bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2025–10–19
twenty papers selected by
TJ Krzystek
  1. Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
  2. Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
  3. Exposure to the organochlorine pesticide cis-chlordane induces ALS-like mitochondrial perturbations in stem cell-derived motor neurons.
  4. OPTN protects retinal ganglion cells and ameliorates neuroinflammation in optic neuropathies.
  5. Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.
  6. Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.
  7. Assessment of Lysosome Trafficking in iPSC-Derived Neurons Utilizing Time-Lapse Videos and TrackMate ImageJ Plugin Software.
  8. C9orf72 hexanucleotide repeat expansions impair microglial response in ALS.
  9. Vesicular transport in the autophagic route: RAB GTPases as pivotal regulators of autophagy.
  10. Immunostaining Methods for Lysosomal Storage Disorder Research.
  11. Rapamycin and Autophagy: Potential Therapeutic Approach for Parkinson's Disease Treatment.
  12. Visualization of lysosomal membrane proteins by cryo electron tomography.
  13. Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy.
  14. Protocol for purifying primitive neural progenitors by fractional passaging to generate astroglia-enriched cerebral organoid.
  15. The role of small GTPases in Alzheimer's disease tau pathologies.
  16. Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis.
  17. Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
  18. 3D cultured human medium spiny neurons functionally integrate and rescue motor deficits in Huntington's disease mice.
  19. Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
  20. Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
  1. Methods Mol Biol. 2026 ;2976 47-60
    Monitoring Autophagosome Biogenesis and Degradation in Mammalian Cells Using LC3B Blotting.
    Jennifer E Palmer, David C Rubinsztein.
      Autophagy is a conserved lysosomal degradation pathway that recycles protein aggregates and damaged organelles to maintain cytoplasmic quality control. Measuring the amount of the lipid-conjugated autophagic protein LC3B-II is a useful way to test whether a particular perturbation affects autophagy. However, the level of LC3B-II is affected by factors that alter either the rate of autophagosome biogenesis or degradation. Consequently, the same steady-state LC3B-II level can be reached by opposing autophagic fluxes. It is thus essential when measuring LC3B-II to perform the assay both in the absence and presence of a lysosomal inhibitor, enabling measurement of the rate of synthesis independent of its degradation. LC3B-II is also a small protein that can be challenging to detect by western blotting. In this chapter, we will provide a method for the efficient western blotting of LC3B-II and guidance as to the interpretation of the results.
    Keywords:  ATG8; Autophagy; Bafilomycin A1; LC3B; SDS-PAGE; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_5
  2. Acta Neuropathol. 2025 Oct 14. 150(1): 43
    Genetic and proteomic analysis identifies BAG3 as an amyloid-responsive regulator of neuronal proteostasis.
    Zachary M Augur, Garrett M Fogo, Mason R Arbery, Yi-Chen Hsieh, Nalini R Rao, Kritika Goyal, Emily Dexter, David A Bennett, Jeffrey N Savas, Andrew M Stern, Tracy L Young-Pearse.
      The autophagy-lysosome pathway (ALP) and the ubiquitin-proteasome system (UPS) are the primary protein degradative mechanisms maintaining proteostasis in neurons. However, the impact of human genetic variation on these pathways and the role of BAG3 are poorly understood, particularly in the context of Alzheimer's disease, where proteostatic dysfunction is a defining hallmark. We utilized a large panel of iPSCs from deeply phenotyped cohorts to interrogate genetic contributions to baseline autophagic flux and UPS activity in human neurons, and protein turnover was assessed using SILAC-based quantitative proteomics. Across this panel of neurons, we observed substantial inter-individual differences in autophagic flux, which was inversely correlated with UPS activity. This reciprocal relationship extended to tau homeostasis, where higher autophagic flux resulted in reduced accumulation of aggregated, phosphorylated tau. Proteomic analyses revealed that global protein turnover dynamics stratified based on degradation pathway activity and could predict pathway-specific substrate dependencies. Interestingly, Bcl-2-associated athanogene 3 (BAG3), an important member of the chaperone-assisted selective autophagy pathway, emerged as a dynamically regulated autophagy chaperone, responsive to pharmacological inhibition of both the UPS and ALP. BAG3 knockout in neurons decreased autophagic flux and increased levels of high-molecular-weight phosphorylated tau. Notably, familial AD mutations and Aβ exposure induced BAG3 expression in neurons, while elevated BAG3 levels in human brain tissue were associated with higher neuropathological burden and disease progression. Our findings identify BAG3 as a key modulator of proteostasis in human neurons. Its regulation across genetic backgrounds and pathological stimuli suggests a central role in maintaining degradation activities in Alzheimer's disease and related disorders.
    Keywords:  Alzheimer’s disease; Amyloid-beta; Autophagy; BAG3; Neurons; Ubiquitin proteasome system; iPSC
    DOI:  https://doi.org/10.1007/s00401-025-02947-7
  3. PLoS One. 2025 ;20(10): e0332422
    Exposure to the organochlorine pesticide cis-chlordane induces ALS-like mitochondrial perturbations in stem cell-derived motor neurons.
    Oliver Clackson, Muhammad Reza Hamid, Adithi Wijesekera, Daniel Kulick, Alison Linsley O'Neil.
      Amyotrophic Lateral Sclerosis (ALS) is a debilitating and incurable neurodegenerative disease with unsolved etiology. Due to the large proportion of patients lacking direct disease inheritance, understanding the environmental factors that contribute to ALS development is of high priority. Epidemiological studies have implicated pesticides and other environmental exposures as possible contributors to ALS pathogenesis. Recently, our group determined that the organochlorine pesticide cis-chlordane is toxic to human motor neurons in a dose-dependent manner, causing an ALS-like phenotype in culture and animals with a mode of action independent of its known GABAA antagonism. Here, we aimed to characterize downstream motor neuron phenotypes associated with cis-chlordane treatment. We performed bulk RNA sequencing, live imaging, immunofluorescent labeling, and real-time metabolic assays on stem cell-derived motor neurons to assess chlordane-associated phenotypes in vitro. We demonstrate that cis-chlordane treatment causes a highly altered mitochondrial phenotype in motor neurons, including increased production of reactive oxygen species, decreased oxygen consumption rate and ATP production, and loss of mitochondrial membrane potential. We further implicate cis-chlordane as a possible mediator of potent motor neuron damage, with exposure to the pesticide inducing mitochondrial phenotypes akin to those seen in ALS. Our findings contribute to the growing body of evidence that future studies of investigating the role of pesticides in ALS development should focus on organochlorine molecules.
    DOI:  https://doi.org/10.1371/journal.pone.0332422
  4. Commun Biol. 2025 Oct 16. 8(1): 1475
    OPTN protects retinal ganglion cells and ameliorates neuroinflammation in optic neuropathies.
    Qinglong Wang, Yiqi Wang, Yi Da Douglas Jiang, Ryan Donahue, Gaby Cao, Weixuan Yan, Hong Guo, Zhenghui Li, Jenna Liang, Jin Hao, Yi Lu, Fengfeng Bei, Qianbin Wang, Feng Tian.
      Optineurin (OPTN) is an adaptor protein that plays a crucial role in many cellular pathways, including NF-κB signaling, programmed cell death, and vesicular trafficking. OPTN dysfunction has been implicated in the pathogenesis of several diseases, such as primary open angle glaucoma (POAG), amyotrophic lateral sclerosis (ALS). While mutations of OPTN seem to be predominantly loss-of-function in ALS, only gain-of-function mechanisms have been reported in POAG. Here, we demonstrate that OPTN knockout in the retina contributes to short-term astrogliosis, retinal ganglion cell (RGC) loss and long-term microglial activation. Moreover, OPTN loss of function does not exacerbate RGC death induced by ocular hypertension. Integrated bioinformatics and immunofluorescence analyses reveal that OPTN dysfunction leads to neuropeptide Y (NPY) downregulation and CHOP upregulation. Overexpression of wild-type OPTN in a hypertension glaucoma model prevents the RGC loss and attenuates microglial activation. Together, our findings highlight a neuroprotective role for OPTN as a key neuroimmune modulator.
    DOI:  https://doi.org/10.1038/s42003-025-08534-6
  5. Methods Mol Biol. 2026 ;2976 25-34
    Assessing Perturbations in Lysosome Health by Flow Cytometry in iPSC-Derived Human Neuron Cultures.
    Kirstin O McDonald, Stephanie M Hughes, Indranil Basak.
      Fluorescent molecular probes have frequently been used to monitor lysosomal health, localization, abundance, and movement through the detection of acidic organelles and lysosomal enzyme activity. Flow cytometry technology provides rapid and accurate analysis of single cells (neurons) or particles (lysosomes) in suspension through laser detection. Herein, we describe how to detect lysosomes via LysoTracker™ and Magic Red® Cathepsin Activity assays in iPSC-derived human neuron cultures by flow cytometry.
    Keywords:  Flow cytometry; Fluorescent probes; Lysosome acidity; Lysosome function; iPSC-derived neuron cultures
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_3
  6. Methods Mol Biol. 2026 ;2976 189-207
    Modeling Lysosomal Disease in Dictyostelium discoideum: Examining the Trafficking and Secretion of Lysosomal Enzymes.
    William D Kim, Robert J Huber.
      Non-mammalian models are powerful systems for enhancing our understanding of lysosomal function and lysosomal storage diseases. The social amoeba Dictyostelium discoideum is an excellent model organism for studying lysosomal function, as its genome encodes many proteins associated with lysosomal disease. Methods for gene knockout are straightforward in D. discoideum and include restriction enzyme-mediated integration (REMI) mutagenesis, homologous recombination via the Cre-loxP system, and CRISPR/Cas9-mediated gene editing, which collectively allow researchers to study protein function (e.g., lysosomal enzymes) in a genetically tractable biomedical model system. Additionally, activity assays for conserved lysosomal enzymes are well-established in D. discoideum. In this chapter, we outline methods for studying the intracellular localization and secretion of conserved lysosomal proteins in D. discoideum.
    Keywords:  Dictyostelium discoideum; Enzyme; Immunofluorescence; Lysosomes; Model organism; Neurodegeneration; Secretion; Trafficking; Western blotting
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_14
  7. Methods Mol Biol. 2026 ;2976 135-149
    Assessment of Lysosome Trafficking in iPSC-Derived Neurons Utilizing Time-Lapse Videos and TrackMate ImageJ Plugin Software.
    Oluwatobi N Eboda, Indranil Basak, Stephanie M Hughes.
      In order to maintain cell homeostasis, lysosomes must be trafficked throughout the cell and associated with different compartments for the delivery and breakdown of macromolecules. This process is especially important in polar cells, such as neurons, that require the trafficking of lysosomes to distal locations to perform their functions. Here, we employed LysoTracker DND-99, a live-cell fluorometric dye that stains acidic organelles, to label lysosomes prior to time-lapse imaging on a Nikon A1 confocal microscope and subsequent assessment of total distance travelled using the TrackMate ImageJ plugin.
    Keywords:  ImageJ; LysoTracker; Lysosome trafficking; TrackMate
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_11
  8. Nat Neurosci. 2025 Oct 14.
    C9orf72 hexanucleotide repeat expansions impair microglial response in ALS.
    Pegah Masrori, Baukje Bijnens, Laura Fumagalli, Kristofer Davie, Suresh Kumar Poovathingal, Tim Meese, Annet Storm, Nicole Hersmus, Raheem Fazal, Diede van den Biggelaar, Bob Asselbergh, Roxane Gruel, Johanna Van Den Daele, Heidi Denton, Paula Polanco Miquel, Simona Manzella, Winnok H De Vos, Siddhartan Chandran, Ludo Van Den Bosch, Dietmar Rudolf Thal, Renzo Mancuso, Philip Van Damme.
      Microglia and neuroinflammation are involved in amyotrophic lateral sclerosis (ALS), but the precise underlying molecular mechanisms remain elusive. We generated single-nuclei transcriptomes from the spinal cord and motor cortex of patients with sporadic ALS (sALS) and C9orf72 ALS (C9-ALS). Here we confirmed that C9orf72 is highly expressed in microglia and observed that the hexanucleotide repeat expansion (HRE) results in haploinsufficiency. Whereas sALS microglia transitioned toward disease-associated cell states, C9orf72 HRE microglia exhibited a diminished response, with alterations in endolysosomal pathways. We confirmed these observations using a human microglia xenograft model, in which C9orf72 mutations led to a reduced activation. We also confirmed the endolysosomal alterations in C9orf72 HRE and C9orf72-deficient induced pluripotent stem cell (iPSC)-derived microglia. We also found a diminished response of C9orf72 HRE astrocytes and provided a map of dysregulated ligand-receptor pairs in microglia and astrocytes. Our data highlight variations in the cellular substrate of sporadic and inherited forms of ALS, which have implications for patient stratification and selection of appropriate treatments.
    DOI:  https://doi.org/10.1038/s41593-025-02075-1
  9. Biochem J. 2025 Oct 17. 482(20): 1531-1544
    Vesicular transport in the autophagic route: RAB GTPases as pivotal regulators of autophagy.
    Romina Abba, María Isabel Colombo.
      Autophagy is recognized as one of the two main intracellular recycling pathways that play an essential role in cellular homeostasis by maintaining accurate energy levels and carrying out quality control functions. One of the major autophagic mechanisms, the so-called macroautophagy, is involved in the lysosomal degradation of different cytoplasmic components, such as long-lived proteins and damaged or dysfunctional organelles. Numerous studies have demonstrated that participation of intracellular membrane trafficking events is key for the progression of autophagy. In this review, we will focus on the small GTPases of the RAS-related in brain protein family, which have a crucial role in vesicular transport.
    Keywords:  RAB GTPases; autophagy; membrane traffic
    DOI:  https://doi.org/10.1042/BCJ20253092
  10. Methods Mol Biol. 2026 ;2976 227-235
    Immunostaining Methods for Lysosomal Storage Disorder Research.
    Ewa A Ziółkowska, Sophie H Wang, Hemanth R Nelvagal, Jonathan D Cooper.
      Immunostaining enables the detection of proteins and antigens in histological preparations due to specific antigen-antibody interactions. Such visualization can be performed by immunoperoxidase methods, but more recently, we have switched to immunofluorescence methods that offer superior sensitivity and simultaneous detection of multiple antigens. Key to this approach is a counterstaining method that masks tissue histofluorescence and the autofluorescent storage material that accumulates in the disorders we study. Such methods allow the analysis of the structural organization of lysosomes and the impact of their dysfunction on cells and tissues. In this chapter, we present an optimized staining protocol that can serve as a standard procedure in histological examinations, ensuring high quality and consistency of quantitative results.
    Keywords:  Histology; Immunofluorescence; Immunohistochemistry; Primary antibody; Secondary antibody; Staining; TrueBlack®
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_16
  11. CNS Neurol Disord Drug Targets. 2025 Oct 14.
    Rapamycin and Autophagy: Potential Therapeutic Approach for Parkinson's Disease Treatment.
    Ahsas Goyal, Anshika Kumari, Aanchal Verma, Neetu Agrawal.
      Parkinson's disease (PD) is a chronic, progressive neurodegenerative disorder marked by the degeneration of dopaminergic neurons in the substantia nigra, leading to characteristic motor symptoms such as bradykinesia, tremor, and rigidity, as well as a range of non-motor manifestations including cognitive impairment, mood disturbances and autonomic dysfunction. Among the multiple cellular mechanisms implicated in PD, the dysregulation of autophagy has gained significant attention in recent years. Autophagy is a crucial intracellular degradation pathway responsible for the removal of misfolded proteins and damaged organelles, processes that are particularly relevant in neurodegenerative diseases. Impairment of autophagic flux contributes to the accumulation of toxic protein aggregates and cellular stress in PD. Rapamycin, a compound originally isolated from Streptomyces hygroscopicus, is a well-established inhibitor of the mechanistic target of rapamycin (mTOR), a central regulator of autophagy. Preclinical studies have shown that rapamycin can stimulate autophagic pathways by suppressing mTOR signalling, leading to increased expression of autophagy markers. These effects have been associated with reduced neuronal damage, improved motor performance and decreased accumulation of pathological proteins in PD models. This review provides an overview of current preclinical research on rapamycin's neuroprotective potential in PD through autophagy enhancement. Although findings are promising, translating these outcomes into clinical practice necessitates a thorough understanding of rapamycin's pharmacodynamics, optimal dosing strategies, potential side effects and long-term safety. Further research is essential to establish its therapeutic viability in human populations.
    Keywords:  Rapamycin; autophagy; mTOR and Parkinson's disease.; neuroprotection
    DOI:  https://doi.org/10.2174/0118715273401017250918141227
  12. Nat Commun. 2025 Oct 17. 16(1): 9234
    Visualization of lysosomal membrane proteins by cryo electron tomography.
    Bridget M McVeigh, José J De Jesús-Pérez, Dirk H Siepe, Prerana Gogoi, Shrawan Kumar Mageswaran, Marian Kalocsay, Elaine M Mihelc, Vera Y Moiseenkova-Bell.
      Lysosomes are essential organelles for cellular homeostasis and signaling, with dysfunction linked to neurological disorders, lysosomal storage diseases, and cancer. While proteomics has advanced our understanding of lysosomal composition, the structural characterization of lysosomal membrane proteins in their native environment remains a significant challenge. Here, we developed a cryo electron tomography workflow to visualize lysosomal membrane proteins within intact, native lysosomal membranes. We isolated endolysosomes by independently targeting two lysosomal membrane proteins, transient receptor potential mucolipin 1 and transmembrane protein 192, enriching organelles that exhibited the expected morphology and proteomic composition of the endolysosomal system. Sub-tomogram averaging enabled the structural refinement of key membrane and membrane-associated proteins, including V-ATPase, Flotillin, and Clathrin, directly within the lysosomal membrane, revealing their heterogeneous distribution across endolysosomal organelles. By integrating proteomics with structural biology, our workflow establishes a powerful platform for studying lysosomal membrane protein function in health and disease, paving the way for future discoveries in membrane-associated lysosomal mechanisms.
    DOI:  https://doi.org/10.1038/s41467-025-64314-0
  13. EMBO Mol Med. 2025 Oct 15.
    Isoginkgetin antagonizes ALS pathologies in its animal and patient iPSC models via PINK1-Parkin-dependent mitophagy.
    Ang Li, Sen Huang, Shu-Qin Cao, Jinyi Lin, Linping Zhao, Feng Yu, Miaodan Huang, Lele Yang, Jiaqi Xin, Jing Wen, Lingli Yan, Ke Zhang, Maoyuan Jiang, Weidong Le, Peng Li, Yong U Liu, Dajiang Qin, Jiahong Lu, Guang Lu, Hanming Shen, Xiaoli Yao, Evandro F Fang, Huanxing Su.
      Damaged mitochondria initiate mitochondrial dysfunction-associated senescence, which is considered to be a critical cause for amyotrophic lateral sclerosis (ALS). Thus, mitophagic elimination of damaged mitochondria provides a promising strategy in ALS treatment. Here, through screening of a large natural compound library (n = 9555), we have identified isoginkgetin (ISO), a bioflavonoid from Ginkgo biloba, as a robust and specific mitophagy inducer. ISO enhances PINK1-Parkin-dependent mitophagy via stabilization of the PINK1/TOM complex. In a translational perspective, ISO antagonizes ALS pathology in C. elegans and mouse models; intriguingly, ISO improves mitochondrial function and antagonizes motor neuron pathologies in three ALS patient-derived induced pluripotent stem cell systems (C9, SOD1, and TDP-43), highlighting a potential broad application to ALS patients of different genetic background. At the molecular level, ISO inhibits ALS pathologies in a PINK1-Parkin-dependent manner, as depletion or inhibition of PINK1 or Parkin blunts its benefits. These results support the hypothesis that mitochondrial dysfunction is a driver of ALS pathology and that defective mitophagy is a druggable therapeutic target for ALS.
    Keywords:  Amyotrophic Lateral Sclerosis; Drug Screening; Isoginkgetin; Mitophagy; PINK1-Parkin
    DOI:  https://doi.org/10.1038/s44321-025-00323-2
  14. STAR Protoc. 2025 Oct 13. pii: S2666-1667(25)00548-9. [Epub ahead of print]6(4): 104142
    Protocol for purifying primitive neural progenitors by fractional passaging to generate astroglia-enriched cerebral organoid.
    Kushal Aluru, Rui Dang, Mengmeng Jin, Peng Jiang.
      Human pluripotent stem cell (hPSC)-derived primitive neural progenitor cells (pNPCs) and cerebral organoids provide a powerful platform for studying human brain development. Here, we present a fractional passaging protocol that yields nearly pure PAX6+/SOX2+ pNPCs. We describe steps for neuroepithelial differentiation, purification, and production of astroglia-enriched organoids. We then detail procedures for downstream analysis. The organoids contain over 15% astrocytes alongside maturing neurons, providing a scalable and reproducible system to model human glial-neuronal interactions in development and disease. For complete details on the use and execution of this protocol, please refer to Dang et al.1.
    Keywords:  Neuroscience; Organoids; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2025.104142
  15. Front Cell Neurosci. 2025 ;19 1650400
    The role of small GTPases in Alzheimer's disease tau pathologies.
    Peter Hoegy, Yan-Hua Chen, Qun Lu.
      Microtubule-associated protein (MAP) tau stabilizes neuronal microtubules in axonal transport and contributes to healthy synapses. In Alzheimer's disease (AD), tau proteins become hyperphosphorylated, reduce microtubule binding, and aggregate into paired helical filaments (PHFs) in neurofibrillary tangles (NFTs). Although the steps of this dysregulation of tau are well established, the mechanisms by which each step is regulated remain incompletely understood. Misfolded protein aggregates, such as amyloid β-peptides (Aβ), are degraded by autophagy and lysosomal pathways, in which small GTPases play essential roles. However, how tau aggregates and spreads from nerve cells and whether small GTPases similarly play pivotal roles are not as clear. Here we review the recent evidence to propose that small GTPases are important in tau protein posttranslational phosphorylation, aggregation, and clearance. As such, small GTPases may prove to be important therapeutic targets that can reduce the AD tau burden.
    Keywords:  Alzheimer’s disease; aggregation; clearance; hyperphosphorylation; microtubule-associated protein tau; neurofibrillary tangles; propagation; small GTPases
    DOI:  https://doi.org/10.3389/fncel.2025.1650400
  16. J Cell Biol. 2025 Dec 01. pii: e202503068. [Epub ahead of print]224(12):
    Coro1A and TRIM67 collaborate in netrin-dependent neuronal morphogenesis.
    Chris T Ho, Elliot B Evans, Kimberly Lukasik, Ellen C O'Shaughnessy, Aneri Shah, Chih-Hsuan Hsu, Brenda Temple, James E Bear, Stephanie L Gupton.
      Neuronal morphogenesis depends on extracellular guidance cues accurately instructing intracellular cytoskeletal remodeling. Here, we describe a novel role of the actin binding protein coronin 1A (Coro1A) in neuronal morphogenesis, where it mediates responses to the axon guidance cue netrin-1. We found that Coro1A localizes to growth cones and filopodial structures and is required for netrin-dependent axon turning, branching, and corpus callosum development. We previously discovered that Coro1A interacts with TRIM67, a brain-enriched E3 ubiquitin ligase that binds the netrin receptor DCC, and is also required for netrin-mediated neuronal morphogenesis. Loss of Coro1A and loss of TRIM67 shared similar phenotypes, suggesting that they may function together in the same netrin pathway. A Coro1A mutant deficient in binding TRIM67 was unable to rescue loss of Coro1A phenotypes, indicating that the interaction between Coro1A and TRIM67 is required for netrin responses. Together, our findings reveal that Coro1A is required for proper neuronal morphogenesis, where it collaborates with TRIM67 downstream of netrin.
    DOI:  https://doi.org/10.1083/jcb.202503068
  17. Methods Mol Biol. 2026 ;2976 1-10
    Determining Lysosomal Enzyme Activity Using Fluorogenic Probes.
    Etienne Sauvageau, Stephane Lefrancois.
      Lysosomes are responsible for a number of cellular functions, including the degradation of various biological molecules. Soluble enzymes within the lysosomal lumen are required to perform this function. Lysosomal activity can be disrupted in a variety of diseases, and measuring the activity of specific enzymes can be performed. In this chapter, we detail how lysosomal enzyme activity can be measured either in cell lysates or intact cells. This can be used to study fundamental cell biology or the effect of therapeutics targeting lysosomal function.
    Keywords:  Batten disease; Fluorogenic substrates; Lysosomal enzyme activity; Lysosomes
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_1
  18. J Clin Invest. 2025 Oct 15. pii: e187941. [Epub ahead of print]135(20):
    3D cultured human medium spiny neurons functionally integrate and rescue motor deficits in Huntington's disease mice.
    Yuting Mei, Yuan Xu, Xinyue Zhang, Ban Feng, Yingying Zhou, Qian Cheng, Yuan Li, Xingsheng Peng, Mengnan Wu, Lianshun Xie, Lei Xiao, Wenhao Zhou, Yuejun Chen, Man Xiong.
      Dysfunction of striatal medium spiny neurons (MSNs) is implicated in several neurological disorders, including Huntington's disease (HD). Despite progress in characterizing MSN pathology in HD, mechanisms underlying MSN susceptibility remain unknown, driving the need for MSNs derived from human pluripotent stem cells (hPSCs), especially subtypes in research and therapy. Here, we established a scalable 3D-default culture system to produce striatal MSNs efficiently from hPSCs by activation of the endogenous sonic hedgehog (SHH) pathway. These cells expressed canonical markers of striatal progenitors and dopamine D1 (D1) and dopamine D2 (D2) MSNs and presented dynamic specification and transcriptional signatures that closely resemble endogenous MSNs at single-cell resolution, both in vitro and post-transplantation in HD mice with quinolinic acid (QA) lesions. Grafted human cells survived and matured into D1-/D2-like MSNs and projected axons to endogenous targets including globus pallidus externus, globus pallidus internus, and substantia nigra pars reticulata to reconstruct the basal ganglia pathways. Functionally, they displayed spontaneous synaptic currents, received regulation from host cortex and thalamus, and were modulated by dopamine to either enhance or reduce neuronal excitability, similar to the endogenous D1-/D2-MSNs, subsequently improving behavior in QA-lesioned HD mice. Our study presents a method for generating authentic MSNs, providing a reliable cell source for HD cell therapy, mechanistic studies, and drug screening.
    Keywords:  Cell biology; Embryonic stem cells; Neurodegeneration; Stem cell transplantation; Stem cells; Transplantation
    DOI:  https://doi.org/10.1172/JCI187941
  19. Nat Cell Biol. 2025 Oct;27(10): 1688-1707
    Chaperone-mediated autophagy regulates neuronal activity by sex-specific remodelling of the synaptic proteome.
    Rabia R Khawaja, Ernesto Griego, Kristen Lindenau, Asma Salek, Jessica Gambardella, Aurora Scrivo, Hannah R Monday, Mathieu Bourdenx, Jesús Madero-Pérez, Zohaib N Khan, Bhakti Chavda, Ronald Cutler, Sarah Graff, Simone Sidoli, Gaetano Santulli, Laura Santambrogio, Inmaculada Tasset, Susmita Kaushik, Li Gan, Pablo E Castillo, Ana Maria Cuervo.
      Chaperone-mediated autophagy (CMA) declines in ageing and neurodegenerative diseases. Loss of CMA in neurons leads to neurodegeneration and behavioural changes in mice but the role of CMA in neuronal physiology is largely unknown. Here we show that CMA deficiency causes neuronal hyperactivity, increased seizure susceptibility and disrupted calcium homeostasis. Pre-synaptic neurotransmitter release and NMDA receptor-mediated transmission were enhanced in CMA-deficient females, whereas males exhibited elevated post-synaptic AMPA-receptor activity. Comparative quantitative proteomics revealed sexual dimorphism in the synaptic proteins degraded by CMA, with preferential remodelling of the pre-synaptic proteome in females and the post-synaptic proteome in males. We demonstrate that genetic or pharmacological CMA activation in old mice and an Alzheimer's disease mouse model restores synaptic protein levels, reduces neuronal hyperexcitability and seizure susceptibility, and normalizes neurotransmission. Our findings unveil a role for CMA in regulating neuronal excitability and highlight this pathway as a potential target for mitigating age-related neuronal decline.
    DOI:  https://doi.org/10.1038/s41556-025-01771-1
  20. Methods Mol Biol. 2026 ;2976 85-102
    Lysosomal Immunoprecipitation (LysoIP) from Cells and Tissues for Metabolomic and Proteomic Analyses.
    Nouf N Laqtom, Monther Abu-Remaileh.
      Lysosomes, known for degrading biomolecules and damaged cellular components, are now recognized as signaling hubs for nutrient sensing and metabolic adaptation, and their dysfunction is implicated in diseases including cancer and neurodegeneration. To understand the composition of the lysosome, the dynamic behavior of its contents, and its specific roles in health and disease, we describe a lysosomal immunoprecipitation method, termed "LysoIP," that enables the isolation of intact lysosomes from cultured cells and mouse tissues. This method utilizes a lysosome-localized 3xHA epitope tag (LysoTag) and a simple, yet robust organelle immunoprecipitation workflow. Isolated lysosomes are extracted with optimized buffers to allow the efficient retrieval of lysosomal proteins, polar metabolites, and lipids, maintaining compatibility with downstream liquid chromatography and mass spectrometry (LC-MS) analyses.
    Keywords:  LC-MS analyses; LysoIP; LysoTag; LysoTag mouse; Lysosomes; Metabolomics; Proteomics; TMEM192
    DOI:  https://doi.org/10.1007/978-1-0716-4844-5_8
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