bims-axbals 2026-05-31 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–05–31
nineteen papers selected by
TJ Krzystek

Striatal neuron excitability is regulated by huntingtin in the adult brain.
The C9orf72/SMCR8 complex maintains microglial homeostasis via RAB8A ESCRT-mediated lysosomal repair.
Huntingtin polyglutamine expansions misdirect axonal transport by perturbing motor and adaptor recruitment.
A human corticospinal organoid-slice connectoid model informs enhancer strategies for post-injury axon regrowth.
From Axonal Growth to Neurodegeneration: The Dual Role of Neurofilament Dynamics in Health and Disease.
CHCHD2 and CHCHD10 promoted autophagic clearance of protein aggregates via GABARAPs.
Reevaluating the role of beta2-microglobulin: new insights on selective vulnerability in ALS pathology.
Human induced pluripotent stem cell-derived cortical grafts rebuild motor circuits after brain injury.
Characterisation of the axon initial segment and intrinsic excitability in the sub-acute phase post-ischaemic stroke.
Membrane ATG8ylation in secretory autophagy.
A transport-independent role for SLC25A12 in mitochondrial stress signalling.
Human brain slice culture model of STXBP1 encephalopathy reveals disruption of neuronal network activity and changes in gene expression.
Characterization of a new mitophagy reporter uncovers requirement of BNIP3 for hypoxia-induced mitophagy in Drosophila.
Optimized multiplex immunofluorescent protocols for simultaneous in situ identification of α-motoneuron subtypes.
Advances in small extracellular vesicle analysis.
Detection of mitochondrial bioenergetics using a novel bimodal 3D microelectrode array (MEA)-based biosensor.
Iron overload inhibits neuronal extracellular vesicles secretion via SNARE dysfunction.
Mitochondria-lysosome coupling contributes to lysosome acidification and aging.
Proteomic profiling of cytoskeletal interactomes using MT-ID and Act-ID.

eNeuro. 2026 May 28. pii: ENEURO.0269-25.2026. [Epub ahead of print]

Striatal neuron excitability is regulated by huntingtin in the adult brain.

Jessica C Barron, Meghan L Greenland, Samantha J Carew, Fatemeh Ashrafganjoie, Emily P Hurley, Christiana Meta Kennedy, Firoozeh Nafar, Craig S Moore, Matthew P Parsons.

Huntington's disease (HD) is a hereditary neurodegenerative disease that typically presents during midlife and is characterized by a combination of motor, cognitive and psychiatric symptoms. HD is fatal and arises from a mutation in the huntingtin (HTT) gene, which results in decreased neuronal health followed by brain atrophy, with spiny projection neurons (SPNs) of the striatum being especially vulnerable to degeneration. HTT loss-of-function, caused by haploinsufficiency of the wild-type HTT gene (wtHTT), is an important feature of HD pathophysiology that has previously been understudied compared to mutant HTT gain-of-function mechanisms. wtHTT is essential for nervous system development and functions as a scaffolding protein to support many vital cellular functions including axonal transport, autophagy and synaptic plasticity. Here, we examined the consequences of wtHTT deletion in the adult cortex and striatum by conditionally inactivating wtHTT in 2-4-month-old male and female Htt fl/fl mice. wtHTT loss of function decreased intrinsic neuronal excitability within SPNs and produced a neuroinflammatory response in these mice, while tissue organization, spine morphology and motor behaviour remained unaffected. Results presented here provide additional evidence that wtHTT is vital for maintaining neuronal health in the adult brain and highlight some potential adverse consequences of non-selective HTT-lowering for the treatment of HD.Significance Statement Huntington's disease (HD) is a fatal brain disease caused by a mutation in the huntingtin (HTT) gene. Despite many ongoing clinical trials, there are currently no disease-modifying therapeutics available for HD patients. HTT-lowering therapeutics have shown promise as potential treatments for HD, however, many are non-selective for HTT and lower levels of mutant and wild-type HTT (wtHTT) proteins. Here, we sought to determine the consequences of wtHTT deletion in the adult cortex and striatum, regions particularly vulnerable to neurodegeneration in HD. wtHTT conditional knockout decreased neuronal excitability and produced an inflammatory response, while tissue organization, spine dynamics and motor phenotypes remained unaffected. Data presented here provide additional evidence that wtHTT is essential for neuronal health in the adult brain.

DOI: https://doi.org/10.1523/ENEURO.0269-25.2026
EMBO J. 2026 May 29.

The C9orf72/SMCR8 complex maintains microglial homeostasis via RAB8A ESCRT-mediated lysosomal repair.

Shan Li, Shidong Xu, Feng Li, Qirui Zhao, Penghui Zhang, Qinghua Guan, Xiangxiang Sun, Jundong Bi, Hu Xiao, Yiyuli Tang, Cheng Peng, Qingfeng Chen, Yonghua Wang, Mei Yang.

Microglia are critical regulators of neuroinflammation and neurodegeneration. Haploinsufficiency of C9orf72, the most frequently mutated gene in amyotrophic lateral sclerosis and frontotemporal dementia, has been linked to autophagy-lysosomal pathway defects, but the role of C9orf72 in microglia remains unclear. Here, we identify the C9orf72/SMCR8 complex as a key regulator of microglial homeostasis through promoting lysosomal membrane repair. Loss of C9orf72 and SMCR8 in mice causes age‑dependent neuroinflammation and microgliosis, with microglia adopting a disease-associated state. In aged brain and spinal cord tissue, microglia display lysosomal damage marked by galectin‑3 accumulation. Using a lysosomotropic agent to induce lysosomal damage in microglia, we find that C9orf72/SMCR8-deficient cells accumulate damaged lysosomes and show defective recruitment of phosphorylated RAB8A and the Endosomal Sorting Complexes Required for Transport (ESCRT) machinery to damaged lysosomes. Notably, mutant microglia accumulate GTP‑bound RAB8A, which becomes hyperphosphorylated and mislocalized to RAB7-positive, LAMP1-negative vesicles. The GTPase-activating activity of the C9orf72/SMCR8 complex is essential for lysosomal repair. Our findings reveal that the C9orf72/SMCR8 complex coordinates RAB8A-ESCRT-mediated lysosomal repair to safeguard microglial homeostasis and limit neuroinflammation.

DOI: https://doi.org/10.1038/s44318-026-00817-w
iScience. 2026 Jun 19. 29(6): 115748

Huntingtin polyglutamine expansions misdirect axonal transport by perturbing motor and adaptor recruitment.

Emily N P Prowse, Brooke A Turkalj, Muriel Sébastien, Lale Gursu, Daniel Beaudet, Jia Feng, Chengqian Zhou, Heidi M McBride, Gary J Brouhard, Mahmoud A Pouladi, Adam G Hendricks.

  Huntington's disease is caused by polyglutamine (polyQ) expansions in huntingtin (HTT). PolyQ lengths >35Q lead to neurodegeneration, and longer repeats correspond to earlier onset of symptoms. HTT scaffolds kinesin-1 and dynein to organelles directly and through adaptors. We tracked BDNF vesicles, mitochondria, and lysosomes in stem-cell-derived neurons engineered to express HTT with polyQ lengths of 30, 45, 65, and 81. BDNF endosomes were more motile in HTT-45Q and HTT-65Q neurons and misdirected toward the distal tip in HTT-81Q neurons. Under neuroinflammatory stress, polyQ expansions resulted in fewer BDNF cargoes and more lysosomes. We next isolated BDNF endosomes from neurons and counted the associated motors and adaptors. We found BDNF endosomes associated with greater numbers of kinesin-1 and HAP1 molecules in HTT-81Q neurons. Together, these results show that polyQ expansions in HTT alter the motors and adaptors recruited to cargoes, resulting in dysregulated transport and responses to neuroinflammatory stress.

Keywords:  Cellular neuroscience; Molecular network; Molecular neuroscience

DOI:  https://doi.org/10.1016/j.isci.2026.115748
Cell Rep. 2026 May 26. pii: S2211-1247(26)00477-8. [Epub ahead of print]45(6): 117399

A human corticospinal organoid-slice connectoid model informs enhancer strategies for post-injury axon regrowth.

George M Gibbons, Tanja Fuchsberger, Mai Abdelgawad, Stefano L Giandomenico, Kornélia Szebényi, Veselina Petrova, Lea M D Wenger, Daniel N Olschewski, Jeremi Chabros, Leila Muresan, Rachael C Feord, Muhammad Asif, James W Fawcett, Susanna B Mierau, Ole Paulsen, Madeline A Lancaster, András Lakatos.

  Axon elongation in the mammalian central nervous system (CNS) declines during development, limiting regenerative capacity after birth. Intrinsic regulators of this process are promising repair targets, as immature axons can regrow in tissues otherwise not conducive to regeneration. Yet the precise timing and mechanisms underlying the cessation of axon growth in the human CNS remain unresolved. Here, we developed a three-dimensional human corticospinal motor organoid-slice connectoid platform mimicking the developmental axon elongation program and its subsequent restriction through maturation. Cortical and spinal slices establish functional connections while remaining spatially segregated, enabling cortical cell-type-specific observations without direct confounding effects by spinal cells. Using single-cell transcriptomics, computational analyses, axon regrowth assays, and live imaging, we identified transcriptional alterations contributing to decreased axon growth in maturing human cortical projection neurons. We further demonstrate that this decline can be reversed using compounds and repurposable drugs targeting a maturation-associated transcriptional shift, promoting post-injury axon repair.

Keywords:  CP: neuroscience; CP: stem cell research; amyotrophic lateral sclerosis; brain and spinal cord organoid; connectoid; corticospinal injury; developmental axon growth; drug screening; human axon repair failure; regeneration; single-cell genomics; spinal cord injury

DOI:  https://doi.org/10.1016/j.celrep.2026.117399
NeuroSci. 2026 May 09. pii: 58. [Epub ahead of print]7(3):

From Axonal Growth to Neurodegeneration: The Dual Role of Neurofilament Dynamics in Health and Disease.

Yikang An, Hongying Lan, Jialong Xiong, Ruoyan Jing, Dongjin Gu, Haoyang Zhang, Xinping Liu, Qi Zhao, Feng Wang.

  Neurofilaments (NFs) are the predominant type IV intermediate filaments in differentiated neurons, functioning not just as static scaffolds, but as active drivers of radial axonal growth and nerve conduction velocity. While their physical properties are well characterized, a critical gap remains in synthesizing how their dynamic assembly and developmental subunit switching directly dictate neurodegenerative outcomes. This review breaks down the molecular architecture and stepwise kinetic assembly of NFs, detailing their role in polarized transport and the formation of a protective viscoelastic gel network within axons. We specifically highlight the physiological expression switching of early subunits, such as alpha-internexin and peripherin, during neuronal maturation, a process often overlooked in traditional structural reviews. By examining how specific gene mutations and aberrant hyperphosphorylation trigger axonal transport jams and protein aggregation, we map the direct pathways leading to amyotrophic lateral sclerosis (ALS) and Charcot-Marie-Tooth (CMT) disease. Finally, we emphasize that a precise mechanistic decoding of NF structural dynamics and their pathological disruption is essential for understanding the fundamental etiology of these neurodegenerative conditions.

Keywords:  Charcot–Marie–Tooth disease; aberrant phosphorylation; amyotrophic lateral sclerosis; assembly mechanisms; axonal growth; expression switching; intermediate filaments; neurodegenerative diseases; neurofilaments; protein aggregation

DOI:  https://doi.org/10.3390/neurosci7030058
Autophagy. 2026 May 25.

CHCHD2 and CHCHD10 promoted autophagic clearance of protein aggregates via GABARAPs.

Zhou Wei, Maggie Zhang, Willcyn Tang, Brijesh Kumar Singh, Zhang Zhiwei, Zhou Lei, Jaron Goh Kim Wee, Faith Tan Rui En, Huang Jingxiu, Sun Qiaoyang, Xiao Bin, Gupta Priyanka, Alfred Sun Xuyang, Zeng Li, Shen Han-Ming, Tan Eng King.

  Mutations in mitochondrial protein CHCHD2 and its paralog CHCHD10 were identified in patients with Parkinson disease (PD), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD) or Alzheimer disease (AD). CHCHD2 and CHCHD10 mutations caused neurodegeneration in model animals as seen in patients, but their pathophysiological roles remain elusive. Here we reported a direct role of CHCHD2 and CHCHD10 in autophagy. We identified a protein complex composing of CHCHD2-CHCHD10-C1QBP/p32-Atg8-family proteins (ATG8s), in which each molecule interacted with another. CHCHD2, CHCHD10 and C1QBP/p32 associated with ATG8s, preferentially, GABARAPs. Disease-associated CHCHD2 and CHCHD10 mutations exhibited varied interaction with ATG8s. By binding to GABARAPs, CHCHD2 and CHCHD10 underwent autophagic degradation, and recruited the ULK1 complex. Autophagy initiation defects occurred upon transient knockdown of CHCHD2, and also in human iPSC-derived CHCHD2-/- or CHCHD2T61I dopaminergic neurons. Importantly, CHCHD2 and CHCHD10 promoted autophagy. CHCHD2 reduced protein aggregates in cells and toxic SNCA/α-synuclein species in mouse striatum. Our study thus revealed mitochondrial proteins CHCHD2 and CHCHD10 as both autophagy substrates and autophagy activators and laid groundwork for therapy targeting patients with neurodegeneration.

Keywords:  Aggregates; CHCHD10; CHCHD2; GABARAPs; autophagy; neurodegeneration

DOI:  https://doi.org/10.1080/15548627.2026.2678427
Acta Neuropathol. 2026 May 29. pii: 63. [Epub ahead of print]151(1):

Reevaluating the role of beta2-microglobulin: new insights on selective vulnerability in ALS pathology.

Melanie Leboeuf, Jik Nijssen, Laura Helen Comley, Julio Cesar Aguila Benitez, Irene Mei, Silvia Gómez Alcalde, Ramón A Muñoz de Bustillo-Alfaro, Vlad Radoi, Susanne Nichterwitz, Christoph Schweingruber, Abraham Acevedo Arozena, Eva Hedlund, Staffan Cullheim.

  Amyotrophic lateral sclerosis (ALS) is characterized by the selective loss of motor neurons (MNs). Why these neurons are particularly vulnerable in ALS remains unclear, as does why certain MN groups remain resistant throughout the disease course. We investigated the role of the human leukocyte antigens (HLAs) and beta2-microglobulin (β2m) in MN susceptibility to ALS, given their reported involvement in both prolonging and shortening disease progression. Loss of HLAs in ALS has also been shown to increase MNs vulnerability to toxicity exerted by activated astrocytes. RNA sequencing of control tissues demonstrated that disease-resistant oculomotor neurons (OMNs) and Onuf's MNs exhibited β2m and HLA mRNA levels comparable to those of vulnerable spinal MNs, suggesting that baseline differences in these transcripts do not explain the differential vulnerabilities of these MN groups. However, HLA protein levels showed an inverse correlation with spinal MN size, with the large MNs, those lost early in ALS, displaying the lowest HLA expression. HLA protein levels were also reduced in spinal MNs from end-stage ALS patient tissues, while remaining relatively unchanged in OMNs. In contrast, spinal MNs uniquely exhibited significant upregulation of β2m and HLA-C transcripts during disease, likely reflecting a protective compensatory response. Together, these findings suggest that β2m and HLAs may contribute to spinal MN vulnerability in ALS. To assess their functional role, β2m knockout mice were crossbred with SOD1G93A ALS mice. Loss of β2m did not alter life span of the ALS mice, but led to partial preservation of lumbrical muscle innervation that was insufficient to maintain motor function. Analysis of GFAP immunoreactivity revealed marked neuroinflammation activation in the spinal cords of β2m knockout mice. As these mice retain normal MN numbers and life-span, this indicates that loss of functional MHC-I, even in the presence of astrocyte activation, is insufficient to cause MN disease. Furthermore, β2m knockout significantly increased GFAP activation in SOD1G93A mice, but did not further exacerbate disease progression, suggesting that loss of functional MHC-I does not necessarily render MNs more vulnerable to astrocyte toxicity. Overall, these findings indicate that β2m and HLAs are dynamically regulated in ALS, and may influence MN vulnerability, but they are not major disease modifiers in ALS.

Keywords:  Amyotrophic lateral sclerosis; Beta2-microglobulin; MHC-I; Motor neuron; Selective vulnerability

DOI:  https://doi.org/10.1007/s00401-026-03024-3
Neural Regen Res. 2026 May 14.

Human induced pluripotent stem cell-derived cortical grafts rebuild motor circuits after brain injury.

Myles D Gladen, Zane R Lybrand.

  Traumatic brain injury causes irreversible neuronal loss, and no existing therapy can replace lost cortical tissue and restore its circuitry. We investigated whether human induced pluripotent stem cell-derived cortical organoids could not only repair motor deficits but also reveal how grafted neurons integrate into the adult brain. Cortical organoids were generated via a modified dual-SMAD inhibition protocol and transplanted into the motor cortex of NOD-SCID mice after controlled cortical impact. Mice receiving grafts achieved full recovery of contralateral forelimb motor function within 28 days, while non-transplanted controls showed persistent deficits. Grafts are selectively projected to the canonical efferent motor pathway targets bilaterally, with minimal off-target integration. Strikingly, we identified abundant perinuclear synaptic puncta in host neurons that colocalized with graft-derived axons, human-specific cytoplasmic labeling, and the excitatory synapse marker post-synaptic density protein 95. These structures, present in both local and long-range motor-associated regions, provide the first structural evidence of graft-derived synaptic input directly onto host neuronal cell bodies, suggesting specific organoid cell types are integrating into the host network. Our findings establish that human induced pluripotent stem cell-derived cortical organoids can restore motor function after traumatic brain injury and reveal a cellular integration signature that advances understanding of how transplanted human neurons connect with the injured adult brain.

Keywords:  central nervous system; cerebral organoids; induced pluripotent stem cells; motor recovery; network establishment; regeneration; structural synapse formation; traumatic brain injury

DOI:  https://doi.org/10.4103/NRR.NRR-D-25-01272
Brain Commun. 2026 ;8(3): fcag160

Characterisation of the axon initial segment and intrinsic excitability in the sub-acute phase post-ischaemic stroke.

Emily S King, Jamie L Beros, Hakuei Fujiyama, Aidan Bindoff, John N J Reynolds, Alexander D Tang.

  Stroke is a leading cause of disability, and stroke-induced changes in cortical excitability are thought to impede functional recovery. Identifying cellular targets that contribute to maladaptive excitability holds great potential for the development of therapeutic interventions to improve stroke outcomes. One potential target is the axon initial segment, the specialized neuronal domain where action potentials are initiated. In the acute phase post-stroke, neurons in the peri-infarct zone have previously been shown to have abnormal axon initial segment structural properties, which may contribute to altered neuronal excitability. However, whether this continues into the sub-acute phase post-stroke, a period with heightened plasticity and when physical rehabilitation typically begins, is unknown. We induced a photothrombotic unilateral ischaemic stroke to the right motor cortex of 13-week-old mice alongside adeno-associated virus labelling of layer 2/3 and layer 5 pyramidal neurons in the peri-infarct zone and contralesional motor cortex. Immunofluorescence staining for Ankyrin-G and whole-cell patch clamp electrophysiology measures were made at 28 days post-stroke to assess changes in axon initial segment structure and function. Additionally, we investigated potential hemispheric-, cortical layer-, and sex-dependent differences in axon initial segment and intrinsic excitability properties. Our results show that normal axon initial segment structure is preserved in the sub-acute phase post-stroke. However, we found that stroke increased action potential half-width and membrane capacitance across both hemispheres and sexes. Additionally, stroke-injured male mice showed hyperpolarized action potential thresholds but reduced maximum spike firing frequencies in the contralesional hemisphere and reduced evoked spike firing frequencies across both hemispheres, while stroke-injured females showed reduced action potential amplitudes and maximum spike firing frequencies in the peri-infarct zone but increased action potential amplitudes in the contralesional hemisphere, along with preserved maximum and evoked firing frequencies in this region. Our results show that despite the preservation of normal axon initial segment structure, changes to intrinsic excitability contribute to the abnormal cortical excitability observed in the sub-acute phase post-stroke. We also provide evidence that stroke induces sex-dependent differences in neuronal function. These findings suggest that intrinsic mechanisms should be considered as a cellular target for stroke therapies and emphasize the importance of considering sex as a biological variable in studies of post-stroke neuronal plasticity and in the development of targeted therapeutic interventions.

Keywords:  axon initial segment; intrinsic plasticity; ischaemic stroke; sub-acute phase post-stroke

DOI:  https://doi.org/10.1093/braincomms/fcag160
Autophagy. 2026 May 24. 1-16

Membrane ATG8ylation in secretory autophagy.

Jayanta Debnath, Andrew M Leidal.

  Mammalian Atg8-family (ATG8) proteins are crucial for macroautophagic/autophagic degradation in the lysosome and facilitate non-degradative processes including multiple distinct forms of unconventional protein secretion. These secretion pathways, collectively termed secretory autophagy, depend upon ATG8 conjugated to membranes to both specify and traffic molecules for extracellular release. Here, we review the current understanding of how membrane ATG8ylation supports secretory autophagy, and propose a cell biological framework for classifying the growing repertoire of secretory autophagy pathways based on membrane ATG8ylation at discrete intracellular vesicular intermediates. Finally, we detail the emerging roles of these pathways in physiology and disease.Abbreviations: Aβ, amyloid-β; Acb1, acyl-coA-binding 1; ALS, amyotrophic lateral sclerosis; APP, amyloid beta precursor protein; APEX2, ascorbate peroxidase; ATG, autophagy related; AWOL, autophagosome-mediated exit without lysis; BafA1, bafilomycin A1; BirA*, mutant BirA biotin ligase; BMI, body-mass index; CASM, ATG8 conjugation at single membranes; DAMPs, danger/damage-associated molecular patterns; DBI, diazepam binding inhibitor, acyl-CoA binding protein; DSS, dextran sodium sulfate; ER, endoplasmic reticulum; ERGIC, endoplasmic reticulum intermediate compartment; ESCRT, endosomal complexes required for transport; EVs, extracellular vesicles; EVPs, extracellular vesicles and particles; HMGB1, high mobility group box 1; IDE, insulin degrading enzyme; IFNB, interferon beta; ILV, intralumenal vesicles; LANDO, LC3-associated endocytosis; LAP, LC3-associated phagocytosis; LIR, LC3 interacting region; LDELS, LC3-dependent EV loading and secretion; LLOMe, L-leucyl-L-leucine methyl ester hydrobromide; M2, influenza A virus matrix 2, MAD, migratory autolysosome disposal; miRNAs, microRNAs; M-MDSC, monocytic myeloid derived suppressor cells; MVEs, multivesicular endosomes; PAMPs, pathogen-associated molecular patterns; P-bodies, processing bodies; PE, phosphatidylethanolamine; PD, Parkinson disease; PS, phosphatidylserine; RBPs, RNA binding proteins; R-EV, RAB22A-induced extracellular vesicle; SLC2A1, solute carrier family 2 member 1; TFRC, transferrin receptor; TGN, trans-Golgi network; TMED10, transmembrane p24 trafficking protein 10; THU, TMED10-channeled unconventional secretion; SALI, secretory autophagy during lysosome inhibition; SCF, SKP1-CUL1-F-box; SNAREs, soluble NSF attachment protein receptors.

Keywords:  ATG8ylation; extracellular vesicles and particles; noncanonical autophagy; secretory autophagy; unconventional protein secretion

DOI:  https://doi.org/10.1080/15548627.2026.2676796
Nat Cell Biol. 2026 May 27.

A transport-independent role for SLC25A12 in mitochondrial stress signalling.

Liu Jiang, Lan Li, Jialiang Guan, Ying Liu.

Mitochondria are central hubs for energy production and cellular adaptation to stress. When mitochondria are damaged, cells activate protective signalling pathways to restore homeostasis and ensure survival. One such pathway, known as the integrated stress response (ISR), reduces overall protein synthesis while enhancing the production of stress-responsive proteins. The mitochondrial carriers SLC25A12 and SLC25A13 transport similar metabolites but are expressed in different tissues and linked to distinct genetic diseases. Here we show that SLC25A12 plays a previously unrecognized role in stress signalling that is independent of its transport activity. SLC25A12 interacts with the mitochondrial protease OMA1, enabling activation of ISR during mitochondrial damage. This signalling function is disrupted by a disease-linked mutation but preserved in transport-deficient variants. Our findings reveal SLC25A12 as a dual-function mitochondrial protein, acting as both a metabolite transporter and a regulator of stress signalling, and suggest that defective ISR activation may contribute to certain SLC25A12-associated pathologies.

DOI: https://doi.org/10.1038/s41556-026-01973-1
Exp Neurol. 2026 May 28. pii: S0014-4886(26)00220-7. [Epub ahead of print] 115855

Human brain slice culture model of STXBP1 encephalopathy reveals disruption of neuronal network activity and changes in gene expression.

Faye McLeod, Anna Dimtsi, Michael A Savage, Yen Chi Tan, Ann Hedley, Andrew J Trevelyan, Gavin J Clowry.

  STXBP1 haploinsufficiency, a major genetic cause of developmental and epileptic encephalopathies, exhibits variability in clinical severity and remains poorly understood mechanistically. Although STXBP1 encodes a core presynaptic protein essential for SNARE-mediated neurotransmitter release, evidence from rodent models and patient-derived neurons indicates that its deficiency produces far broader molecular and cellular disruptions across multiple neurodevelopmental processes. Understanding how these widespread perturbations contribute to STXBP1 encephalopathy requires integrative approaches that extend beyond single-phenotype assays. Here, we used foetal human organotypic cortical cultures that preserve tissue architecture including the transient subplate, a critical hub in early cortical network development. Human cultures (15-18 post-conception weeks) retaining intact subplate, cortical plate, and progenitor zone organisation, were subjected to shRNA-mediated STXBP1 knockdown. We combined live calcium imaging, targeted transcriptomics, protein expression validation, and neurite-growth assays to assess functional and structural outcomes. STXBP1 knockdown disrupted spontaneous subplate neuronal network activity observed by calcium imaging at 14 days in vitro, reducing signal amplitude and synchronicity, indicating impaired early circuit function. Transcriptomic profiling revealed dysregulation of gene expression involved in synaptogenesis, ion transport, and extracellular matrix organisation. Protein-level analyses confirmed alterations in key synaptic components. At the cellular level, neurons exhibited shortened neurites and accumulation of the axon-guidance receptor EPHA4 at growth cones, suggesting defects in early connectivity. These findings expand the mechanistic framework of STXBP1 encephalopathy beyond synaptic dysfunction to encompass coordinated molecular, structural and network-level pathology during a critical window of cortical development, highlighting convergent pathways that may inform early therapeutic strategies.

Keywords:  Circuit function; Ephrin signalling; Human organotypic slices; Neurodevelopment; STXBP1; Transcriptomics

DOI:  https://doi.org/10.1016/j.expneurol.2026.115855
Res Sq. 2026 May 11. pii: rs.3.rs-9407058. [Epub ahead of print]

Characterization of a new mitophagy reporter uncovers requirement of BNIP3 for hypoxia-induced mitophagy in Drosophila.

Hubert Osei Acheampong, Emily Rozich, Ryan Insolera.

BACKGROUND: Mitophagy is the cellular removal of unwanted mitochondria via the lysosome. Given the importance of this process to energy demanding tissues, mitophagy defects have been linked to various metabolic and neurodegenerative diseases. Mitophagy assessment tools are important for evaluating and quantifying mitophagy flux, which are useful in studying mitophagy pathways, mechanisms, and dysfunction. Mitophagy reporters are commonly used reagents to examine endpoint mitophagy flux. Following the generation of a new mitophagy reporter, mitoSRAI (mitochondrial Signal Retaining Autophagy Indicator), we introduced this reporter as a transgene into Drosophila melanogaster (Dm). We hypothesized that mitoSRAI will be capable of measuring mitophagic flux through microscopic visualization of the TOLLES:YPet fluorescence ratios, and biochemically through the relative persistence of TOLLES proteins in the lysosomes following YPet degradation.
RESULTS: We found that when we express the mitoSRAI reporter in the Dm larval muscle wall and examine mitoSRAI flux by inducing mitophagy via hypoxia, we observe a significant increase in TOLLES only fluorescent signals and bands by confocal imaging and western blotting respectively. Complementarily, the readout of mitoSRAI is sensitive to conditions of mitophagy inhibition under hypoxia. To validate our results, we compared mitoSRAI to a similarly constructed reporter, matrix-QC, and found that mitoSRAI is less responsive to neuronal and fat body mitophagy flux manipulations.
CONCLUSION: Overall, our work characterizes the strengths and weaknesses of the application of the mitoSRAI reporter in Dm. We demonstrate with the mitoSRAI reporter that BNIP3 is an important mediator for hypoxia-induced mitophagy in Dm.

DOI: https://doi.org/10.21203/rs.3.rs-9407058/v1
J Neurosci Methods. 2026 May 28. pii: S0165-0270(26)00145-7. [Epub ahead of print] 110815

Optimized multiplex immunofluorescent protocols for simultaneous in situ identification of α-motoneuron subtypes.

Teresa L Garrett, Lee Wintermute, Maggie Armitage, Shelby Ward, Kalin Gerber, Sherif M Elbasiouny.

   BACKGROUND: Accurate identification of α-motoneuron (α-MN) subtypes - slow (S), fast fatigue-resistant (FR), fast fatigue-intermediate (FI), and fast fatigable (FF) - is essential for studying motor circuit organization and selective vulnerability in neurodegenerative disease. While electrophysiological approaches can distinguish these subtypes, existing immunohistochemical (IHC) methods lack the ability to simultaneously identify all four α-MN classes in situ, particularly the FI subtype, limiting their utility for large-scale or tissue-based analyses.
NEW METHOD: Here, we present novel multiplex immunofluorescent strategies that enables simultaneous in situ identification of S, FR, FI, and FF α-MN subtypes, including intermediate populations, within single sections of mouse lumbar spinal cord. This approach integrates a combinatorial marker framework with optimized co-labeling conditions to resolve subtype-specific molecular signatures, including FI MNs, which have not been previously distinguishable using standard IHC methods.
RESULTS: We establish a systematic validation pipeline demonstrating robust and reproducible subtype classification across multiple protocols, sexes, mouse strains, and disease conditions, including the G93A-SOD mouse model of amyotrophic lateral sclerosis. Labeled populations recapitulate known size distributions and exhibit consistent subtype-specific patterns across lumbar segments, supporting both the accuracy and reproducibility of the method.
CONCLUSIONS: By enabling comprehensive in situ classification of all major α-MN subtypes, this approach represents a substantive refinement of multiplex IF, overcoming key limitations of existing IF methods and enabling analyses of α-MN subtype organization and selective vulnerability that were previously not feasible with standard histological techniques. This framework is broadly applicable to studies of motor system organization, aging, and neurodegenerative disease.

Keywords:  Alpha Motoneuron; Amyotrophic Lateral Sclerosis; Fatigue Intermediate Motoneuron Typing; Lumbar Spinal Cord; Multiplex Immunohistochemistry

DOI:  https://doi.org/10.1016/j.jneumeth.2026.110815
Adv Clin Chem. 2026 ;pii: S0065-2423(26)00017-X. [Epub ahead of print]133 79-125

Advances in small extracellular vesicle analysis.

Wei Zhang, Su Su Thae Hnit, Xin Feng, Yuling Wang.

  Small extracellular vesicles (sEVs) have emerged as crucial mediator of intercellular communication, playing key roles in various physiological and pathological processes. Comprehensive sEV analysis has become increasingly important for understanding its molecular composition, biological functions, and potential applications in disease diagnosis, prognosis, and therapy. This chapter provides an overview of recent advances in sEV analysis, focusing on biomolecules including proteins, nucleic acids, lipids, and glycans. Furthermore, the application of various omics technologies (including proteomics, genomics, transcriptomics, lipidomics, and glycomics) is discussed. These technologies have significantly expanded the understanding of sEV biology and function. By highlighting these latest developments in sEV analysis, this chapter aims to provide a comprehensive review of the current state of this field.

Keywords:  Exosome; Glycans; Lipids; Molecular analysis; Nucleic acids; Omics; Proteins; Small extracellular vesicle

DOI:  https://doi.org/10.1016/bs.acc.2026.01.003
Microsyst Nanoeng. 2026 May 28. pii: 208. [Epub ahead of print]12(1):

Detection of mitochondrial bioenergetics using a novel bimodal 3D microelectrode array (MEA)-based biosensor.

Randall K James, Tatiana C Hostios, Ji Chang, Aakash Patel, Faisal Bin Kashem, Isaac Johnson, Jorge Manrique Castro, James Hickman, Swaminathan Rajaraman.

Developing new label-free paradigms for functional assays in biomedical research has the potential to catalyze efforts in drug discovery and improve the understanding of complex disorders. Mitochondria are an essential organelle in nearly every eukaryotic organism that perform vital functions such as adenosine triphosphate (ATP) production, redox signaling, reactive oxygen species (ROS) homeostasis and regulation of programmed cell death. These activities are regulated by electrophysiological processes that occur in the inner mitochondrial membrane (IMM) and outer mitochondrial membrane (OMM) in response to metabolic demands, making them an important physiological marker for bioenergetic studies. Mitochondria dysfunction is an early pathological biomarker of complex diseases, such as diabetes, neurodegeneration, myopathy, cancer, and cardiovascular disease. Built atop a novel microfabrication strategy for 3D Microelectrode Arrays (MEAs), we demonstrate a 3D mitochondria biosensor capable of bimodal sensing of mitochondrial electrophysiology from the OMM and IMM using electrochemical impedance spectroscopy (EIS) and electrophysiology recordings. Data obtained using EIS displays impedance magnitude and phase characterization of mitochondria isolated from NIH3T3 and induced pluripotent stem cells (iPSC) models, these measurements represent the major functional outputs of cellular respiration and electron transport chain (ETC) activity through the detection of conductive and capacitive properties of the IMM. Additionally, time-resolved electrophysiological recordings from an NIH3T3 derived mitochondrial pellet captured sub-millisecond voltage transients, establishing a complementary real-time electrophysiological profile of mitochondrial membrane activity that can be attributed voltage dependent anion channel (VDAC) gating or IMM potential dynamics.

DOI: https://doi.org/10.1038/s41378-026-01275-4
Neurochem Int. 2026 May 26. pii: S0197-0186(26)00083-5. [Epub ahead of print]198 106192

Iron overload inhibits neuronal extracellular vesicles secretion via SNARE dysfunction.

Xiaoting Wang, Yang Li, Xiaoqing Mi, Tongtong Yang, Yun Li, Jinjie Zhang, Fengju Jia, Junxia Xie, Ning Song.

  Iron deposition in the brain is observed in several neurodegenerative diseases and aging. Extracellular vesicles (EVs), including exosomes, are critical mediators of intercellular communication. Neuron-derived EVs are recently recognized as promising biomarkers in the plasma for neurological disorders. However, it remains unclear whether and how neuronal iron overload affects EVs (exosomes) secretion. In this study, we used L1 cell adhesion molecule (L1CAM) to label neuron-derived EVs and observed a significant reduction in L1CAM-positive vesicle concentration in the plasma of mice with brain iron deposition. Iron-reduced EVs secretion was also observed in PC12 cells with ferric ammonium citrate treatment. We then reported the inhibition of iron overload on exosomes secretion was independent of cell proliferation, exosomal trafficking pathway and lysosomal degradation pathway, however, probably via disrupting SNARE complex function. Finally, we observed that neuron-derived EVs are more abundant in the plasma of mice during ageing. Our results indicate that iron overload inhibits neuronal EVs secretion both in vivo and in vitro, potentially through dysfunction of the SNARE complex.

Keywords:  Extracellualr vesicles; Iron; Neuron; SNARE

DOI:  https://doi.org/10.1016/j.neuint.2026.106192
Mol Cell. 2026 May 29. pii: S1097-2765(26)00310-2. [Epub ahead of print]

Mitochondria-lysosome coupling contributes to lysosome acidification and aging.

Qingqing Liu, Seungmin Yoo, Zhixin A Zhang, Liying Li, Hetian Su, Lingraj Vannur, Alexandra C Wooldredge, Jun-Wei B Hughes, Pierre-Yves Desprez, Nan Hao, Gordon Lithgow, Julie K Andersen, Malene Hansen, Judith Campisi, Chuankai Zhou.

  Nearly all cellular processes are pH dependent. The acidic pH inside the lysosome (vacuole in yeast) is essential for cellular content degradation, signaling, and autophagy. Defects in lysosome/vacuole acidification are a conserved hallmark of aging and age-related diseases. Traditionally, the lysosome/vacuole is thought to import free protons (H⁺) from the surrounding neutral cytosol. Here, we uncovered a conserved lysosome/vacuole acidification mechanism from yeast to human involving lysosomal/vacuolar uptake of H+ pumped out by mitochondrial electron transport chain through mitochondria-lysosomes/vacuoles membrane contacts. Aging/senescence-associated disruption of mitochondria-lysosome/vacuole contacts causes lysosomal/vacuolar de-acidification, which can be reversed by either expressing an engineered linker to connect these two organelles or through an asymmetry-dependent rejuvenation process in daughter cells. Preserving lysosomal acidification in senescent human cells prevents the induction of major senescence-associated secretory phenotype factors and restores autophagic flux. These findings reshape our current understanding of the mechanisms underlying lysosomal/vacuolar (de-)acidification in both young and aged/senescent cells.

Keywords:  Mito-Vac/Lyso contacts; SASP; aging; autophagy; cellular senescence; mitochondria; proton; vacuolar/lysosomal acidification

DOI:  https://doi.org/10.1016/j.molcel.2026.05.004
bioRxiv. 2026 May 13. pii: 2026.05.12.724647. [Epub ahead of print]

Proteomic profiling of cytoskeletal interactomes using MT-ID and Act-ID.

Hannah Neiswender, Jessica E Pride, Rajalakshmi Veeranan-Karmegam, Phylicia Allen, Jamesia Henderson, Mary E Lowe, Eric A Vitriol, Kathryn E Bollinger, Graydon B Gonsalvez.

The microtubule and actin cytoskeletons form dynamic, interconnected networks that are critical for eukaryotic cell function. These networks govern intracellular organization, cargo transport, cell migration, and tissue morphogenesis. Microtubules and actin filaments are regulated by diverse binding proteins that control many aspects of their function. However, identifying cytoskeletal-interacting proteins has been challenging due to the transient and weak nature of many interactions and the disruption of native architecture by conventional biochemical approaches. These limitations suggest that numerous physiologically relevant cytoskeletal regulators remain undiscovered. Identifying these factors requires novel and sensitive methodologies that can capture cytoskeletal interactions under native cellular conditions. Here, we present MT-ID and Act-ID, powerful proximity-labeling tools for identifying microtubule and actin-interacting proteins, respectively. MT-ID employs the microtubule-binding domain of MAP7 (EMTB) fused to TurboID, a highly active promiscuous biotin ligase. Act-ID utilizes the actin-binding domain of ITPKA (F-tractin) similarly fused to TurboID. We validate both approaches by successfully identifying numerous known cytoskeletal regulators and discovering potentially novel interacting proteins. Functional characterization reveals that LIMCH1 is a previously unrecognized microtubule-associated protein whose depletion increases microtubule density. Additionally, we identify FBXO30 as a novel actin-interacting protein, with its loss promoting increased focal adhesion formation. MT-ID and Act-ID will be useful not only to identify cytoskeletal interacting proteins but also to define changes to the cytoskeletal interactome when cells are exposed to changing physiological conditions.

DOI: https://doi.org/10.64898/2026.05.12.724647