bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2026–04–26
33 papers selected by
TJ Krzystek



  1. PLoS One. 2026 ;21(4): e0345890
      Sequestosome 1/p62 (hereafter referred to as p62) is a multifunctional protein that orchestrates various cellular stress response pathways including autophagy, proteasome-mediated degradation, antioxidant defense, nutrient sensing, and inflammatory signaling. Mutations in distinct functional domains of p62 are linked with the neurodegenerative disease amyotrophic lateral sclerosis (ALS), underscoring its importance in neural cells. Neurons and astrocytes, two key cell types in the brain, perform distinct roles in brain physiology and thus encounter a unique landscape of cellular stress. However, how p62 is regulated in these cell types in response to various stress modalities remains largely unexplored. Several functions for p62 depend on its engagement with ubiquitinated substrates. Thus, we investigated how the regulation of p62-ubiquitin conjugates differs between neurons and astrocytes exposed to two stress modalities: lysosomal membrane damage and metabolic stress. Lysosomal damage triggered ubiquitin-dependent assembly of p62 puncta in both neurons and astrocytes. In contrast, nutrient deprivation elicited different responses between neurons and astrocytes. Neurons formed p62-ubiquitin structures more prominently and displayed a greater dependence on ubiquitin for p62 clustering. Together, these findings reveal cell-type-specific and stress-specific regulation of p62-ubiquitin conjugates, indicating that neurons and astrocytes can deploy distinct quality control strategies.
    DOI:  https://doi.org/10.1371/journal.pone.0345890
  2. Front Med (Lausanne). 2026 ;13 1792336
      Amyotrophic Lateral Sclerosis (ALS) is a rapidly progressive neurodegenerative disease characterized by the selective loss of upper and lower motor neurons, leading to muscle weakness, paralysis, and ultimately respiratory failure. The multifactorial etiology of ALS, encompassing genetic mutations, protein aggregation, oxidative stress, excitotoxicity, and dysregulated RNA metabolism, has hindered the development of effective therapies. Traditional animal and 2D cell models have provided important mechanistic insights but often fail to fully capture the human-specific and multicellular aspects of disease pathophysiology. Recent advances in induced pluripotent stem cell (iPSC)-derived organoids offer a promising human-based platform for ALS research, enabling the generation of disease-relevant neural and neuromuscular subtypes in three-dimensional architectures. These models recapitulate key pathological features, including protein mis-localization, neuromuscular junction defects, synaptic impairments, and glial contributions to motor neuron degeneration, while also serving as platforms for drug screening and mechanistic studies. Importantly, spinal and neuromuscular organoids bridge the gap between simplified in vitro systems and the complex human nervous system, providing a unique framework to study ALS pathogenesis. This review provides a comprehensive overview of the various differentiation protocols, experimental strategies and key results obtained to date, with a primary focus on validating and benchmarking organoid models, while also highlighting their limitations, emerging clinical applications, translational potential, and opportunities for personalized therapeutic discovery.
    Keywords:  ALS; disease modeling; neuromuscular organoids; spinal organoids; tridimensional cell cultures
    DOI:  https://doi.org/10.3389/fmed.2026.1792336
  3. J Neurosci. 2026 Apr 22. pii: e0917252026. [Epub ahead of print]46(16):
      
    Keywords:  Parkinson's disease; autophagy; lysosomes; neurodegeneration; synapses
    DOI:  https://doi.org/10.1523/JNEUROSCI.0917-25.2026
  4. Annu Rev Genomics Hum Genet. 2026 Apr 21.
      TDP-43 is an RNA-binding protein that regulates multiple aspects of RNA processing, and its mislocalization from the nucleus to the cytoplasm is a defining feature of amyotrophic lateral sclerosis (ALS). While both loss- and gain-of-function mechanisms contribute to disease, the discovery of cryptic splicing has shed light on the downstream consequences of TDP-43 nuclear clearance for neuronal health. Here, we highlight how loss of nuclear TDP-43 can drive a cascade of events that lead to the impairment of cellular proteostasis and result in a positive feedback loop that perpetuates neuronal dysfunction. This sustains the appearance of cryptic splicing events in genes that are involved in key pathways for the maintenance of axonal homeostasis and synaptic transmission. In contrast to their detrimental effects on neuronal health, cryptic splicing mechanisms may be harnessed to develop novel therapeutic strategies, unprecedentedly expanding the availability of therapeutic avenues for TDP-43 proteinopathies.
    DOI:  https://doi.org/10.1146/annurev-genom-022024-011307
  5. PNAS Nexus. 2026 Apr;5(4): pgag112
      Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common cause of familial Parkinson's disease, yet the molecular mechanism governing LRRK2 activation remains incompletely understood. LRRK2 is a large multidomain enzyme whose kinase activity is regulated by intramolecular interactions and by its Ras of complex proteins (ROC) GTPase domain. Here, we combine cryo-electron microscopy, X-ray crystallography, and structure-guided biochemical perturbations to define how ROC conformational switching regulates LRRK2 activation. Cryo-EM reconstructions reveal that monomeric full-length LRRK2 samples three distinct conformational states-autoinhibited, intermediate, and activated-indicating that large-scale activation-associated rearrangements can occur through an intrinsic intramolecular pathway, independently of Rab29 binding, higher-order oligomerization, or membrane association. A 1.6-Å crystal structure of an extended ROC construct reveals intrinsic conformational plasticity within the GTPase switch regions that likely underlies these transitions. Structure-guided disulfide engineering identifies a functional coupling between residue R1441 and switch II that directly modulates GTPase activity in both isolated ROC and full-length LRRK2. Disruption of this coupling phenocopies the disease-associated R1441H mutation. Together, these findings establish ROC as a dynamic conformational engine that drives a multistep intramolecular activation mechanism in LRRK2, providing mechanistic insight into how pathogenic mutations promote aberrant kinase activation.
    Keywords:  GTPase; LRRK2; Parkinson's disease; cryo-EM; kinase
    DOI:  https://doi.org/10.1093/pnasnexus/pgag112
  6. Amyotroph Lateral Scler Frontotemporal Degener. 2026 Apr 25. 1-10
      Amyotrophic lateral sclerosis (ALS) is marked by substantial clinical heterogeneity. This heterogeneity has impacted clinical trials by obscuring treatment effects and causing inefficiency. In this review, we summarize potential approaches for addressing heterogeneity in ALS via patient stratification and cohort enrichment methods and highlight potential challenges and limitations. These categories include stratification based on genetics, clinical characteristics (e.g. pattern of weakness, ALS Functional Rating Scale rates of progression), wet biomarkers (e.g. neurofilament light chain), neuroimaging, and novel methods employing statistical modeling or machine learning. These stratification methods have yet to be fully leveraged in clinical trial design. But these strategies must be employed thoughtfully and judiciously due to potential issues stratification can introduce. Future clinical trials should explore how participant stratification and cohort enrichment strategies may improve our ability to identify treatment effects, which may ultimately aid in the quest to establish more personalized medicine for persons with ALS.
    Keywords:  Amyotrophic lateral sclerosis; clinical research; clinical trials; motor neuron disease; pharmacotherapy
    DOI:  https://doi.org/10.1080/21678421.2026.2659128
  7. NPJ Parkinsons Dis. 2026 Apr 24.
      RAB12 is a small GTPase and a validated substrate of LRRK2, a kinase genetically linked to Parkinson's disease (PD). While RAB12-LRRK2 signaling has been implicated in ciliogenesis and immune regulation, the neuronal function of RAB12 remains largely unexplored. Here, we investigated the role of RAB12 in synaptic physiology using Rab12 knockout (KO) mice. Rab12 KO mice developed normally but exhibited increased locomotor activity in adulthood. Electrophysiological recordings from striatal slices revealed enhanced presynaptic release probability and increased excitatory drive onto medium spiny neurons. Consistently, live-cell imaging of cultured cortical neurons revealed that Rab12 deletion facilitated, while Rab12 overexpression inhibited, synaptic vesicle exocytosis. Biochemical fractionation showed enrichment of RAB12 in synaptic vesicle-associated fractions containing presynaptic components. Proteomic analysis of Rab12 KO striatal synaptosomes further identified alterations in proteins involved in synaptic membrane trafficking pathways. Together, these findings establish RAB12 as a negative regulator of synaptic vesicle exocytosis and excitatory neurotransmission in vivo. Our study defines a physiological role for RAB12 in synaptic function and provides a basis for future investigation into how LRRK2-dependent RAB12 signaling may contribute to neuronal dysfunction in PD.
    DOI:  https://doi.org/10.1038/s41531-026-01353-4
  8. J Cell Biol. 2026 May 04. pii: e202604009. [Epub ahead of print]225(5):
      Several mechanisms repair damaged lysosomal membranes, but how can lysosomes prevent membrane failure in the first place? Kim et al. (https://doi.org/10.1083/jcb.202509180) uncover a rapid response whereby TMEM63A-dependent ion efflux relieves membrane tension, buying time for slower repair mechanisms to engage.
    DOI:  https://doi.org/10.1083/jcb.202604009
  9. iScience. 2026 May 15. 29(5): 115530
      Localized protein translation enables spatially restricted cellular dynamics, particularly in neurons, where specific mRNAs are translated in axons and dendrites far from the cell body. Translating ribosome affinity purification (TRAP) has been used to study axonal translation in rodents and cell-type-specific translation in Drosophila, but existing protocols are not optimized for axons, where material is extremely limited. Here, we present a highly sensitive TRAP protocol for isolating ribosome-bound mRNAs from low-input samples, enabling recovery of axonal mRNAs from Drosophila larval and adult (leg) motor neurons. RNA-seq identified axonally translated transcripts, including mRNAs encoding ribosomal and mitochondrial proteins, similar to those reported in axons of other species, indicating conservation of axonal translation in Drosophila. This low-input method enables analysis of local translation with Drosophila genetics across developmental stages, genetic backgrounds, and disease models, and can be adapted for rare genotypes, other tissues and model systems requiring high sensitivity.
    Keywords:  genetics; molecular biology; neuroscience
    DOI:  https://doi.org/10.1016/j.isci.2026.115530
  10. iScience. 2026 Apr 17. 29(4): 115479
      Recurrent hydatidiform moles (RHMs) are a gestational disorder primarily caused by maternal-effect loss-of-function mutations in NLRP7. This study established an in vitro model of NLRP7 mutation using patient-derived induced pluripotent stem cells (iPSCs) and integrated high-content imaging (HCI) with artificial intelligence (AI) to dissect mutation-induced multi-organellar dysfunction. Multiparametric HCI enabled synchronous capture of cellular and subcellular phenotypes, including mitochondrial function and lysosomal distribution. We developed a bio-inspired AI framework (BioVision-Segmentation) that combines a hybrid Transformer-Voronoi architecture for segmentation with mutual information analysis to quantify organelle interactions. Our findings reveal that NLRP7 mutations disrupt the lysosome-mitochondria crosstalk hub, leading to energy metabolism dysregulation, reactive oxygen species (ROS) accumulation, and organelle spatial distribution defects. Furthermore, transcriptome sequencing corroborated these findings. This study elucidates the organellar pathogenesis of RHMs and provides a technological platform for exploring therapeutic targets, facilitating a shift toward early embryo protection strategies.
    Keywords:  Biological sciences; Machine learning; Methodology in biological sciences
    DOI:  https://doi.org/10.1016/j.isci.2026.115479
  11. iScience. 2026 May 15. 29(5): 115529
      Human induced pluripotent stem cell (iPSC)-derived neurons provide a platform for modeling brain disorders. Among disease-relevant cellular phenotypes, impaired neurite outgrowth has emerged as an indicator reflecting key aspects of neurological disease pathophysiology. We conducted a high-throughput phenotypic screening of over 21,000 small molecules to identify compounds that enhance neurite outgrowth in iPSC-derived neurons, and we identified three bioactive compounds sharing a common indazole scaffold. Notably, one of these compounds selectively targets TNIK, a kinase involved in neuronal development. Scaffold expansion led to the discovery of thienopyridone derivatives with potent neurite-promoting activity. Two thienopyridone compounds were further validated in a human neural organoid model, in which their neurite outgrowth-promoting effects were reproducibly confirmed. Transcriptomic profiling revealed activation of signaling pathways associated with neurotrophic stimulation. These findings identify thienopyridones as a scaffold for neuritogenic small molecules, suggesting their potential as a therapeutic strategy for brain disorders and for promoting neural regeneration.
    Keywords:  biological sciences; neuroscience; pharmacology; stem cells research
    DOI:  https://doi.org/10.1016/j.isci.2026.115529
  12. Biochim Biophys Acta Mol Basis Dis. 2026 Apr 22. pii: S0925-4439(26)00136-5. [Epub ahead of print] 168273
      Ceroid lipofuscinosis neuronal 5 (CLN5) disease is a subtype of neuronal ceroid lipofuscinosis (NCL, commonly known as Batten disease) that is caused by mutations in the CLN5 gene. While 70 distinct CLN5 disease-causing mutations have been documented, the pathological effects of these mutations are largely unknown. In this study, we used the model eukaryote Dictyostelium discoideum to examine the molecular and cellular effects of five CLN5 disease-causing mutations (p.Cys77Tyr, p.Trp158Ser, p.Tyr209Asp, p.Glu303*, and p.Tyr343*). We used informatics tools to show that the five mutations alter the predicted structure of the protein. We then introduced these mutations into Dictyostelium Cln5 and examined their effects on the localization and secretion of the protein, as well as proteostasis and lysosomal activity. We observed that the mutations alter the cellular distribution of Cln5 and intracellular catabolic mechanisms, including 20S proteasome-mediated protein degradation and lysosomal enzyme-mediated breakdown. The mutations also affect vesicles within the endo-lysosome pathway and the release of Cln5 and other lysosomal enzymes from cells, which impacts extracellular enzyme activity. Finally, while cell proliferation and aggregation were not affected by mutated Cln5, loss of the signal peptide in Cln5 delayed aggregation, suggesting an extracellular role for the protein. This study, which is the first to comprehensively examine the effects of the p.Cys77Tyr, p.Trp158Ser, p.Tyr209Asp, p.Glu303*, and p.Tyr343* mutations on cellular function, enhances our understanding of the effects of mutations in CLN5 on endo-lysosomal trafficking and lysosomal biology, as well as the pathological mechanisms underlying CLN5 disease.
    Keywords:  CLN5; Catabolism; Dictyostelium; Enzyme activity; Lysosomes; Mutation; Secretion
    DOI:  https://doi.org/10.1016/j.bbadis.2026.168273
  13. FEBS Lett. 2026 Apr 24.
      Lysosomes play a critical role in the quality control of cellular organelles. However, lysosomal membranes can be damaged under a variety of conditions, leading to the onset of various diseases. Damaged lysosomes are selectively cleared via a ubiquitin-dependent mechanism, but the molecular mechanisms underlying this process have not been adequately elucidated. In this study, we found that RNF115 is a lysosomal damage-responsive ubiquitin ligase that undergoes massive translocation from the cytosol to the p62/SQSTM1-positive puncta around ruptured lysosomes. In accordance with the changes in its distribution, the depletion of RNF115 delayed the removal of Gal3 from damaged lysosomes during the restoration process following lysosomal damage. These observations suggest that RNF115 is responsible for the clearance of damaged lysosomes.
    Keywords:  BAG6; E3 ubiquitin ligase; RNF115; autophagy; lysophagy; lysosomal membrane damage; lysosome
    DOI:  https://doi.org/10.1002/1873-3468.70346
  14. Cell Rep Med. 2026 Apr 20. pii: S2666-3791(26)00175-8. [Epub ahead of print] 102758
      Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive loss of motor neurons. Insufficiency of neurotrophic factors is suspected to underlie the disease, but direct evidence remains scarce. In this study, we discover that brain-derived neurotrophic factor (BDNF) val/met mutation, which results in a decrease in BDNF secretion, reduces survival time of ALS patients in two separate cohorts. Using a knockin mouse model of the ALS causal gene FUSR521C, we demonstrate that BDNF haploinsufficiency leads to shortened lifespan, accelerated motor dysfunctions, and exacerbated motor neuron death. Importantly, activation of the BDNF receptor TrkB by an agonistic antibody effectively rescues these ALS-associated phenotypes. In additional ALS mouse models, TrkB activation antibody also shows superior therapeutic effects compared to current ALS medication riluzole. Our data indicate that insufficient BDNF could be a crucial contributing factor for ALS progression, and activation of BDNF-TrkB pathway may represent a promising therapeutic strategy against ALS.
    Keywords:  ALS, BDNF, V66M, FUS, TrkB antibody, B90-1
    DOI:  https://doi.org/10.1016/j.xcrm.2026.102758
  15. Life Sci Alliance. 2026 Jul;pii: e202503551. [Epub ahead of print]9(7):
      The clinical and genetic diversity of Parkinson's disease (PD) makes it challenging to identify common mechanisms across different forms. To search for such shared pathways, we performed transcriptomic analysis of iPSC-derived dopaminergic neurons from patients with LRRK2 or Parkin mutations. We discovered a convergent gene expression signature in both genetic backgrounds, indicating a shift away from developmental and proliferative programs (e.g., Wnt/β-catenin signaling, cell cycle) and toward pathways of mature neuronal function (e.g., synaptic transmission, potassium channels). This shift was particularly pronounced in LRRK2 neurons, which showed enhanced markers of synaptic maturation. Concurrently, PD neurons exhibited down-regulation of gene programs supporting axon growth and structural development and upregulated TRAIL (TNF-related apoptosis-inducing ligand) apoptotic pathway. Our findings suggest that in these hereditary forms of PD, distinct mutations may propel neurons toward a similar state of premature specialization coupled with impaired structural development and increased vulnerability, revealing a potential common path in disease pathogenesis.
    DOI:  https://doi.org/10.26508/lsa.202503551
  16. Mol Neurobiol. 2026 Apr 21. pii: 573. [Epub ahead of print]63(1):
      Beta-hydroxybutyrate (D-BHB), a ketone body with neuroprotective properties, has been previously shown to increase the autophagic flux in neurons, but its effect on autophagy induction in astrocytes remains unexplored. Astrocyte functionality is essential for neuronal support, and this includes an optimal degradation and recycling of organelles and other cellular components through autophagy. The present study shows that D-BHB exposure to cultured astrocytes elevates the conjugated form of microtubule-associated protein 1A/1B-light chain 3 (LC3-II) levels, increases the number of autophagosomes, and reduces sequestosome-1 (SQSTM1/p62) protein content. D-BHB also enhanced the phosphorylation of AMP-activated kinase (AMPK) and Unc-51-like autophagy activating kinase 1 (ULK1), suggesting increased autophagy initiation. In addition, D-BHB induced the activation of the transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, increasing the abundance of the lysosomal-associated membrane protein 1 (LAMP1) and lysosomal number. Pharmacological inhibition of Sirtuin 1 (SIRT1) and AMPK/ULK1 activity abated D-BHB-induced increase in LC3-II and SQSTM1/p62 degradation, suggesting that D-BHB-mediated autophagy activation is dependent on the SIRT1/AMPK/ULK1 pathway. Also, results show that D-BHB induction of the autophagy-lysosomal axis improves astrocyte survival under oxygen-glucose deprivation (OGD). Together, the present data suggest that astrocytes, acting as targets of D-BHB, might contribute to the neuroprotective effects of ketone bodies against acute brain injury.
    Keywords:  AMP kinase; Ketone bodies; Lysosome biogenesis; mTORC1
    DOI:  https://doi.org/10.1007/s12035-026-05852-6
  17. Proc Natl Acad Sci U S A. 2026 Apr 28. 123(17): e2536562123
      Nerve growth factor (NGF) is a member of the neurotrophin family, essential for neuronal survival and phenotypic maintenance. However, in vitro, its function can be disrupted by oxidative posttranslational modifications such as tyrosine nitration. Nitrated NGF (NO2NGF) has been shown to have a gain-of-toxic, pro-apoptotic, activity in motoneuron cultures. Herein, we report the generation and characterization of monoclonal antibodies (mAbs) that specifically recognize NO2NGF to unravel its formation in vivo. Using hybridoma technology, we produced mAbs with high affinity and selectivity for NO2NGF, as demonstrated immunochemically and by surface plasmon resonance. The antibodies elicit neutralizing capacity to NO2NGF in neuronal cells. Nitrated Tyr52 within the NGF48-58 sequence was identified as the primary antigenic determinant by crystallographic analysis of antibody:peptide complexes at atomic resolution, peptide-based epitope mapping and molecular dynamics simulations, whereas local sequence NGF motifs around the nitrated tyrosine are important for protein specificity. The antibodies revealed NO2NGF accumulation in amyotrophic lateral sclerosis (ALS) rodent models and human subjects. Indeed, immunofluorescence showed selective accumulation of NO2NGF in spinal cord regions undergoing motor neuron degeneration, as well as in sciatic nerves and neuromuscular junctions. Our findings establish NGF nitration as an oxidative hallmark in ALS and demonstrate that monoclonal antibodies targeting this chemical modification are powerful tools for both mechanistic studies and biomarkers development. This work proposes a link between neurotrophin nitration and neurodegenerative disease progression and opens avenues for therapeutic exploration along the peroxynitrite-tyrosine nitration pathway.
    Keywords:  ALS; crystallography; monoclonal antibodies; nerve growth factor; tyrosine nitration
    DOI:  https://doi.org/10.1073/pnas.2536562123
  18. Mol Ther Nucleic Acids. 2026 Jun 16. 37(2): 102922
      Central nervous system (CNS) projection neurons' failure to repair or regenerate injured axons has devastating consequences for those who have sustained CNS injuries. Thus, there is a need for translatable factors capable of promoting long-distance axon regeneration in the CNS. We hypothesized that supporting lysosomes in injured neurons by supplementing their structural factors through gene therapy may foster axon regeneration. To test our hypothesis, we selected Atp6v0c for experimental regulation because it plays roles in lysosomal acidification and the degradation of misfolded proteins in response to endoplasmic reticulum (ER) stress in injured neurons. We tested this in a rodent optic nerve crush (ONC) model of traumatic optic neuropathy (TON), in which injured prototypical CNS projection neurons, the retinal ganglion cells (RGCs), do not regenerate damaged axons and eventually degenerate. Atp6v0c transgene expression was achieved using intravitreally injected adeno-associated virus serotype 2 (AAV2), which transduces the RGCs. For benchmarking, we compared efficacy to AAV2 targeting of prominent regulators of axon regeneration, Pten, and Klf9. We found that Atp6v0c transgene promoted RGC survival and long-distance axon regeneration, comparable to targeting Pten and Klf9. Thus, Atp6v0c is an axon regeneration-promoting factor with potential for treating CNS injury and disease.
    Keywords:  MT: Oligonucleotides: Therapies and Applications; axon regeneration; gene therapy; optic nerve injury; retinal ganglion cell
    DOI:  https://doi.org/10.1016/j.omtn.2026.102922
  19. Biofabrication. 2026 Apr 24.
      Engineered three-dimensional (3D) neural constructs hold significant promise for repairing neural tissue damage and recapitulating the human brain in vitro for disease modeling and drug screening applications. However, most current 3D neural models, including freestanding organoids and dense bioprinted neural constructs, lack the architectural and functional organization required to emulate the cerebral cortex, which comprises grey matter regions rich in neuronal cell bodies and white matter tracts formed by long-range axonal projections. This architectural mismatch limits the models' ability to support functional connectivity analysis, predict in vivo behavior, and achieve effective integration with host tissue. In this study, we present an extruded droplet-on-demand (X-DoD) bioprinting technique that enables deterministic spatial patterning of droplets containing human induced pluripotent stem cells (iPSCs) encapsulated within an extracellular matrix (ECM)-based hydrogel and embedded in a permissive, low-concentration hydrogel bulk that supports diffusion. Using a 5×5 droplet array pattern, we demonstrate that after 30 days of differentiation into cortical neurons and formation of 3D neuronal networks, the micron-scale, cell-body-dense droplets (microtissues) remain localized at their initial droplet sites and are interconnected by millimeter-scale neurite projections. This defined grey-white matter-like organization enables functional analysis via calcium imaging and seamless integration with custom electronic devices for advanced neurophysiological interrogation. Calcium imaging and electrical recordings revealed temporally preserved, propagating network activity, with network excitability dynamically modulated by treatment with GABA antagonist bicuculline. Altogether, the X-DoD bioprinting platform offers a powerful and adaptable approach for engineering spatially organized 3D neural networks with tunable connectivity, providing a robust tool for studying brain function, disease modeling, and future therapeutic applications.
    Keywords:  3D bioprinting; 3D neural network; ECM hydrogel; Extruded droplet-on-demand; Functional connectivity; cortical neuron; iPSC
    DOI:  https://doi.org/10.1088/1758-5090/ae647c
  20. PLoS One. 2026 ;21(4): e0346157
      While the uptake of cargos via endocytosis and the subsequent trafficking through the cell is crucial for normal cellular function and tightly regulated, the study of this bears challenges. Most studies of Rab GTPases, the primary coordinators of endocytic progression, rely on ectopic expression of fluorescently tagged proteins via transient transfection. Previous studies already showed that the design of the fluorescent tag as well as the unpredictable nature of transient transfection can cause problems. Even though the pitfalls of overexpression have been reported for several research fields, the consequences of overexpression on endocytic trafficking are under-reported. To highlight the importance of working with endogenous levels of proteins to draw conclusions about endosome colocalization and identity, we present an example where the colocalization of two endosomal regulators/markers, Rab11 and LAMP1, varied drastically when these proteins were analyzed at their endogenous levels or following ectopic expression. When both proteins were ectopically expressed, up to 90% colocalization was observed. However, when analyzed at the endogenous level no colocalization was detectable. This study shows how important vesicular trafficking perturbation can occur following ectopic expression of endosomal proteins.
    DOI:  https://doi.org/10.1371/journal.pone.0346157
  21. J Vis Exp. 2026 Apr 03.
      Extracellular vesicles (EVs), particularly small extracellular vesicles (sEVs) defined as vesicles smaller than 200 nm, serve as essential mediators of intercellular communication and released by nearly all cell types into various biological fluids. The infrapatellar fat pad (IPFP) is closely associated with the development and progression of knee osteoarthritis (OA). However, standardized methods for isolating sEVs directly from IPFP explants remain limited. We present a reproducible protocol for the isolation and characterization of sEVs derived from IPFP tissues obtained from OA patients. Conditioned medium collected from IPFP tissue cultures is sequentially cleared of cells and debris by low-speed centrifugation, followed by ultracentrifugation to enrich vesicles within the sEV size range. The resulting vesicles are characterized according to minimal information for studies of extracellular vesicles (MISEV2023) recommendations using a set of representative protein markers across distinct functional categories: CD63 as a membrane-associated tetraspanin, ALIX and TSG101 as cytosolic proteins involved in biogenesis mediated by the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, and Calnexin as an endoplasmic reticulum-resident protein to monitor potential non-vesicular contamination. This workflow yields well-defined sEV preparations suitable for downstream applications such as functional assays or proteomic profiling in osteoarthritis research.
    DOI:  https://doi.org/10.3791/70518
  22. Nucleic Acids Res. 2026 Apr 13. pii: gkag354. [Epub ahead of print]54(7):
      Inhibition of messenger RNA translation is a common feature in proteostatic stress cellular responses. Puromycin, a widely used compound for studying translation, disrupts protein synthesis by mimicking the 3' end of aminoacyl-transfer RNAs. Despite its extensive use as a research tool to probe the connection between translation activity and various physiological and pathological states, the cellular response associated with puromycin-induced translation stress remains incompletely understood. Here, we used electron tomography and topology analysis to define the effects of puromycin on the translation machinery in situ. We show that puromycin-treated neuronal cells exhibit an accumulation of eIF5A-bound ribosomes in a translationally inactive "idle" state, and thereby defining a broader role of eIF5A in ribosome homeostasis. Additionally, the idle ribosomes formed dimeric complexes mediated by ribosomal RNA expansion segments, suggesting an evolved mechanism involving these regions in translational hibernating and protecting idle ribosomes. We further show that the hibernating disome formation is not unique to puromycin administration but represents a conserved mechanism as a response to different cellular stressors including endoplasmic reticulum stress and amino acid depletion. Collectively, our findings illuminate distinct states of mammalian ribosome hibernation and dimerization, providing new insights into the relationship of cellular stress and the dynamic regulation of ribosomal activity.
    DOI:  https://doi.org/10.1093/nar/gkag354
  23. Front Endocrinol (Lausanne). 2026 ;17 1794600
      Lysosomes, as organelles with degradative, secretory and signaling functions in eukaryotic cells, play a pivotal role in maintaining cellular energy homeostasis and biological recycling processes. In recent years, lysosomal dysfunction has garnered extensive attention from scholars for its implications in neurodegenerative and autoimmune diseases. however, its role in the occurrence and progression of diabetes mellitus and its complications remains to be further explored. Therefore, this article summarizes the research progress on lysosomal dysfunction in diabetes and its complications, hoping to highlight a promising therapeutic direction.
    Keywords:  autophagy; diabetes; diabetic complications; lysosomal; lysosomal dysfunction
    DOI:  https://doi.org/10.3389/fendo.2026.1794600
  24. J Huntingtons Dis. 2026 Apr 20. 18796397261443137
      Somatic instability (SI) of the CAG tract in HTT is a major driver of neurodegeneration of Spiny Projection Neurons (SPNs), the primary neuronal subtype affected in Huntington's disease (HD). SPNs can accumulate hundreds of CAG repeats during a patient's lifetime, and once the expansion exceeds ∼150 CAGs, they acquire distinct, cell-autonomous transcriptional alterations that ultimately contribute to degeneration. Here, we developed the "HD-Phase-Model", a mathematical framework designed to identify "super-expanded" SPNs without repeat sizing, by leveraging the only available single-nucleus HD post-mortem dataset that provides both transcriptional profile and matched HTT CAG sizes. After validating model performance on the test data, we applied it to independent single-nucleus datasets lacking CAG sizing information and across multiple brain regions. In all cases, the model consistently detected SPNs populations with convergent transcriptional dysregulation signatures indicative of extreme CAG expansion.Importantly, although the model was trained on caudate SPNs, we observed highly similar dysregulation patterns in putamen and accumbens, while no evidence of super-expansion was found in SPNs from Alzheimer's and Parkinson's disease donors.Together, these findings demonstrate that transcriptional profiles alone can serve as predictors of HTT CAG size, enabling systematic identification of super-expanded SPNs and providing insights into HD-specific neurodegenerative mechanisms.
    Keywords:  cell-autonomous transcriptional dysregulation; mathematical modelling; single-nucleus transcriptome and matched HTT CAG size; somatic instability; spiny projection neurons
    DOI:  https://doi.org/10.1177/18796397261443137
  25. BMC Biol. 2026 Apr 21.
      Phase separation has emerged as a key mechanism of cellular compartmentalization, complementing classical membrane-bound organelles. In addition to forming membraneless compartments that provide confined environments for biochemical reactions, phase separation actively organizes and regulates membrane vesicles. Protein condensates interact with vesicular organelles through defined modes of communication, influencing their assembly, trafficking, and turnover. This review highlights recent advances in understanding how phase separation contributes to vesicle biology, including synaptic vesicle cycling, COPII-mediated transport, and endocytic vesicle organization, processes essential for maintaining physiological homeostasis and efficient intracellular transport.
    Keywords:  Membraneless condensates; Membranous organelles; Phase separation; Vesicle trafficking
    DOI:  https://doi.org/10.1186/s12915-026-02607-6
  26. Epilepsy Curr. 2026 Apr 20. 15357597261443067
      Epilepsy has traditionally been viewed as a disorder involving neuronal hyperexcitability and brain network dysfunction. However, growing evidence indicates that recurrent seizures are associated with widespread disturbances in brain homeostasis, including metabolic stress, neuroinflammation, vascular dysregulation, and sleep disruption. These processes extend beyond neurons and involve brain-wide clearance mechanisms that have received limited attention in epilepsy research. The glymphatic system is a specialized pathway that facilitates cerebrospinal fluid-interstitial fluid exchange and promotes the clearance of metabolic waste and neurotoxic solutes from the brain. Glymphatic transport depends on astrocytic aquaporin-4 channels and is strongly modulated by sleep-wake state, which is highly relevant to epilepsy given the close bidirectional relationship between seizures and sleep disturbances. Impaired glymphatic clearance has been linked to protein accumulation, neuroinflammation, and cognitive decline during aging and in neurodegenerative diseases, suggesting that similar mechanisms may contribute to epilepsy-related disease progression. In this review, we summarize current knowledge of glymphatic anatomy and physiology, focusing on advances in neuroimaging. We then synthesize emerging evidence demonstrating glymphatic dysfunction across multiple epilepsy syndromes. We discuss the clinical implications of impaired cerebral waste clearance for disease burden, treatment outcomes, and cognitive dysfunction and highlight potential therapeutic strategies aimed at modulating glymphatic function. Finally, we address the ongoing debates regarding glymphatic mechanisms, imaging biomarkers, and causal relationships in epilepsy. Collectively, the available data suggest that glymphatic system dysfunction represents a system-level abnormality in epilepsy, offering a complementary framework that integrates the metabolic, vascular, and sleep-related aspects of epileptic brain dysfunction.
    Keywords:  cognitive dysfunction; epilepsy; glymphatic system; neuroimaging
    DOI:  https://doi.org/10.1177/15357597261443067
  27. J Cell Biol. 2026 May 04. pii: e202603182. [Epub ahead of print]225(5):
      Bridge-like lipid-transport protein 2 (BLTP2) transfers lipids at membrane contact sites, but its precise localization is unclear. Dziurdzik et al. and Dai et al. identify and characterize a conserved contact site between the endoplasmic reticulum and the plasma membrane mediated by BLTP2.
    DOI:  https://doi.org/10.1083/jcb.202603182
  28. Methods Mol Biol. 2026 ;3023 149-164
      Stem cell-derived organoids research provides an exciting model and technology to develop new therapies for difficult and intractable diseases. Human-induced pluripotent stem cells (hiPSCs) have the potential to differentiate into organ-like three-dimensional structures, especially when it is challenging to obtain patient samples for this purpose. This chapter outlines a comprehensive differentiation protocol for the generation and manipulation of retinal organoids derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and hiPSCs. The method involves culturing PSCs, differentiating them into retinal organoids, and in vitro transduction of organoids with adeno-associated virus (AAV) vectors. AAVs can be used to label a desired population, to test novel promoters, and to test gene therapies. We will also describe key steps following organoid transduction, which entail collection, immunostaining, imaging and preservation for molecular biology readouts. The protocol provides a detailed guide for researchers working in the field of retinal biology and gene therapy.
    Keywords:  Adeno-associated virus (AAV) vectors; Differentiation; Gene therapy; Pluripotent stem cells; Retinal organoids; Transduction
    DOI:  https://doi.org/10.1007/978-1-0716-5198-8_10
  29. Sci Rep. 2026 Apr 20. pii: 12892. [Epub ahead of print]16(1):
      
    Keywords:   lux ; Bioluminescence; Microscopy; Neurons
    DOI:  https://doi.org/10.1038/s41598-026-46211-8
  30. Autophagy. 2026 May;22(5): 877-880
      Skeletal muscle is a fundamental tissue as it is found throughout the body, sustains posture, and produces movement. Yet, skeletal muscle disorders, such as myopathies, affect a large percentage of the population, degrading an individual's quality of life. A recent study links myopathy progression to the decline in chaperone-mediated autophagy that occurs during aging. Underscoring the importance of a balanced CMA pathway in maintaining skeletal muscle function and integrity, the study also provides mechanistic insights into the pathways that are dysregulated due to defective CMA and presents an approach to reverse the age-dependent decline in this process.Abbreviations: ATP2A1, ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 1; CMA, chaperone-mediated autophagy; HSPA8, heat shock protein family A (Hsp70) member 8; LAMP2A, lysosomal associated membrane protein 2A.
    Keywords:  Aging; calcium homeostasis; chaperone-mediated autophagy; mitochondrial homeostasis; myopathy; skeletal muscle
    DOI:  https://doi.org/10.1080/15548627.2026.2627051
  31. Nat Commun. 2026 Apr 23.
      Extracellular vesicles (EVs) are nanoscale particles secreted by cells that carry diverse biomolecules reflecting their cell of origin. Single-EV imaging approaches have enabled precise characterization of heterogeneous EV populations; however, their broader application is limited by low-throughput workflows and cumbersome EV isolation procedures. Here, we introduce a streamlined, high-throughput imaging platform capable of analyzing protein expression of individual intact EVs directly from unprocessed biological samples at the single-vesicle level. Our approach employs a functionalized glass surface optimized for high-throughput single-EV imaging, facilitating specific capture of EVs and enabling integration with existing automation technologies. We evaluate the platform's analytical capabilities by characterizing various recombinant EV samples and demonstrate its clinical utility by analyzing EVs in a total of 191 human plasma samples with high-throughput efficiency. This technology will offer a pathway for high-precision and large-scale characterization of EVs in clinical samples.
    DOI:  https://doi.org/10.1038/s41467-026-72179-0
  32. Glia. 2026 Jun;74(6): e70163
      Microglial cells are key players in maintaining brain homeostasis and responding to pathological conditions. Their multifaceted roles in health and disease have garnered significant attention in the context of neurodegeneration. In recent years, single-cell transcriptomic techniques have provided unprecedented insights into microglial heterogeneity, revealing distinct subpopulations and gene expression patterns associated with neuroprotection or neurotoxicity. Here, we dissect the transcriptomic landscape of microglia by leveraging human single-nuclei RNA sequencing datasets from multiple neurodegenerative conditions, including Amyotrophic Lateral Sclerosis, frontotemporal dementia, Alzheimer's disease, aging, and Parkinson's disease. This integrative analysis identifies distinct microglial subpopulations, reflecting functional heterogeneity across diseases and reveals a shared cross-disease microglial transcriptional program associated with inflammatory and neurodegenerative processes. Using a machine learning framework, we further demonstrate that this transcriptional program enables robust discrimination between neurodegenerative and control samples. Experimental validation in primary microglia isolated from a mouse model of Niemann-Pick disease type C, also known as juvenile Alzheimer's disease, supports the conservation of key components of this program and highlights Spp1 as a biomarker of disease-associated microglia states. Overall, this study provides an improved portrait of microglia transcriptional remodeling across neurodegenerative disorders and offers a framework for identifying conserved molecular features that may inform therapeutic strategies aimed at modulating microglial activity to mitigate disease progression and foster neuroprotection.
    Keywords:  Niemann‐Pick; SPP1; genomics; macrophages; microglia; neurodegeneration
    DOI:  https://doi.org/10.1002/glia.70163
  33. J Histochem Cytochem. 2026 Apr 24. 221554261433071
      Forebrain organoids (FOs) closely replicate key features of human brain, but often develop necrotic cores due to oxygen diffusion limits, resulting in disassembly. While dynamic culture devices can mitigate this, they add variability and diverge from adult cerebral static environment. Inspired by allometric scaling principles of brain growth, we developed a uniform, static culture protocol applying a 1-day transient high-dose Fibroblast Growth Factor 2 (100 ng/ml) treatment before neural induction. This acted as a proliferative stimulus, promoting long-term viability and structural integrity. Using human embryonic stem cells, early-FOs with diameters of 500-1000 µm achieved an 83.33% survival rate at 20 days in vitro (DIV). Area and volume increased significantly during 60 DIV culture period, but between 30 and 60 DIV they plateaued, indicating a transition from neural development to maturation. Weight increased until 30 DIV, but it significantly dropped between 30 and 60 DIV, possibly reflecting the formation of lumen-like structures. At 60 DIV, immunofluorescence revealed organized PAX6+ ventricle-like structures, where SOX2 marked neural progenitors, TUJ1 and MAP2 indicated mature neurons, GFAP identified astrocytes, and SYN1 highlighted emerging synaptic networks. This scalable protocol supports robust FOs generation, providing optimization and practical improvement within established frameworks to advance precision medicine.
    Keywords:  3D model; FGF2; aging; allometry; developmental stages; human forebrain organoids; neural markers; nourishment diffusion; reproducibility
    DOI:  https://doi.org/10.1369/00221554261433071