysosomes for degradation. We found that lipophagy levels were low in most fly tissues, except for the prothoracic gland (PG) during larval development. Therefore, we performed a small-scale screening in the PG to identify regulators of lipophagy. We discovered that the loss of nmd, a gene encoding a mitochondrial AAA-ATPase, led to developmental failure and reduced lipophagy in the PG. Further studies indicated that nmd was not only required for lipophagy but also essential for general macroautophagy/autophagy in both PG and fat body tissues. Autophagy was induced but blocked at the autophagosome-lysosome fusion stage upon nmd reduction. Additionally, nmd interacted with mitochondrial protein import machinery, such as Tom20, Tom40, and the import cargo, such as Idh. Loss of nmd decreased protein import into mitochondria. Similar to the loss of nmd, reduction of Tom20 or Tom40 also resulted in reduced lipophagy in the PG. In adult flies, reducing nmd expression in the eyes caused lipid droplet accumulation and severe degeneration during aging. Overexpression of bmm, a triglyceride lipase, reduced lipid droplets in the eye but did not rescue the eye degeneration caused by the reduction of nmd.
    Keywords:  Drosophila; lipophagy; mitochondrial protein import; neuronal homeostasis; nmd; prothoracic gland
    DOI:  https://doi.org/10.1080/15548627.2025.2522124
  38. Nat Cell Biol. 2025 Jun 26.
    A lysosomal surveillance response to stress extends healthspan.
    Terytty Yang Li, Arwen W Gao, Rendan Yang, Yu Sun, Yuxuan Lei, Xiaoxu Li, Lin Chen, Yasmine J Liu, Rachel N Arey, Kimberly Morales, Raya B Liu, Wenzheng Wang, Ang Zhou, Tong-Jin Zhao, Weisha Li, Amélia Lalou, Qi Wang, Tanes Lima, Riekelt H Houtkooper, Johan Auwerx.
      Lysosomes are cytoplasmic organelles central for the degradation of macromolecules to maintain cellular homoeostasis and health. However, how lysosomal activity can be boosted to counteract ageing and ageing-related diseases remains elusive. Here we reveal that silencing specific vacuolar H+-ATPase subunits (for example, vha-6), which are essential for intestinal lumen acidification in Caenorhabditis elegans, extends lifespan by ~60%. This longevity phenotype can be explained by an adaptive transcriptional response typified by induction of a set of transcripts involved in lysosomal function and proteolysis, which we termed the lysosomal surveillance response (LySR). LySR activation is characterized by boosted lysosomal activity and enhanced clearance of protein aggregates in worm models of Alzheimer's disease, Huntington's disease and amyotrophic lateral sclerosis, thereby improving fitness. The GATA transcription factor ELT-2 governs the LySR programme and its associated beneficial effects. Activating the LySR pathway may therefore represent an attractive mechanism to reduce proteotoxicity and, as such, potentially extend healthspan.
    DOI:  https://doi.org/10.1038/s41556-025-01693-y
  39. JACS Au. 2025 Jun 23. 5(6): 2475-2490
    Endogenous SNAP-Tagging of Munc13‑1 for Monitoring Synapse Nanoarchitecture.
    Maria Kowald, Sylvestre P J T Bachollet, Fritz Benseler, Maria Steinecker, Moritz Boll, Sofia Kaushik, Tolga Soykan, Siqi Sun, Ramona Birke, Dragana Ilic, Nils Brose, Hanna Hörnberg, Martin Lehmann, Silvio O Rizzoli, Johannes Broichhagen, Noa Lipstein.
      Synaptic function is governed by highly regulated protein machineries, whose abundance and spatial localization change continually. Studies to determine dynamic changes in synaptic protein nanoarchitecture typically rely on immunolabeling or on the expression of fluorescent proteins. The former employs chemical fluorophores and signal amplification but requires fixation. The latter enables monitoring of proteins by live microscopy but uses suboptimal fluorophores. Self-labeling tags have been introduced to combine the advantages of these two approaches, and here we introduce a knock-in mouse line where the essential presynaptic protein Munc13-1 is endogenously fused to the self-labeling SNAP tag. We demonstrate efficient Munc13-1-SNAP labeling in fixed cultured neurons and in brain sections by various SNAP dyes, as well as by a novel far-red and cell impermeable compound, SBG-SiR-d12. We introduce and characterize SBG-SiR-d12 as a highly efficient dye for SNAP-tag labeling of extracellular epitopes and of intracellular proteins such as Munc13-1 in fixed and permeabilized tissue. Finally, we show that Munc13-1-SNAP can be labeled in living neurons and monitored through live-cell imaging using confocal and super resolution microscopy. We conclude that the Unc13aSNAP mouse line is a useful tool for labeling the presynaptic compartment and for the analysis of presynaptic nanoarchitectural dynamics, with potential for wide adoption.
    Keywords:  Active zone; Munc13-1; SBG-SiR-d12; SNAP tag; Synapse; Unc13a
    DOI:  https://doi.org/10.1021/jacsau.4c00946
  40. bioRxiv. 2025 Apr 23. pii: 2025.04.21.649822. [Epub ahead of print]
    TNIP1 and Autophagy Receptors regulate STING Signaling.
    Eric N Bunker, Tara D Fischer, Peng-Peng Zhu, François Le Guerroué, Richard J Youle.
      Activation of the cGAS-STING pathway stimulates innate immune signaling as well as LC3B lipidation and ubiquitylation at Golgi-related vesicles upon STING trafficking. Although ubiquitylation at these subcellular sites has been associated with regulating NF-κB-related innate immune signaling, the mechanisms of Golgi-localized polyubiquitin chain regulation of immune signaling is not well understood. We report here that the ubiquitin- and LC3B-binding proteins, TNIP1 and autophagy receptors p62, NBR1, NDP52, TAX1BP1, and OPTN associate with STING-induced ubiquitin and LC3B-labeled vesicles, and that p62 and NBR1 act redundantly in spatial clustering of the LC3B-labeled vesicles in the perinuclear region. We also find that while TBK1 kinase activity is not required for the recruitment of TNIP1 and the autophagy receptors, it also plays a role in sequestration of the LC3B-labeled vesicles. The ubiquitin binding domains, rather than the LC3B-interacting regions, of TNIP1 and OPTN are specifically important for their recruitment to Ub/LC3B-associated perinuclear vesicles, while OPTN is also recruited through a TBK1-dependent mechanism. Functionally, we find that TNIP1 and OPTN play a role in STING-mediated innate immune signaling, with TNIP1 acting as a significant negative regulator of both NF-κB- and Interferon-mediated gene expression. Together, these results highlight autophagy-independent mechanisms of autophagy receptors and TNIP1 with unanticipated roles in regulating STING-mediated innate immunity.
    DOI:  https://doi.org/10.1101/2025.04.21.649822
  41. Mol Cell Biol. 2025 Jun 22. 1-10
    Nuclear Roles for Canonically Lysosomal Proteases.
    Anna E Enneking, Marc M Khorey, Laura E Edgington-Mitchell.
      While the cysteine proteases legumain and cathepsins have traditionally been known as "lysosomal" proteases, there is increasing evidence to suggest that they also contribute to a wide range of extralysosomal processes, including in the nucleus. This review aims to provide a comprehensive overview of the current knowledge regarding the translocation of these proteases to the nucleus and their functions on arrival. We discuss possible mechanisms for transporting these proteases to the nucleus, including the presence of a nuclear localization signal sequence or hitchhiking on other proteins that possess this sequence. This transport requires the proteases to first reach the cytosol, which may occur via direct cytosolic translation of truncated proteases or downstream of lysosomal membrane permeabilization. We also discuss the evidence for functions of these proteases upon arrival to the nucleus, including cell cycle progression, cell differentiation, cell death, immune regulation, and epigenetic regulation. As protease substrate profiling methods continue to improve, it is anticipated that many new nuclear substrates and interacting partners will be identified to reveal additional functions for nuclear proteases.
    Keywords:  Protease; cathepsin; legumain; lysosome; nucleus; proteolysis; trafficking
    DOI:  https://doi.org/10.1080/10985549.2025.2519158
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