bims-axbals 2026-09-20 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–09–20
24 papers selected by
TJ Krzystek

A guide to selecting high-performing antibodies for TMEM175 (UniProt ID: Q9BSA9) for use in western blot, immunoprecipitation, and immunofluorescence.
Hyperactive KIF5A in Neurodegeneration.
Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures.
A guide to selecting high-performing antibodies for DJ-1 ( PARK7) (Q99497) for use in western blot, immunoprecipitation, and immunofluorescence.
A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence.
A guide to selecting high-performing renewable antibodies for KIF5A (Q12840) across western blot, immunoprecipitation, and immunofluorescence.
A guide to selecting high-performing antibodies for Netrin-1 (UniProt ID: O95631) for use in western blot and immunoprecipitation.
Peripheral TDP-43 pathology in amyotrophic lateral sclerosis: toward a systemic proteinopathy.
Lipids regulate export of lysosomal enzymes from the endoplasmic reticulum.
Loss of interruption in the HTT CAG repeat is associated with somatic expansion and loss of medium spiny neurons in Huntington's disease.
Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro Phenotyping.
Inhibition of mitochondrial dynamics in Dictyostelium discoideum through the overexpression of FszA and FszB proteins.
Neuro-glial lipid imbalance in a Drosophila model of amyotrophic lateral sclerosis 8.
Mitochondria-Lysosome Quality Flux in Aging Mesenchymal Stem/Stromal Cells: Quality-Control Bottlenecks and Therapeutic Opportunities for Regenerative Decline.
Cholesterol maintains the degradative capacity of lysosomes during clearance and recycling of dysfunctional mitochondria.
A Standardized Protocol for Generating iPSC-Derived Human Microglia for Functional Genomic Assays.
Human iPSC-Derived Brain Organoids: A Disease-Oriented Evaluation of Modeling Fidelity.
Visualizing synaptic disruptions in the release and regulation of dopamine hotspots in Huntington's Disease.
DFCP1-containing ER microdomains mediate lysosomal membrane repair.
Lysosomal dysfunction in vascular smooth muscle cells: Mechanisms and therapeutic targets in vascular disease.
Using Brain Organoids to Explore Human Neurobiology.
Parallelized bright-field and fluorescence imaging platform for organoids.
A suite of fluorescent timers for visualizing protein spatiotemporal dynamics in plants.
Cryo-electron tomography of functionally validated synapses in human induced neurons.

F1000Res. 2026 ;15 942

A guide to selecting high-performing antibodies for TMEM175 (UniProt ID: Q9BSA9) for use in western blot, immunoprecipitation, and immunofluorescence.

Vera Ruíz Moleón, Charles Alende, Maryam Fothouhi, Sara González Bolívar, Riham Ayoubi, Vincent Francis, Peter S McPherson, Carl Laflamme.

  TMEM175 is the pore-forming subunit of a lysosomal K+ channel complex that regulates lysosomal pH stability and membrane potential. To further investigate its cellular functions and implications in neurodegenerative diseases, antibody reagents are needed. Here we have characterized six TMEM175 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  Endosomal/lysosomal proton channel TMEM175; Potassium channel TMEM175; Q9BSA9; TMEM175; Transmembrane protein 175; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.182354.1
Cytoskeleton (Hoboken). 2026 Sep 14. e70204

Hyperactive KIF5A in Neurodegeneration.

David Villarroel-Campos, James N Sleigh.

  The highly polarised morphology of neurons and the sheer length of their axons make transport of cargoes throughout the cell a formidable task. Decades of evidence obtained from genetic studies on patients and animal models highlight deficits in axonal transport as a recurrent cause, or early contributing factor, in a plethora of neurodegenerative diseases. Axonal transport abnormalities usually manifest as a slowing of cargo trafficked by molecular motors along microtubules; however, hyperactivation of motors can also lead to disease. That is the case for the kinesin-1 protein KIF5A, in which hyperactive mutations are linked to amyotrophic lateral sclerosis (ALS) and neonatal intractable myoclonus (NEIMY). In this Perspective, we summarise the latest insights into the impact of KIF5A hyperactivity, such as loss of autoinhibition, aggregation, altered cargo binding and microtubule damage. We conclude by discussing possible strategies to counteract these disruptions, with an emphasis on the necessity of restoring axonal transport to physiological levels, a key requirement to maintain neuronal homeostasis.

Keywords:  amyotrophic lateral sclerosis (ALS); axonal transport; kinesin; microtubules; motor neuron; motor protein

DOI:  https://doi.org/10.1002/cm.70204
Bio Protoc. 2026 Sep 05. 16(17): e5801

Quantitative Analysis of Axonal Degeneration and TDP-43 Aggregation in Compartmentalized Human iPSC-Derived Motor Neuron-Myotube Co-cultures.

Anand Ganapathy Subramaniam, Lucas Keniger de Andrade Gensas, Tal Gradus-Pery, Eran Perlson.

  Amyotrophic lateral sclerosis (ALS) is characterized by early and spatially restricted pathology in motor axons, including distal degeneration and accumulation of aggregation-prone proteins such as TDP-43. However, a major limitation in the field has been the lack of approaches that enable robust, quantitative, and compartment-specific analysis of these early axonal events, particularly in human-relevant systems. Here, we describe an integrated experimental and analytical framework that enables quantitative dissection of axonal degeneration and protein aggregation, specifically within distal motor axons. By combining compartmentalized human co-cultures with a dedicated image analysis strategy, this approach enables selective and quantitative analysis of pathological processes specifically within axons, independent of surrounding tissues such as muscle and other cellular compartments. This framework captures both structural degeneration and protein aggregation dynamics at subcellular resolution, enabling spatially resolved quantitative analysis of disease-relevant changes along axons. Importantly, the analytical framework is not limited to TDP-43 but is broadly applicable to diverse aggregation-prone proteins, thereby providing a generalizable platform to study axonal pathology across neurodegenerative diseases. Together, this work provides a scalable approach for investigating axonal pathology as an early and measurable feature of neurodegeneration, with potential applications in mechanistic studies and therapeutic targeting in ALS and related disorders. Key features • Compartmentalized human induced pluripotent stem cell (iPSC)-derived motor neuron-myotube co-cultures for modeling distal axonal pathology. • Microfluidic separation of somatic and distal axonal compartments enabling spatial perturbation and analysis. • Quantitative imaging of neurofilament heavy chain (NFH)-associated axonal degeneration and pTDP-43 accumulation. • Semi-automated workflow for a reproducible, scalable, and modular pipeline for image quantification.

Keywords:  Amyotrophic lateral sclerosis; Automated image analysis; Axonal degeneration; Human iPSC-derived motor neuron–myotube co-culture; Microfluidic co-culture; TDP-43 aggregation

DOI:  https://doi.org/10.21769/BioProtoc.5801
F1000Res. 2026 ;15 989

A guide to selecting high-performing antibodies for DJ-1 ( PARK7) (Q99497) for use in western blot, immunoprecipitation, and immunofluorescence.

CongYao Zha, Charles Alende, Maryam Fotouhi, Sara González Bolívar, Riham Ayoubi, Vincent Francis, Peter S McPherson, Carl Laflamme.

  DJ-1 is a multifunctional protein that plays a pivotal role in cellular protection against oxidative stress and neurodegeneration. Mutations in the PARK7 gene are associated with early-onset familial Parkinson's disease. Here we have characterized sixteen DJ-1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  DJ-1; PARK7; Parkinson disease protein 7; UniProt ID Q99497; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.182633.1
F1000Res. 2026 ;15 1021

A guide to selecting high-performing antibodies for Syntenin-1 (O00560) for use in western blot, immunoprecipitation, and immunofluorescence.

Vera Ruíz Moleón, Charles Alende, Maryam Fothouhi, Sara González Bolívar, Riham Ayoubi, Vincent Francis, Peter S McPherson, Carl Laflamme.

  Syntenin-1 is the Syndecan-binding protein 1 and a PDZ domain-containing adaptor protein that regulates diverse cellular processes through its interactions with transmembrane receptors, cytoskeletal components, and signaling molecules. Here we have characterized twelve Syntenin-1 commercial antibodies for western blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  MDA-9; Melanoma differentiation-associated protein 9; SDCBP; Syndecan-binding protein 1; Syntenin-1; UniProt ID O00560; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.183125.1
F1000Res. 2026 ;15 1030

A guide to selecting high-performing renewable antibodies for KIF5A (Q12840) across western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Kinesin Family Member 5A (KIF5A) is an important protein for anterograde cellular transport. We characterized ten commercially available research antibodies against KIF5A for use in western blot, immunoprecipitation, and immunofluorescence using a standardized workflow. Antibody performance was assessed by comparing signal in a wild-type cell line and its corresponding knockout derivative. This work is part of a broader collaborative public-good initiative to improve biomedical research by systematically evaluating commercial antibodies against human proteins and openly sharing the data as a resource for the scientific community. We encourage readers to use this report as a guide for selecting antibodies best suited to their specific applications.

Keywords:  KIF5A; Kinesin Family Member 5A; Kinesin heavy chain isoform 5A; UniProt ID Q12840; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.184178.1
F1000Res. 2026 ;15 1129

A guide to selecting high-performing antibodies for Netrin-1 (UniProt ID: O95631) for use in western blot and immunoprecipitation.

NeuroSGC/YCharOS/EDDU collaborative group and ABIF consortium

  Netrin-1 is a secreted protein that regulates cell migration and survival, controlling axonal guidance during development, morphogenesis, apoptosis, angiogenesis, inflammation and cancer progression. Here we have characterized fourteen Netrin-1 commercial antibodies for western blot and immunoprecipitation using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. These studies are part of a larger, collaborative initiative seeking to address antibody reproducibility issues by characterizing commercially available antibodies for human proteins and publishing the results openly as a resource for the scientific community. While the use of antibodies and protocols vary between laboratories, we encourage readers to use this report as a guide to select the most appropriate antibodies for their specific needs.

Keywords:  NTN1; Netrin-1; O95631; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.184099.1
Acta Neuropathol. 2026 Sep 15. pii: 34. [Epub ahead of print]152(1):

Peripheral TDP-43 pathology in amyotrophic lateral sclerosis: toward a systemic proteinopathy.

Philippe Codron, Marion Miranda, Maelle Garnier, Shiyang He, Julien Gouju, Julien Cassereau, Pascal Leblanc, Franck Letournel.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by progressive degeneration of upper and lower motor neurons. Cytoplasmic accumulation of phosphorylated TAR DNA-binding protein 43 (pTDP-43) is the pathological hallmark of most ALS cases. While ALS has traditionally been viewed as a disease confined to the brain, spinal cord and motor nerves, recent studies have reported pTDP-43 pathology in multiple other tissues (skeletal and cardiac muscle, skin, minor salivary glands, gastrointestinal tract and lymph nodes), referred to as peripheral pathology. The detection of pTDP-43 beyond the nervous system suggests that ALS-associated TDP-43 proteinopathy may be more widespread than previously recognized and raises fundamental questions regarding the spatial and temporal landscape of ALS pathology. Peripheral pTDP-43 accumulation may reflect a systemic biological susceptibility affecting multiple tissues, propagation of pathological TDP-43 species between anatomical compartments, or a combination of both mechanisms. While its biological significance is yet to be determined, the presence of pTDP-43 in peripheral tissues broadens the current conceptual framework of ALS. It may also provide new opportunities for pathology-based biomarkers, therapeutic monitoring, and mechanistic studies aimed at understanding disease initiation and progression. However, current evidence is derived from small and methodologically heterogeneous cohorts, and peripheral pTDP-43 pathology is not restricted to ALS, emphasizing the need for larger standardized studies.

Keywords:  Amyotrophic lateral sclerosis; Biomarkers; Peripheral pathology; Phosphorylated TDP-43; Proteinopathy; Skeletal muscle; TDP-43

DOI:  https://doi.org/10.1007/s00401-026-03086-3
Proc Natl Acad Sci U S A. 2026 Sep 22. 123(38): e2616684123

Lipids regulate export of lysosomal enzymes from the endoplasmic reticulum.

Baolong Xia, Myeonghoon Han, Isaac Park, Norbert Perrimon.

  Lysosomal enzymes are synthesized in the Endoplasmic Reticulum (ER) and transported to lysosomes to execute their functions. Deficiencies in lysosomal enzymes or components of the lysosomal transport machinery result in lysosomal storage disorders. While mannose-6-phosphate mediated lysosomal enzymes sorting in the Golgi has been extensively characterized, the mechanisms governing their export from the ER remain elusive. Here, we show that de novo lipogenesis, a metabolic pathway responsible for fatty acid synthesis, regulates lysosomal enzyme transport. Inhibition of de novo lipogenesis leads to the retention of lysosomal enzymes within the ER. Mechanistically, fatty acid derived from de novo lipogenesis is used for Arf1 myristoylation. Myristoylated Arf1 promotes retrograde vesicle trafficking from the Golgi to the ER, thereby maintaining the homeostatic bidirectional flux required for efficient ER export of lysosomal enzymes. Our findings uncover a critical functional link between lipid metabolism and lysosomal enzyme trafficking.

Keywords:  SREBP; de novo lipogenesis; lysosomal enzyme transport; protein myristoylation; proximity labeling

DOI:  https://doi.org/10.1073/pnas.2616684123
Neuron. 2026 Sep 14. pii: S0896-6273(26)00640-9. [Epub ahead of print]

Loss of interruption in the HTT CAG repeat is associated with somatic expansion and loss of medium spiny neurons in Huntington's disease.

Chris Kay, Jessica Levesley, Kert Mätlik, Hailey Findlay Black, Emily Harvey, Stephanie Bortnick, Kyla Javier, Christina Buchanan, Tanushri Soomarooah, Inês Caldeira Brás, Glen Sequiera, Mahmoud Pouladi, Larissa Arning, Hoa H P Nguyen, Richard Roxburgh, Maurice Curtis, Richard L M Faull, Nathaniel Heintz, Michael R Hayden.

  Synonymous loss-of-interruption variants in the expanded CAG repeat sequence of Huntingtin (HTT) accelerate the clinical onset and progression of Huntington's disease (HD). Medium spiny neurons (MSNs) are gradually lost in HD and undergo selective somatic CAG expansion, but it is unclear how somatic expansion relates to MSN pathology. Here, we show that MSNs with large (111-150 CAG) and very large (>150 CAG) somatic expansions are rare in early manifest HD but accumulate in proportion with duration of disease. In patients with the deleterious CAG-CCG loss-of-interruption (CAG-CCG LOI) modifier, the proportion of MSNs with large and very large expansions is increased ∼5-fold despite reduced small somatic expansions in blood, and caudate MSN counts are reduced. Our findings suggest that increased somatic CAG expansion contributes to accelerated striatal MSN pathology and onset of HD but that MSNs with very large genomic CAG expansions can persist among surviving neurons of the HD brain.

Keywords:  Huntington’s disease; caudate nucleus; genetic modifiers; medium spiny neurons; neurodegeneration; repeat interruptions; somatic expansion; somatic instability; striatum; trinucleotide repeat disorders

DOI:  https://doi.org/10.1016/j.neuron.2026.08.010
Curr Protoc. 2026 Sep;6(9): e70449

Morphogen-Directed, High-Throughput Development of hiPSC-Derived Telencephalic Organoids for Comparative In Vitro Phenotyping.

Dosh Whye, Erika M Norabuena, Wardiya Afshar-Saber, Becca Lewis, Taryn J Polanco, Delaney Wood, Mustafa Sahin, Elizabeth D Buttermore.

  Pluripotent stem cell (PSC)-derived telencephalic organoids have long been established as an in vitro cell model to study the molecular and cellular features of the developing cerebral cortex and associated brain structures. Numerous directed organoid differentiation protocols have been developed that recapitulate cell subtype specification, cytoarchitectural organization, and cellular function with a high degree of fidelity. Protocol development has focused on the scalability of this in vitro system, and most organoid culture platforms involve a population-based approach in large vessel sizes. This makes comparative study with multiple cell lines both costly and time-consuming, and lowers the throughput for phenotypic screening. We have established a high-throughput approach for in vitro generation of hiPSC-derived telencephalic organoids that can be used to develop comparative phenotypic assays involving high-content imaging platforms. This article first highlights the use of a scaled-down, miniaturized cell culture volume that is amenable to high-throughput production of multiple 384-well plates. Then, using controlled morphogen cues, 3D hiPSC aggregates are directed into an anterior telencephalic identity and further specified into dorsal pallium or ventral subpallium cell fates, which can additionally be combined into assembloids for modeling excitatory and inhibitory networks or left to develop separately as single organoid subtypes. Finally, we introduce a Support Protocol detailing an in vitro 3D cell painting assay for organoid morpho-phenotypic characterization. © 2026 Wiley Periodicals LLC. Basic Protocol 1: High-throughput 3D aggregation of hiPSCs in morphogen-rich media Basic Protocol 2: Recombinant inhibitory protein-directed induction of telencephalic organoids Basic Protocol 3: Morphogen-guided induction of dorsal telencephalic/pallial organoids Basic Protocol 4: Morphogen-guided induction of ventral telencephalic/subpallial organoids Support Protocol: 3D live cell painting for organoid morphometric phenotyping.

Keywords:  human induced pluripotent stem cells; morphogens; pallium; subpallium; telencephalic organoids

DOI:  https://doi.org/10.1002/cpz1.70449
Front Cell Dev Biol. 2026 ;14 1903771

Inhibition of mitochondrial dynamics in Dictyostelium discoideum through the overexpression of FszA and FszB proteins.

Ashley Ryan, Kari Naylor.

  Mitochondria play a central role in cells through energy production, calcium regulation, and cell death regulation. Dysfunction of mitochondria can impair energy production causing cellular damage which could be detrimental to an organism. Mitochondrial dynamics such as fission, fusion, and motility determine the organelle's structure and can indicate the overall health of the cell. Dictyostelium discoideum, a well-established model for mitochondrial dynamics, contains two GTPase proteins that are predicted to mediate mitochondrial dynamics, FszA and FszB. In this study, we overexpressed GFP tagged FszA and FszB proteins to gain insight into their role in the mitochondrial dynamics of D. discoideum. Through live imaging, we quantified mitochondrial fission and fusion events, localization of the proteins with respect to fission and fusion events, and mitochondrial velocity. Results show that the overexpression of FszA-GFP, FszB-GFP, and GFP-FszB significantly decreased mitochondrial fission and fusion, and overexpressed GFP-FszA significantly decreased mitochondrial fusion compared to the control AX4 strain. Images of GFP-FszA and FszA-GFP strains showed little co-localization with the mitochondria during fission and fusion events, but GFP-FszB and FszB-GFP were localized to the mitochondria during fission events. Overexpression of FszB-GFP and GFP-FszB also significantly decreased mitochondrial velocity. The results of this study give insight into the underlying mechanism behind mitochondrial dynamics and could advance future studies in D. discoideum neurodegeneration models.

Keywords:  Dictyostelium discoideum; FszA; FszB; FtsZ proteins; fission; fusion; mitochondrial dynamics

DOI:  https://doi.org/10.3389/fcell.2026.1903771
Acta Neuropathol Commun. 2026 Jul 27. pii: 189. [Epub ahead of print]14(1):

Neuro-glial lipid imbalance in a Drosophila model of amyotrophic lateral sclerosis 8.

Lovleen Garg, Kriti Chaplot, Gowhith Kuppili, Shweta Tendulkar, Jomon Joseph, Siddhesh Kamat, Girish Ratnaparkhi.

  Membrane Contact sites (MCS) have emerged as physiologically relevant zones that coordinate inter-organelle communication and cellular function. VAPB, an ER-resident MCS tethering protein, plays a central role in regulating MCSs through its numerous protein interactors, thereby influencing cellular homeostasis. A pathogenic missense VAPBP56S mutation causes familial Amyotrophic Lateral Sclerosis 8 (ALS8) in humans, with progressive degeneration of motor neurons. The precise mechanisms underlying the motor neurodegeneration remain poorly understood. In this study, we examine lipid imbalance in the brain of a Drosophila model of ALS8 (VAPBP58S). Specifically, we find that lipid homeostasis is disrupted in an age-dependent manner. Strikingly, cholesterol esters and sphingolipids show an age-dependent increase, while cholesterol shows a decrease. Intriguingly, from a cellular perspective, despite the accumulation of triacylglycerols (TAGs) in the brains of VAPBP58S animals, the increased neutral lipid species do not correlate with lipid droplets (LDs), which are fewer in density and smaller in size. Lipid imbalance and progressive motor dysfunction in VAPBP58S animals can be reversed by expressing VAPBWT, suggesting a relationship between VAPB activity and lipid flux. To uncover VAPB's role in lipid homeostasis, we modulate VAPB activity in neurons and glia to dissect out tissue-specific roles. We find that both cell types contribute to lipid homeostasis in differential ways. In glia, LD flux is strongly dependent on VAPB activity, a dependence further recapitulated in cultured human cell lines, suggesting evolutionary conservation of the regulatory mechanism. Thus, we hypothesize that lipid dysregulation constitutes a critical pathogenic feature of ALS8, with the VAPBP56S allele disrupting lipid homeostasis in the neuro-glial axis.

Keywords:  Cholesterol; Climbing; Glia; Lipid droplets; Neurodegeneration; VAP33A

DOI:  https://doi.org/10.1186/s40478-026-02391-y
Cells. 2026 Aug 27. pii: 1548. [Epub ahead of print]15(17):

Mitochondria-Lysosome Quality Flux in Aging Mesenchymal Stem/Stromal Cells: Quality-Control Bottlenecks and Therapeutic Opportunities for Regenerative Decline.

Lingling Cheng, Gaojie Song.

  Aging mesenchymal stem/stromal cells (MSCs) lose regenerative capacity as redox imbalance, mitochondrial damage, defective organelle quality control and chronic inflammation converge. Yet these processes are commonly considered in isolation, obscuring whether damaged mitochondrial cargo reaches lysosomes and is ultimately degraded. Here, we define mitochondria-lysosome quality flux (MLQF) as an author-proposed, evidence-graded framework that tracks mitochondrial damage from recognition and sorting through lysosomal delivery to terminal lysosomal degradation in aging MSCs. The framework explicitly separates delivery to an acidic compartment from completed degradation and distinguishes direct MSC evidence from cross-model mechanisms and candidate pathways. MSC studies most strongly support macroautophagy-dependent mitophagy, particularly when assessed using dynamic flux reporters. By contrast, mitochondria-derived vesicles and microautophagy-like or piecemeal routes remain incompletely validated in MSCs. Studies in non-MSC systems further show that mitochondria-lysosome contact sites can support lysosomal acidification, although their contribution to natural MSC aging remains unresolved. By locating rate-limiting defects across this continuum, MLQF provides a testable basis for linking incomplete mitochondrial clearance to inflammatory signaling, lineage drift and regenerative decline, and for selecting bottleneck-matched interventions.

Keywords:  cellular senescence; mesenchymal stem/stromal cells; mitochondrial quality control; mitochondria–lysosome quality flux; mitophagy

DOI:  https://doi.org/10.3390/cells15171548
Nat Commun. 2026 09 16. pii: 9647. [Epub ahead of print]17(1):

Cholesterol maintains the degradative capacity of lysosomes during clearance and recycling of dysfunctional mitochondria.

Haoning Yang, Koji Matsuhisa, Meng Wei, Wataru Nishi, Dylan Hong Zheng Koh, Aik Yong Sim, Isabelle Bonne, Yasunori Saheki.

Efficient clearance and recycling of dysfunctional mitochondria through the robust catabolic activity of lysosomes are essential for cellular health. However, how membrane lipids contribute to maintaining the degradative capacity of lysosomes remains poorly understood. Here, we show that cholesterol plays a critical role in preserving the functional integrity of degradative lysosomes. Clearance of damaged mitochondria by degradative lysosomes is tightly coupled with the acute accumulation of phosphatidylinositol 4-phosphate (PI4P) on the lysosomal surface via PI4KIIα activity. This PI4P accumulation activates oxysterol-binding protein (OSBP)-mediated cholesterol transport from the endoplasmic reticulum (ER) to lysosomal membranes. The resulting efflux of cholesterol from the ER activates sterol regulatory element-binding protein 2 (SREBP-2), enhancing cholesterol production. Sustained cholesterol accumulation on lysosomal membranes maintains lysosomal acidity and membrane integrity for efficient mitochondrial degradation. This degradation process then leads to the release of free fatty acids and their recycling and storage through the formation of DGAT1-dependent lipid droplets. These findings uncover a key phosphoinositide-regulated cholesterol transport pathway that promotes the clearance and recycling of dysfunctional mitochondria, a process whose impairment is closely linked to neurodegeneration.

DOI: https://doi.org/10.1038/s41467-026-77423-1
Methods Mol Biol. 2027 ;3074 85-103

A Standardized Protocol for Generating iPSC-Derived Human Microglia for Functional Genomic Assays.

Jason Ngo, Emily Lee, Marta Olah, Falak Sher.

  Human induced pluripotent stem cell (iPSC)-derived microglia (iMG) provide an in vitro experimental system for studying human microglial biology, neuroinflammation, and genetic risk mechanisms associated with neurological disease. This chapter describes a standardized, scalable, and reproducible protocol for the differentiation of human iPSCs into functional microglia-like cells, with particular emphasis on applications in transcriptional and epigenomic network analysis. The protocol supports high-viability floating iMG production, compatibility with pooled CRISPR perturbation approaches, and downstream multiomic profiling, including single-cell RNA sequencing, chromatin accessibility assays, and proteomics. Detailed procedures are provided for iPSC maintenance, hematopoietic progenitor cell generation, microglial maturation, functional genomics integration, and quality control.

Keywords:  ATAC-seq; CRISPR screening; Epigenomics; Functional genomics; Perturb-seq; Single-cell RNA sequencing; Stem cell differentiation; Transcriptional networks; iPSC-derived microglia

DOI:  https://doi.org/10.1007/978-1-0716-5539-9_6
Eur J Neurosci. 2026 Sep;64(5): e70687

Human iPSC-Derived Brain Organoids: A Disease-Oriented Evaluation of Modeling Fidelity.

Bishwa R Pokharel, Abigail Dickerson, Sanjana G Shenoy, Mira S Ramasamy, Niska Majumdar, Paul P Cook, Shaw M Akula.

  Two-dimensional (2D) cell culture systems and conventional animal models have been crucial in the study of central nervous system (CNS) pathology. However, they are limited in their abilities to elucidate human-specific neurobiology and disease etiology. Human induced pluripotent stem cell (iPSC)-derived brain organoids have emerged as a complementary platform that offers a three-dimensional (3D) structure and human-specific environment. They exhibit cellular heterogeneity, apical-basal polarity, and the ability to perform tissue-specific functions such as absorption, secretion, and electrophysiological activity. This review critically evaluates the use of iPSC-derived brain organoids as a disease-focused approach to study neuroinflammation, neurodegeneration, infection, and aging. Organoids provide a model for early pathogenic events, cellular interactions, and human-specific responses, providing insights into disease hallmarks such as tau and amyloid pathology, the vulnerability of dopaminergic neurons, and host-pathogen interactions. However, they are limited by developmental immaturity, inadequate systemic integration, and heterogeneity across models, which hinders their ability to represent late-stage disease progression and human level physiology. Therefore, their primary use lies in modeling human-specific early disease mechanisms, while integration with animal models and in vitro systems is crucial for progressing translational neuroscience.

Keywords:  CNS; brain; iPSC; organoids

DOI:  https://doi.org/10.1111/ejn.70687
J Neurosci. 2026 Sep 15. pii: e1862242026. [Epub ahead of print]

Visualizing synaptic disruptions in the release and regulation of dopamine hotspots in Huntington's Disease.

Sarah J Yang, Natsumi Komatsu, Brendan Butler, Jackson Travis Del Bonis O'Donnell, Francesca Giordani, Ashvin Irrinki, David V Schaffer, Markita P Landry.

Dopamine neuromodulation is a critical process that facilitates learning, motivation, and motor control. Disruption of these processes has been implicated in several neurodegenerative disorders including Huntington's Disease (HD). While dopaminergic signaling is a therapeutic target for treating physical and psychiatric HD symptoms, the mechanism by which dopaminergic dysfunction occurs during HD is unknown. New tools for the visualization of dopamine dynamics at the spatiotemporal resolution of neuromodulator release (ms) and dopaminergic boutons (µm) provide a richer understanding of how dopamine signaling is disrupted in HD. Here we employ near-infrared fluorescent catecholamine nanosensors (nIRCats) to image dopamine release within the striatum of R6/2 Huntington's Disease model mice of either sex. We find that dorsal striatal dopamine release decreases with progressive degeneration and that these deficits are primarily driven by a decrease in the number of nIRCat imaged dopamine release sites, termed dopamine hotspots, combined with decreased release fidelity. Using nIRCat's high spatial resolution, we track individual dopamine hotspots over repeated stimulations and pharmacological applications to measure dopamine release fidelity from individual sites. Compellingly, we found that D2-receptor (D2R) antagonist sulpiride drives increased fidelity of dopamine hotspot activity in wild type striatum but not in late-disease HD striatum, suggesting that D2R regulation of dopamine release is compromised in late HD. These findings, enabled by nIRCats, provide more detailed insights into how dopamine release is disrupted and dysregulated during Huntington's Disease.Significance statement Huntington's Disease (HD) is a neurodegenerative disorder with no cure. Dopamine signaling is known to deteriorate in HD but has not been studied at the level of individual release sites. Here, we image dopamine release from individual dopamine release sites in R6/2 HD mouse brain slices containing the striatum with novel dopamine nanosensors. We find that dopamine release site number and release fidelity are decreased in late HD. Furthermore, we demonstrate that D2-receptor signaling may be altered in late disease R6/2 HD mice, and that these disruptions are likely to drive decreased dopamine release fidelity over multiple stimulations. These findings suggest dopaminergic neurons projecting to the striatum as a potential therapeutic target for HD treatment to complement more commonly targeted medium spiny neurons.

DOI: https://doi.org/10.1523/JNEUROSCI.1862-24.2026
Nat Cell Biol. 2026 Sep 15.

DFCP1-containing ER microdomains mediate lysosomal membrane repair.

Maja Radulovic, Sascha Pust, Athanasios Kournoutis, Di Chen, M Isabel Giner, Qijian Liang, Santosh Phuyal, Thomas J Böddeker, Katy McCarron, Eric Herrmann, Kevin Rose, Sebastian W Schultz, Andreas Brech, James H Hurley, Claudio Bussi, Luis Bonet-Ponce, Maximiliano G Gutierrez, Camilla Raiborg, Harald Stenmark.

Lysosomal membrane integrity is essential for preserving cellular homeostasis in response to different stressors. Upon lysosomal membrane permeabilization, cells activate several mechanisms for lysosomal membrane repair, including ESCRT proteins, phosphatidylinositol 4-phosphate (PI4P)-dependent lipid transfer from the endoplasmic reticulum (ER) and conjugation of ATG8 family proteins to single membranes (CASM). The interplay between these pathways and the regulation of the lipid transfer machinery remain incompletely understood. Here we show that phosphatidylinositol 3-phosphate (PI3P)-containing ER domains play a major role in lysosomal membrane repair. PI3P is formed on lysosome-proximal ER domains by the phosphatidylinositol 3-kinase PIK3C3/VPS34 in response to membrane damage, and inhibition or depletion of PIK3C3 inhibits lysosome repair. Mechanistically, the ATPase DFCP1/ZFYVE1 accumulates on lysosome-proximal ER domains by its PI3P binding, triggered by Ca2+ efflux from lysosomes and requiring the ULK1 kinase complex and ER proteins of the VAP family. Downstream of CASM, PI4P, ESCRTs and PI3P, DFCP1 promotes focal accumulation of the lipid channel VPS13C on ER domains proximal to damaged lysosomes to promote their repair. The function and dynamics of DFCP1 depend on its ability to bind and hydrolyse ATP, and absence of DFCP1 compromises cellular resistance to vacuolar damage induced by Listeria monocytogenes. We conclude that DFCP1 mediates concentration of the ER-associated lipid transport machinery at damaged lysosomes to promote their sealing in response to Ca2+ flux and PIK3C3 activation.

DOI: https://doi.org/10.1038/s41556-026-02062-z
Atherosclerosis. 2026 Sep 14. pii: S0021-9150(26)01267-0. [Epub ahead of print]422 121901

Lysosomal dysfunction in vascular smooth muscle cells: Mechanisms and therapeutic targets in vascular disease.

Qi Zhu, Wa Du, Chenran Wang, Matthew Corriere, Yanbo Fan.

  Vascular smooth muscle cell (VSMC) dysfunction is a common feature of atherosclerosis, aortic aneurysms, vascular calcification, and other vascular diseases. In VSMCs, lysosomes play an essential role in autophagic degradation, endocytic cargo processing, intracellular recycling, and cellular stress responses. Under pathological conditions, lysosomal dysfunction promotes foam cell formation, osteogenic differentiation, and extracellular matrix remodeling in VSMCs, thereby contributing to vascular diseases. This review summarizes current evidence on the role of lysosomal dysfunction in VSMC biology, its contribution to vascular diseases, and the molecular mechanisms involved. We also discuss strategies to restore lysosomal function and the challenges of targeting lysosomal pathways in VSMCs. A better understanding of VSMC-specific lysosomal regulation may facilitate the development of therapeutic strategies for vascular diseases.

Keywords:  Aortic aneurysm; Atherosclerosis; Autophagy; Lysosome dysfunction; Vascular calcification; Vascular disease; Vascular smooth muscle cells

DOI:  https://doi.org/10.1016/j.atherosclerosis.2026.121901
Cell Mol Neurobiol. 2026 Sep 18. pii: 141. [Epub ahead of print]46(1):

Using Brain Organoids to Explore Human Neurobiology.

Hany E Marei, Anwarul Hasan.

  One of the major challenges in neuroscience is understanding how the human brain develops into a highly organized and functionally integrated organ, because many developmental processes cannot be investigated directly in humans and are not fully recapitulated in animal models. Brain organoids derived from human pluripotent stem cells have emerged as powerful three-dimensional (3D) experimental models that recapitulate key features of human neurodevelopment, including regional patterning, cellular diversification, and neural circuit assembly. Recent advances in long-term organoid culture, organoid bioengineering, vascularization, assembloid technology, transplantation, multi-omics, and artificial intelligence have substantially expanded the applications of brain organoids to investigate human neurodevelopment, model neurological disorders, support drug discovery, and advance therapeutic development. Integration of single-cell and spatial multi-omics with computational approaches has enabled robust molecular benchmarking, assessment of developmental fidelity, and evaluation of organoid reproducibility, facilitating direct comparison with primary human fetal brain tissue. However, current organoid systems remain limited owing to incomplete cellular and tissue complexity, inter-organoid variability, limited vascularization, limited functional maturation, and incomplete physiological integration. Continued advances in tissue engineering, computational biology, and standardized differentiation protocols are expected to enhance the biological fidelity and translational utility of brain organoids for basic and translational neuroscience, as well as mechanistic and translational research.

Keywords:  3D in vitro models; Brain organoids; Cellular heterogeneity; Chromatin architecture; Clonal lineage tracing; Cortical progenitors; Corticogenesis; Developmental trajectories; Disease modeling platforms; Epigenetic maturation; Functional maturation; Human neurodevelopment; Human–rodent chimeric models; In vivo integration; Increasingly complex neural network activity; Mechanistic neuroscience; Multi-omic analysis; NMDA receptor development; Neural circuit formation; Neural stem cells; Neurodevelopmental disorders; Organoid transplantation; Outer radial glia; Pluripotent stem cells; Synaptic maturation; Transcriptional regulation

DOI:  https://doi.org/10.1007/s10571-026-01808-5
Cell Rep Methods. 2026 Sep 15. pii: S2667-2375(26)00302-4. [Epub ahead of print] 101601

Parallelized bright-field and fluorescence imaging platform for organoids.

Kanghyun Kim, Rubal Singla, Amey Chaware, Jieun Park, Ina Klockner, Josh Lerner, Kevin Li, Fanghong Shen, Clay Dugo, Paul Reamey, Aurélien Bègue, Mark Harfouche, Abhishek Mishra, Christopher A McPherson, Jason L Stein, Roarke Horstmeyer.

  Organoid viability, maturation, and growth are commonly assayed through bright-field and fluorescence microscopy using a single-objective lens. However, standard imaging systems pose significant limitations for high-throughput applications, particularly in large-scale experiments requiring simultaneous imaging of organoids, necessitating tools that can rapidly and consistently capture organoid features while minimizing disturbances to culture conditions. Here, we present a multi-camera array scanner (MCAS) that parallelizes imaging through the simultaneous use of 48 objective lenses and sensors, resulting in a 98% reduction in acquisition times compared to commercial high-content imagers. We demonstrate and validate this system in multiple-well plate formats, in both 2D and 3D neural cell cultures, and in bright field and fluorescence. The MCAS improves the efficiency for measuring organoid growth rates, assessing responses to morphogens and drugs, and measuring viral transduction rates. Together, these findings establish the MCAS as a scalable and versatile imaging platform for rapid phenotyping in organoid research.

Keywords:  CP: imaging; CP: stem cell; automation technology; cortical brain organoids; drug screening; fluorescent imaging; high-throughput imaging; microscopy

DOI:  https://doi.org/10.1016/j.crmeth.2026.101601
Sci Adv. 2026 Sep 18. 12(38): eaef3538

A suite of fluorescent timers for visualizing protein spatiotemporal dynamics in plants.

Yukihiro Nagashima, Xin Qiao, Suji Ye, Qiaochu Shen, Jun Liu, Delui Liu, Fausto Andrés Ortiz-Morea, Xue Ding, Vinita Sharma, Juan Dong, Libo Shan, Hisashi Koiwa.

Fluorescent timers (FTs), including tandem FTs (tdFTs), enable real-time visualization of precise protein localization and turnover within living cells, an essential property for elucidating protein function. In plants, application of FT has been limited largely because of the mismatch between FT maturation kinetics and the slow growth rate of plant cells. Here, we systematically evaluated multiple FT configurations to identify tdFTs optimized for plant systems. Using Arabidopsis thaliana, we tested a range of fluorescent proteins (FPs) differing in maturation times. Functional tdFT expression was confirmed in diverse organelles. Selected tdFT configurations were developed to track protein biogenesis/turnover in autoimmunity, protein transport in abiotic stress, and polarized protein translocation in determination of cell fate. When the large size of tdFT interfered with protein mobility, we developed a bipartite FT, separately tagging proteins with different FPs. This resource provides a framework for selecting FTs to dissect dynamic protein behaviors in plant cells.

DOI: https://doi.org/10.1126/sciadv.aef3538
Structure. 2026 Sep 18. pii: S0969-2126(26)00261-3. [Epub ahead of print]

Cryo-electron tomography of functionally validated synapses in human induced neurons.

Aiden J Houcek, Rong Sun, Spencer J Rothfuss, Oliver R Chalkley, Ege T Kavalali, Qiangjun Zhou.

  Cryo-electron tomography (cryo-ET) has provided unparalleled insights into synaptic architecture, yet its application to human neurons has been limited by sample preparation challenges. Here, we establish an integrated platform that combines human induced neurons (iNs) cultured directly on electron microscopy (EM) grids with functional electrophysiological validation and cryo-ET imaging. Human iNs formed extensive neuronal networks on EM grids, exhibited reliable synaptic transmission and intrinsic excitability, and maintained short-term synaptic plasticity. While iNs exhibited intact synaptic transmission on EM grids, we found that EM grids acted as a current sink for extracellular electrical stimulation. Following vitrification, cryo-ET enabled direct visualization of ultrastructural features of excitatory synapses, including synaptic vesicle pools, active zone proximal vesicles, postsynaptic densities, and associated organelles. Together, our results establish a foundation for structurally and functionally dissecting synaptic organization between human iNs and provide initial insight into cryo-ET imaging of synapses from stem cell-derived human neurons.

Keywords:  cryo-electron tomography; electrophysiology; human induced neurons; synaptic ultrastructure

DOI:  https://doi.org/10.1016/j.str.2026.08.014