bims-axbals 2026-10-25 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–10–25
twenty papers selected by
TJ Krzystek

ClC-7 links PIKfyve inhibition to Rab-dependent LRRK2 activity at lysosomes.
Swollen lysosomes grab Rabs for a CASM-free route to LRRK2 activation.
Drosophila dendrites are resilient to adult-onset deficits in mitochondrial dynamics.
Cryo-EM advances in TDP-43 amyloid fibrils structure determination.
The Lysosomal Clock: Are We Only as Old as Our Lysosomes?
Cytoplasmic TDP-43 Accumulation in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay.
Machine learning image analysis predicts Parkinson disease genotype and mitochondrial lysosomal abnormalities in iPS neurons.
Protein disulphide isomerase protects against ALS/FTD phenotypes associated with cyclin F in zebrafish and cellular models.
TMEM106B risk converges on C-terminal fragment homeostasis: genetics, mechanisms and therapy.
Empty clathrin cages in neurons.
A Cilia-Centered Nicotinamide Adenine Dinucleotide (NAD+) Signaling Network Regulates Hypothalamic Lysosomal Homeostasis and Metabolic Aging.
prickle-mediated epilepsy in flies is a brain-derived neurodevelopmental defect.
Endogenous Protein Tagging in Postmitotic Neurons via Lentiviral and Adeno-associated Virus Delivery.
ARF-1 coordinates cargo sorting at FERARI endosomal recycling hubs.
Development of NanoBRET Cellular Target Engagement Assays in Primary Neurons for Activating Mutants of p21-Activated Kinase 1.
Ghost Neuron preparation unlocks high-resolution structural mapping of intracellular architecture in axons.
mir-57 Influences Posterior Motor Neuron Axon Morphology in Caenorhabditis elegans.
KAT6A-Mutant iPSC-Derived Cortical Neurons Display Precocious Maturation, Dysregulated Electrophysiology, and Altered Morphology.
Metformin improves mitophagy-related pathways and mitochondrial-lysosomal homeostasis in chromosome 21 trisomic fibroblasts.
Protocol for studying membrane protein dynamics and associated synaptic vesicle recruitment on native membrane sheets.

J Cell Biol. 2026 Nov 02. pii: e202511137. [Epub ahead of print]225(11):

ClC-7 links PIKfyve inhibition to Rab-dependent LRRK2 activity at lysosomes.

Devin Clegg, Amanda Bentley-DeSousa, Agnes Roczniak-Ferguson, Shawn M Ferguson.

Increased activity of leucine-rich repeat kinase 2 (LRRK2) confers Parkinson's disease risk. LRRK2 dynamically localizes to lysosomal membranes in response to various stresses, yet the mechanisms by which distinct lysosomal perturbations are communicated to LRRK2 remain unclear. Here, we show that inhibition of the lysosomal lipid kinase PIKfyve promotes LRRK2 recruitment and signaling through a pathway that requires the lysosomal chloride/proton antiporter ClC-7. ClC-7 in turn controls the accumulation of multiple Rab GTPases on lysosomes. LRRK2 signaling under these conditions requires its established Rab-binding surfaces, with Rab12 contributing significantly to this response. This pathway operates independently of CASM. In contrast, lysosomal stresses that induce CASM require both Rab-binding sites on LRRK2 and GABARAP for robust LRRK2 signaling. These findings identify ClC-7-dependent lysosomal remodeling and Rab accumulation as key features linking PIKfyve inhibition to LRRK2 signaling and reveal that distinct lysosomal stresses engage different combinations of Rab and GABARAP inputs to activate LRRK2.

DOI: https://doi.org/10.1083/jcb.202511137
J Cell Biol. 2026 Nov 02. pii: e202610007. [Epub ahead of print]225(11):

Swollen lysosomes grab Rabs for a CASM-free route to LRRK2 activation.

Suzanne R Pfeffer.

Multiple lysosomal stresses activate Parkinson's-linked LRRK2 kinase, and these involve the so-called "CASM" pathway to help recruit LRRK2 to lysosome surfaces. Clegg et al. (https://doi.org/10.1083/jcb.202511137) show that LRRK2, activated upon lysosome swelling caused by PIKfyve inhibition, instead relies solely on Rab GTPase binding for lysosome localization and activation.

DOI: https://doi.org/10.1083/jcb.202610007
J Cell Biol. 2026 Dec 07. pii: e202511010. [Epub ahead of print]225(12):

Drosophila dendrites are resilient to adult-onset deficits in mitochondrial dynamics.

Hailey Q Wang, Demetria G Fortson, Eavan D Burton, Hunter S Whitbeck, Briana M Robles, Kwass Wass, Letitia Bortey, Avi J Adler, Jordan I Kalai, Giovanna Napoleone, Erin L Barnhart.

Mitochondrial transport, fusion, and fission are necessary for neuronal development, but the role of mitochondrial dynamics in neuronal maintenance remains unclear. In this work, we employed functional in vivo imaging of neurons in the Drosophila visual system, "horizontal system" (HS) cells, to determine how adult-onset deficits in mitochondrial dynamics affect mitochondrial localization, local regulation of ATP, and dendrite maintenance. In mature HS neurons, inhibition of mitochondrial transport or fusion depleted mitochondria from the dendrite over time but, surprisingly, had no effect on dendrite morphology. Moreover, adult-restricted mitochondrial mislocalization affected neither visual stimulus-driven dendritic calcium responses nor local, dynamic regulation of ATP levels. In contrast, when induced during development, the same perturbations caused mitochondrial mislocalization, loss of dendrite complexity, abrogation of stimulus-locked calcium responses and ATP fluctuations, and age-dependent dendrite degeneration. Thus, although mitochondrial dynamics are necessary during neuronal development, mature dendrites are capable of maintaining form and function in vivo upon mitochondrial depletion.

DOI: https://doi.org/10.1083/jcb.202511010
Neurobiol Dis. 2026 Oct 17. pii: S0969-9961(26)00405-5. [Epub ahead of print] 107659

Cryo-EM advances in TDP-43 amyloid fibrils structure determination.

Zhengdong Xu, Xi Chen, Wenjuan Yi.

  TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA-binding protein whose pathological aggregation characterizes a spectrum of neurodegenerative disorders known as TDP-43 proteinopathies, including amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD), and chronic traumatic encephalopathy (CTE). Recent breakthroughs in cryo-electron microscopy (cryo-EM) have revolutionized our understanding of these diseases by resolving the atomic structures of TDP-43 amyloid fibrils. These structural insights have been pivotal in elucidating the transition from functional liquid-liquid phase separation (LLPS) to irreversible solid-state aggregation. This review synthesizes key cryo-EM findings from 2019 to 2026, encompassing fibrils formed by the full-length low-complexity domain, disease-relevant core fragments, and filaments extracted directly from patient tissues, as well as the pathological implications of distinct structural polymorphs. These findings not only deepen our understanding of the brain science aspects of the pathogenesis of TDP-43 proteinopathies, but also provide an important structural basis for the development of specific conformational diagnostic methods and targeted therapeutic drugs.

Keywords:  Amyloid; Brain science; Cryo-EM; TDP-43; TDP-43 proteinopathies

DOI:  https://doi.org/10.1016/j.nbd.2026.107659
Traffic. 2026 Dec;27(4): e70056

The Lysosomal Clock: Are We Only as Old as Our Lysosomes?

Alexis Diaz Vegas, Thierry Galli.

Lysosomes are a major organelle involved in intracellular degradation, membrane traffic and metabolic signaling, but how their biochemical composition changes during normal aging remains poorly defined. Puszynska and colleagues now combine lysosome purification with metabolomics and lipidomics across tissues, species and ages to reveal a conserved signature of lysosomal aging characterized by accumulation of glycerophosphodiesters (GPDs) and cystine and remodeling of bis(monoacylglycero)phosphate (BMP). Strikingly, these changes are largely obscured in whole-tissue analyses and resemble metabolic abnormalities associated with lysosomal storage disorders, including CLN3 disease and cystinosis. The signature develops progressively with age, can predict chronological age, and is attenuated by caloric restriction in peripheral tissues but not in the brain. Within the brain, age-dependent metabolite accumulation is particularly prominent in microglia and is also detected in neurons, linking lysosomal aging to cell populations central to neurodegenerative disease. These findings define an organelle-level metabolic clock, suggesting that one might be only as young as one's lysosomes. Establishing whether GPDs, cystine and BMP remodeling merely mark lysosomal age or actively contribute to declining lysosomal degradation and membrane traffic may uncover a metabolic connection between physiological aging, lysosomal storage disorders and vulnerability to late-onset neurodegeneration.

DOI: https://doi.org/10.1111/tra.70056
Can J Neurol Sci. 2026 Oct 21. 1-7

Cytoplasmic TDP-43 Accumulation in Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay.

Alexandre Paquet, Lydia Touzel-Deschênes, Vincent Roy, Jean-Denis Brisson, Jean Mathieu, Nicolas Dupré, Stephan Saikali, François Gros-Louis.

   BACKGROUND: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare, early-onset neurodegenerative disorder characterized by progressive cerebellar ataxia, spasticity and peripheral neuropathy. It is caused by biallelic mutations in the SACS gene, which encodes sacsin, a large protein involved in cytoskeletal organization and mitochondrial homeostasis, and protein quality control. Despite advances in understanding sacsin biology, the molecular mechanisms underlying neurodegeneration in ARSACS remain incompletely understood. Here, we report the first neuropathological evidence of pathological TAR DNA-binding protein 43 (TDP-43) accumulation in ARSACS.
METHODS: Available postmortem brain tissue from a genetically confirmed ARSACS patient homozygous for the Quebec founder mutation (SACS c.8844delT) was analyzed by immunohistochemistry, immunofluorescence and biochemical fractionation.
RESULTS: Neurons in the prefrontal cortex, motor cortex and cerebellum exhibited cytoplasmic phosphorylated dot-like TDP-43 (pTDP-43) aggregates with reduced nuclear localization. Biochemical fractionation further identified detergent-insoluble TDP-43 species in ARSACS tissue. Additional pathological features included increased neurofilament accumulation, vacuole-like neuronal changes, Purkinje cell loss and p62-positive cytoplasmic inclusions. Similar dot-like cytoplasmic pTDP-43 aggregates were observed in a second ARSACS case.
CONCLUSION: These findings identify pTDP-43 as a previously unrecognized pathological feature of ARSACS. While the patient exhibited no clinical or histopathological signs of amyotrophic lateral sclerosis, the presence of atypical pTDP-43 aggregates observed in ARSACS may represent a rare, secondary, early-stage or potentially underrecognized manifestation of TDP-43 pathology rather than a defining hallmark of the disease. Further studies in additional patients and experimental ARSACS models will be required to determine the prevalence and mechanistic basis of TDP-43 abnormalities in this disorder.

Keywords:  ARSACS; TDP-43; ataxia; cerebellum; neurofilaments; neurons

DOI:  https://doi.org/10.1017/cjn.2026.10695
NPJ Parkinsons Dis. 2026 Aug 01. pii: 255. [Epub ahead of print]12(1):

Machine learning image analysis predicts Parkinson disease genotype and mitochondrial lysosomal abnormalities in iPS neurons.

Yan Li, Maximilian Powell, Jessica Chedid, Ratneswary Sutharsan, Adahir Labrador-Garrido, Dad Abu-Bonsrah, Chiara Pavan, Tyra Fraser, Dmitry Ovchinnikov, Melanie Zhong, Ryan Davis, Dario Strbenac, Jennifer A Johnston, Lachlan H Thompson, Deniz Kirik, Clare L Parish, Glenda M Halliday, Carolyn M Sue, Nicolas Dzamko, Gautam Wali.

Mitochondrial and lysosomal dysfunction are central features of Parkinson's disease (PD) across major genetic forms including PRKN, SNCA, and LRRK2. We applied cell morphomics, a machine-learning-based framework combining high-content imaging with quantitative feature extraction, to analyse mitochondrial and lysosomal morphology at single-cell resolution in iPS cell-derived cortical neurons from PD patients and healthy controls (13 lines total). Supervised machine-learning models distinguished PD neurons from controls with high accuracy (AUC = 0.87) and reliably separated individual genotypes. Feature importance and attribution analysis revealed genotype-specific organelle biases in the relative contribution of mitochondrial and lysosomal features, with mitochondrial features dominating classification in PRKN neurons, balanced mitochondrial and lysosomal contributions in SNCA neurons, and a greater lysosomal contribution in LRRK2 neurons. Multi-class models retained strong performance, and findings were reproduced across two independent laboratories using different dyes and imaging conditions. These results demonstrate that morphomics provides a robust and scalable framework to quantify genotype-specific organelle abnormalities in PD neurons and supports its application for cellular stratification and biomarker discovery.

DOI: https://doi.org/10.1038/s41531-026-01458-w
Redox Rep. 2026 Dec 31. 31(1): 2732775

Protein disulphide isomerase protects against ALS/FTD phenotypes associated with cyclin F in zebrafish and cellular models.

Sina Shadfar, Sonam Parakh, Emma Perri, Cyril Jones Jagaraj, Sara Assar Kashani, Keelin Beyers, Audrey M G Ragagnin, Angela S Laird, Julie D Atkin.

   BACKGROUND: Mutations in CCNF, encoding cyclin F, cause a small proportion of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases. Mutant cyclin F mislocalises from the nucleus to the cytoplasm, impairs the ubiquitin proteasome system, disrupts endoplasmic reticulum (ER) to Golgi transport, and triggers ER stress and apoptosis. Protein disulphide isomerase (PDI), a redox-active ER chaperone of the thioredoxin superfamily, protects against ALS phenotypes induced by mutant SOD1, TDP-43, and FUS, but its activity against mutant cyclin F has not been examined.
OBJECTIVES: To test whether PDI overexpression prevents the cellular and behavioural phenotypes induced by the ALS/FTD-associated cyclin FS621G mutation.
METHODS: Neuro-2a cells were co-transfected with mCherry-tagged wild-type or mutant cyclin FS621G and V5-tagged PDI or empty vector. Subcellular localisation, XBP-1 and CHOP activation, proteasome reporter (GFPu) accumulation, nuclear morphology, and cleaved caspase-3 immunoreactivity were assessed by confocal microscopy. Transient transgenic zebrafish larvae generated by mRNA co-injection of cyclin F and PDI were assessed by photomotor response at 48 h post-fertilisation.
RESULTS: Mutant cyclin FS621G induced cytoplasmic mislocalisation, ER stress (assessed by XBP-1 and CHOP activation), proteasome dysfunction (indicated by GFPu accumulation), and apoptosis (assessed by nuclear condensation and caspase-3 activation), all of which were significantly attenuated by PDI co-expression. Zebrafish larvae expressing cyclin FS621G swam significantly shorter distances than uninjected or wildtype controls, and co-expression of PDI restored motor function to near-wildtype levels.
CONCLUSION: PDI protects against ALS/FTD-associated mutant cyclin F in both cellular and in vivo zebrafish models, identifying PDI as a redox-based therapeutic target for CCNF-linked ALS/FTD.

Keywords:  CCNF; ER stress; FTD; MND; ageing; cyclin F; neurodegeneration; proteostasis; zebrafish

DOI:  https://doi.org/10.1080/13510002.2026.2732775
Brain. 2026 Oct 22. pii: awag367. [Epub ahead of print]

TMEM106B risk converges on C-terminal fragment homeostasis: genetics, mechanisms and therapy.

Miranda Lastra Osua, Jolien Perneel, Cristina T Vicente, Rosa Rademakers.

  TMEM106B is a genetic modifier influencing susceptibility to multiple neurodegenerative disorders, including frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP), Alzheimer's disease, Parkinson's disease, and limbic-predominant age-related TDP-43 encephalopathy, as well as contributing to healthy brain aging and neuronal reserve. The gene harbours single nucleotide polymorphisms and structural variants that define risk and protective haplotypes, and despite extensive efforts to identify the functional variant within these haplotypes, consensus remains elusive. In this review, we synthesize current knowledge of TMEM106B genetics from human cohorts and mechanistic studies, highlighting how variants may influence its regulatory network and protein levels, with an emphasis on its aggregation-prone C-terminal domain. We further integrate evidence on the roles of TMEM106B within the endo-lysosomal system and beyond, including insights into protein structure, posttranslational modifications, interactors, proteolytic processing, and fibril formation. These findings are contextualized using data from mouse and induced pluripotent stem cell (iPSC) models, highlighting current gaps in the available models and the advances required to study its functions and cleavage. Finally, we explore therapeutic strategies targeting TMEM106B, focusing on modulation of expression, specific functional pathways, and fibril formation.

Keywords:  FTLD-TDP; TMEM106B; endo-/lysosomal system; neurodegeneration

DOI:  https://doi.org/10.1093/brain/awag367
Curr Opin Neurobiol. 2026 Oct 21. pii: S0959-4388(26)00121-2. [Epub ahead of print]101 103285

Empty clathrin cages in neurons.

Subhojit Roy.

Clathrin has established roles in endocytosis and short-range vesicle trafficking, where clathrin cages envelop membrane-pits on the plasma membrane or vesicles exchanging between Golgi/endosomes, and this canonical role of clathrin is thought to be conserved in all cell types including neurons. However, the polarized architecture of neurons creates unique demands on clathrin recruitment and function, requiring clathrin to operate in presynapses that are far from sites of protein synthesis in the soma and act within the limited membrane area of periactive-zones in a nonconstitutive manner-responding to cues such as neuronal activity. Recent studies have revealed unusual long-lasting clathrin assemblies that are transported along axons and captured at synapses, congregating around synaptic vesicle clusters. Unexpectedly, cryo-electron tomography shows empty cages of clathrin at synapses that do not enclose any membrane. This short review discusses recent findings related to the structure and dynamics of neuronal clathrin and frames some new questions that have emerged.

DOI: https://doi.org/10.1016/j.conb.2026.103285
Aging Cell. 2026 Nov;25(11): e70745

A Cilia-Centered Nicotinamide Adenine Dinucleotide (NAD+) Signaling Network Regulates Hypothalamic Lysosomal Homeostasis and Metabolic Aging.

Se Eun Park, Eun Roh, Jae Woo Park, Seong Jun Kim, So Yeon Jeong, Chae Beom Park, Hyo Sun Lim, Ji Soo Kim, Mun Gyeom Kim, Myeong Ju Kim, Young In Cho, Wontae Jeong, Gil Myoung Kang, Sang-Yeob Kim, Se Hee Min, Min-Seon Kim.

  Nicotinamide adenine dinucleotide (NAD+) is an essential metabolite that regulates diverse cellular metabolic processes. Primary cilia, hair-like organelles on the cell surface, function as critical signaling hubs. Here, we demonstrate that cellular NAD+ levels are key regulators of both primary cilia growth and lysosomal function in hypothalamic neurons. NAD+ depletion induces ciliary shortening and diminishes lysosomal activity in a cilia-dependent manner. Mechanistically, the lysosomal repair complex protein CHMP4B translocates from periciliary satellites to lysosomes during lysosomal stress, a process impaired by ciliary dysgenesis. Disrupted lysosomal recruitment of CHMP4B leads to abnormal lysosomal Ca2+ signaling and lysosomal dysfunction. Aged mice with hypothalamic NAD+ deficiency exhibit shortened cilia and lysosomal stress phenotypes, both of which are restored by calorie restriction (CR). Moreover, CR improves aging-associated metabolic dysfunction, at least in part, through hypothalamic cilia-dependent mechanisms. These findings identify a primary cilium-mediated-NAD+ signaling axis that maintains lysosomal homeostasis in hypothalamic neurons, the disruption of which drives metabolic aging.

Keywords:  CHMP4B; NAD+; TRPML1; aging; calorie restriction; hypothalamic neurons; lysosomal homeostasis; primary cilia

DOI:  https://doi.org/10.1111/acel.70745
J Neurogenet. 2026 Oct 20. 1-16

prickle-mediated epilepsy in flies is a brain-derived neurodevelopmental defect.

Anthony J Lilienthal, Krishna M Nukala, Brady Williquett, J Robert Manak.

  Damaging DNA variants in PRICKLE genes cause epilepsy from flies to humans. While classic work, primarily in flies, has demonstrated that PRICKLE proteins work with other planar cell polarity proteins to polarize epithelial sheets, the function of PRICKLE proteins in the nervous system is still unclear and may play distinct roles in the nervous system of different organisms. We thus sought to compare and contrast Drosophila prickle with PRICKLE genes in vertebrate systems in order to identify unifying roles across species. First, we find that similar to expression of the mouse Prickles, fly prickle is expressed in a wide variety of CNS neurons, in particular post-mitotic maturing neuroblasts and neurons. However, unlike in the mouse, fly prickle is not enriched in the axon initial segment (AIS) but is broadly distributed across the entire length of the expressing neurons. Second, we find that targeted expression of the prickle-spiny-legs (pksple) isoform in a) glutamatergic neurons or b) a region analogous to the human brain stem in pksple mutant animals suppresses seizures, implicating dysfunction of these neurons in seizure progression. Strikingly, targeting prickle expression to only larval/pupal stages can fully rescue adult seizures, demonstrating that this form of epilepsy can be considered a neurodevelopmental defect such that an abnormality that occurs during development leads to a disorder that is late-onset. Finally, targeting pksple expression exclusively in pksple mutant brains suppresses seizures, demonstrating that, similar to humans, prickle-associated epilepsy arises in the brain. Consistent with this result, removal of the head of pksple mutants also significantly reduces seizures, confirming that brain neuronal dysfunction contributes to fly seizures.

Keywords:  Drosophila; epilepsy; neurodevelopment; prickle; seizure circuitry

DOI:  https://doi.org/10.1080/01677063.2026.2742881
Curr Protoc. 2026 Oct;pii: e70492. [Epub ahead of print]6(10):

Endogenous Protein Tagging in Postmitotic Neurons via Lentiviral and Adeno-associated Virus Delivery.

Lorena Benedetti, Hyun Ah Yi, Lihua Wang, Jennifer Lippincott-Schwartz.

  This protocol describes a method for endogenous protein tagging in postmitotic primary neurons using homology-independent targeted integration (HITI), a CRISPR/Cas9-based approach that exploits non-homologous end joining (NHEJ). Unlike homology-directed repair (HDR), HITI does not require cell division, making it uniquely suited for postmitotic neurons. SpCas9 is delivered by a replication-incompetent simian immunodeficiency lentiviral vector (pLenti SIV), while the sgRNA, donor tag cassette, and a fluorescent plasma membrane marker are co-packaged in a single AAV vector. The two vectors are co-delivered at DIV5, and endogenous tagging is assessed at DIV15-23. The workflow is illustrated using Junctophilin-3 (JPH3) tagged with mEmerald at its endogenous locus. Airyscan microscopy imaging of endogenously tagged JPH3 at native expression levels revealed its localization at endoplasmic reticulum-plasma membrane (ER-PM) contact sites and uncovered a periodic distribution of ER-PM junctions along the dendritic arbor, enabling identification of a previously unrecognized ladder-like organization of the ER in neuronal dendrites. Genomic integration of the tag is validated at both 5' and 3' junctions by next-generation sequencing. The protocol can be adapted to other neuronal proteins by redesigning the guide RNA and donor sequence, and supports fluorescent proteins as well as epitope and self-labeling tags. The plasma membrane marker can also be replaced with other fluorescent reporters or markers for intracellular organelles to assess infection efficiency and verify subcellular localization of the protein of interest. The full workflow takes 5-6 weeks. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Protocols included in this article: Basic Protocol 1: sgRNA design and HITI donor vector cloning Basic Protocol 2: Primary rat neuron culture and viral co-transduction Basic Protocol 3: Live-cell imaging of endogenously tagged proteins Support Protocol 1: Production of lentiviral and AAV particles Support Protocol 2: Validation of knock-in by next-generation sequencing (NGS).

Keywords:  AAV; CRISPR/Cas9; HITI; SIV‐based lentiviral vector; endogenous protein tagging; neurons

DOI:  https://doi.org/10.1002/cpz1.70492
J Cell Biol. 2026 Dec 07. pii: e202512157. [Epub ahead of print]225(12):

ARF-1 coordinates cargo sorting at FERARI endosomal recycling hubs.

Jachen A Solinger, Curdin A Franziscus, Trian Nuredini, Dominic L Müller, Staf Clement, Danilo Ritz, Petia Adarska, Francesca Bottanelli, Anne Spang.

Endosomal pathways are central to cellular organization, compartmentalization, and intercellular communication. Independent of the endocytic entry route, sorting endosomes function as critical junctions at which membrane proteins are sorted for recycling or degradation. The multicomponent tether FERARI orchestrates recycling by coordinating Rab GTPase activity with vesicle fusion and fission through a kiss-and-run (KAR) mechanism. Here, we identify the small GTPase ARF-1 as an essential regulator of cargo loading at FERARI-dependent endosomal KAR sites. Cycling of ARF-1 between its active and inactive states is required for efficient cargo capture into recycling vesicles. Our findings support a model in which cycling ARF-1 activity governs the temporal association of cargo with adaptor proteins and complexes, thereby controlling selective uptake into vesicles destined for the plasma membrane. These results uncover a previously unappreciated layer of regulation of endosomal recycling and highlight the interplay between ARF and Rab family GTPases in this process.

DOI: https://doi.org/10.1083/jcb.202512157
ACS Chem Neurosci. 2026 Oct 21. 17(20): 3441-3452

Development of NanoBRET Cellular Target Engagement Assays in Primary Neurons for Activating Mutants of p21-Activated Kinase 1.

Jacob L Capener, Abdiel Badillo-Martinez, Bassel Awada, Zachary W Davis-Gilbert, Thomas W Kramer, Cameron S Blair, Frances M Bashore, Hassan Al-Ali, Alison D Axtman.

  The p21-activated kinases (PAKs) are a group of serine-threonine kinases central to multiple signaling pathways that govern cell survival and proliferation. Aberrant activity of PAK1, the most well characterized member of the PAK family, drives progression of several malignancies and brain disorders, including Alzheimer's disease and neurodevelopmental disorders. Despite growing interest in PAK1 as a drug target for these diseases, there is no assay to evaluate the intracellular target engagement of PAK1 inhibitors. To address this need, we developed first-in-class NanoBRET assays for wild-type PAK1 and a neurodevelopmental disorder-causing gain-of-function PAK1 mutant. Furthermore, we executed our novel PAK1 NanoBRET assay to evaluate target engagement of PAK1 inhibitors in primary hippocampal neurons. To the best of our knowledge, this is the first demonstration of a NanoBRET cellular target engagement assay in primary neurons, thereby increasing the relevance of our work by confirming PAK1 inhibitor binding to the aberrant form of the protein in cerebral cells.

Keywords:  PAK1; cellular target engagement; gain-of-function mutation; kinase inhibitors; nanoBRET; primary neurons

DOI:  https://doi.org/10.1021/acschemneuro.6c00326
Nat Commun. 2026 Sep 23. pii: 11049. [Epub ahead of print]17(1):

Ghost Neuron preparation unlocks high-resolution structural mapping of intracellular architecture in axons.

Satish Bodakuntla, Julia Marelli, Victor Vasquez-Montes, Simon Glauser, Christian Biertümpfel, Naoko Mizuno.

Cellular cryo-electron microscopy (cryo-EM) offers powerful tools to reveal molecular architecture inside cells, but dense cytoplasm often obscures fine molecular features, especially in narrow neuronal projections. Here, we introduce the Ghost Neuron technique, a preparation combining brief hypotonic treatment with controlled mechanical rupture immediately before freezing, facilitating high-contrast imaging of neuronal ultrastructure within its neurite topology. By removing the plasma membrane and reducing cytoplasmic density, the method allows high-resolution structural analysis while preserving the overall geometry of neurites. The method allows structural information to be mapped back onto neuronal architecture. The preparation also releases endogeneous macromolecular complexes onto EM grids, creating particle-rich regions suitable for high-resolution structural analysis. Using Ghost Neuron, we resolved axonal microtubules at 3.59 Å and 3.9 Å resolution, distinguishing expanded and compact lattice states, and mapped these states as interspersed patches along individual microtubules within axons. We also resolved axonal actin filaments and identified cofilin-decorated actin. These results establish Ghost Neuron as a discovery platform for linking molecular structure to neuronal organization.

DOI: https://doi.org/10.1038/s41467-026-77741-4
Eur J Neurosci. 2026 Oct;pii: e70736. [Epub ahead of print]64(8):

mir-57 Influences Posterior Motor Neuron Axon Morphology in Caenorhabditis elegans.

Nethma Waduge, Hannah R Tatnell, Gregory M Davis.

  Organization and specification of the nervous system start in the embryo and are largely controlled by a cluster of transcription factors known as HOX genes, which control body patterning and morphogenesis at the transcriptional level. However, this entire process requires the influence of multiple pathways, including cell signalling, genomic and post-genomic pathways. These include the Wnt (wingless) pathway, which controls neuronal development and polarity, and the small miRNA pathway, which regulates genes post-transcriptionally. In this study, we investigated mir-57, the homolog of human miR-10 in Caenorhabditis elegans and its role in influencing motor neuron function. mir-57 is expressed posteriorly throughout development and enriched in motor neurons. Absence of mir-57 results in decreased movement and abnormal axonal projections of posterior motor neurons. Moreover, the Wnt pathway genes cam-1 and mom-5 are mir-57 targets and show elevated expression at the post-transcriptional level in mir-57 deletion mutants. Taken together this study suggests that mir-57 influences Wnt receptor expression to maintain posterior motor neuron axon morphology.

Keywords:  axons; development; miRNA; motor neurons

DOI:  https://doi.org/10.1111/ejn.70736
Int J Biol Sci. 2026 ;22(15): 8448-8475

KAT6A-Mutant iPSC-Derived Cortical Neurons Display Precocious Maturation, Dysregulated Electrophysiology, and Altered Morphology.

Naama Wiesel-Motiuk, Ashwani Choudhary, Yara Hussein, Utkarsh Tripathi, Wote Amelo Rike, Aviram Shemen, Ritu Nayak, Souvik Chakraborty, Omveer Sharma, Tatiana Rabinski, Michal Stark, Karin Weiss, Gad D Vatine, Shani Stern, Yehuda G Assaraf.

  KAT6A syndrome is a complex neurodevelopmental disorder caused by heterozygous pathogenic KAT6A variants inflicting variable degrees of developmental delay and intellectual disability via a mechanism that remains largely uncharacterized. To further our understanding, we differentiated KAT6A syndrome patient-derived induced pluripotent stem cells into neural progenitor cells and cortical neurons. KAT6A syndrome patient-derived cells exhibit an altered transcriptome, with many of the dysregulated genes associated with autism spectrum disorder. At an early stage of differentiation, KAT6A-mutant cortical neurons show many elevated neurodevelopment-specific genes, including key transcription factors. Detailed electrophysiological measurements uncover that KAT6A-mutant cortical neurons display a hyperexcitability phenotype at an early stage of differentiation, followed by hypoexcitability at a later stage, compared with control neurons. These electrophysiological alterations were accompanied by decreased sodium and potassium currents. Remarkably, early KAT6A-mutant neurons exhibit a markedly lower percentage of GABA-positive neurons and increased spontaneous calcium activity as compared with control neurons. Moreover, multiple morphometric alterations in early-stage KAT6A-mutant cortical neurons indicate premature maturation, including increased neurite outgrowth, arbor complexity and soma size. Consistently, KAT6A knockout SH-SY5Y cells exhibit impaired differentiation capability and dysregulated gene expression. These novel findings establish the deleterious impact of pathogenic KAT6A variants on cortical neuron differentiation and electrophysiological function.

Keywords:  KAT6A; KAT6B; autism spectrum disorder; lysine acetyltransferase; molecular neuroscience; neurodevelopmental syndromes

DOI:  https://doi.org/10.7150/ijbs.134210
Cell Mol Life Sci. 2026 Jul 22. pii: 386. [Epub ahead of print]83(1):

Metformin improves mitophagy-related pathways and mitochondrial-lysosomal homeostasis in chromosome 21 trisomic fibroblasts.

Nunzia Mollo, Rosalba Natale, Morena D'Ariano, Simona Coppola, Daniele Di Meglio, Adriana Limone, Gaetano Calì, Claudia D'Agostino, Nunzia Pastore, Daniela Sarnataro, Simona Paladino, Anna Conti, Lucio Nitsch, Antonella Izzo.

  Mitochondrial dysfunction is a core cellular abnormality in Down syndrome (DS), yet the mechanisms underlying impaired mitochondrial quality control remain poorly defined. Here, we provide an integrated characterization of the autophagy-mitophagy-lysosome axis in human trisomic fetal fibroblasts (DS-HFFs). We show that trisomic cells display a paradoxical combination of elevated basal autophagy/mitophagy-related markers and defective mitochondrial clearance, indicative of a saturated and inefficient quality control system. DS-HFFs exhibit increased steady-state levels of PINK1, PARKIN, OPTN, NDP52, LC3-II, and p62, accumulation of autophagic vacuoles, reduced autophagic flux, and impaired delivery of mitochondria to lysosomes. We further demonstrate that metformin alleviates the saturation of the autophagy-mitophagy pathway by modulating multiple components of the mitochondrial quality control pathway, including attenuation of mTOR-associated signaling, enhancement of autophagic flux and normalization of mitophagy-related protein levels. Crucially, metformin increases mitochondria-lysosome association and rescues lysosomal degradative competence as shown by increased Dq-BSA activity, and increased maturation of Cathepsin D. Together, our findings support mitophagy inefficiency due to pathway saturation as a key pathogenetic mechanism in trisomic cells and identify metformin as a pharmacological tool capable of relieving the proteostatic overload intrinsic to trisomy 21. More broadly, these results align with a model in which aneuploidy imposes a chronic proteostatic burden that compromises organelle quality control.

Keywords:  Metformin; Mitochondrial quality control; Mitophagy; PINK1-PARKIN pathway; Trisomy 21

DOI:  https://doi.org/10.1007/s00018-026-06355-2
STAR Protoc. 2026 Oct 23. pii: S2666-1667(26)00576-9. [Epub ahead of print]7(4): 104923

Protocol for studying membrane protein dynamics and associated synaptic vesicle recruitment on native membrane sheets.

Akshay Kapadia, Anne-Sophie Hafner.

  Plasma membrane sheets provide direct access to the cytosolic face of the cellular membrane while retaining membrane-associated proteins and lipids. Here, we present a protocol for generating and validating sonication-derived membrane sheets from cultured cells, primary neurons, and isolated synaptosomes. We describe procedures for real-time and fixed imaging of membrane protein organization, interactions, associated synaptic vesicle recruitment, and quantitative analysis. This platform is readily adaptable to investigate diverse membrane proteins, lipid-dependent mechanisms, and vesicle tethering events across cellular systems.

Keywords:  Cell Biology; Cell Membrane; Neuroscience

DOI:  https://doi.org/10.1016/j.xpro.2026.104923