bims-axbals 2026-09-27 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2026–09–27
25 papers selected by
TJ Krzystek

AUG-Dependent Translation of Antisense Repeat Transcripts Contributes to Dipeptide Repeat Protein Production in C9ORF72 Expansion Carriers.
Loss of TDP-43 function drives cryptic circular RNAs in neurodegenerative diseases.
What can we learn on ALS pathophysiology from iPSC-derived motor neurons harbouring TARBDP mutations: a systematic review.
Integrative synaptosome multi-omics reveals disrupted synapse organization and localized cryptic transcripts in C9ORF72-frontotemporal dementia.
Restoring multiple TDP-43 cryptic targets, but not solely Unc13a , rescues motor neuron disease.
Defective membrane dynamics in Huntington's disease: A role of Rab11 dysfunction.
TDP-43 dysfunction induces cryptic circular RNAs in ALS/FTD.
Mitochondrial Quality Control Impairment Is a Hallmark of TDP-43G376D ALS Patient-Derived Fibroblasts.
Convergent TBK1 loss of function drives gasdermin E dependent pyroptosis and motor neuron degeneration across ALS genotypes.
Dysregulation of mitochondrial bioenergetics, RNA processing, and cellular stress responses in FUS-ALS: a multi-dataset transcriptomic perspective.
CDKL5 deficiency impairs TBK1-mediated autophagy and clearance of neuronal protein aggregates.
Lysosomal function and homeostasis: From cellular adaptation to disease pathogenesis.
TBK1 overexpression reduces neuronal loss in the P497S UBQLN2 mouse model of ALS/FTD.
Ciliary neurotrophic factor slows axonal transport of signalling endosomes.
Drug screen and machine learning predict neuroprotective agents in a preclinical human model of childhood dementia.
Chronic Contractile Activity Remediates Lysosomal Dysfunction in a Muscle Cell Lysosomal Disease Model.
Microtubule-associated protein 1S phosphorylation regulates dynein-driven transport in the brain and synaptogenesis in neurons.
Mitochonic acid 5 alleviates amyotrophic lateral sclerosis phenotypes via mitochondrial augmentation.
Fluid and Imaging Biomarkers in Huntington's Disease.
Enhanced axonal mitochondrial motility and neural activity-induced energy deficits destabilize synaptic transmission in models of bipolar disorder.
Dynamin-2 promotes Atg9A retrieval from phagophores during autophagy.
miR-196a reduces mutant Huntingtin aggregates by Rad23b-mediated degradation in Huntington's disease.
A lifespan single-cell atlas of the human developing hippocampus benchmarks familial Alzheimer's disease brain organoids.
PP2A and CDK16 antagonistically regulate WIPI2B phosphorylation and neuronal autophagosome biogenesis.
Deep Insights Without Clearing: An Optimized Protocol for Patient-Derived Whole-Organoid Immunofluorescence Imaging.

Cells. 2026 Sep 19. pii: 1701. [Epub ahead of print]15(18):

AUG-Dependent Translation of Antisense Repeat Transcripts Contributes to Dipeptide Repeat Protein Production in C9ORF72 Expansion Carriers.

Sandra Almeida, Yuanzheng Gu, Mark W Kankel.

  A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Bidirectional transcription of the repeat expansion generates sense and antisense RNAs that are translated into dipeptide repeat (DPR) proteins, but the mechanisms of translation initiation remain incompletely understood. Here, we used CRISPR-Cas9 genome editing and steric-blocking antisense oligonucleotides (ASOs) to investigate the role of AUG codons within the antisense repeat RNA. Deletion of an AUG-containing region upstream of the antisense repeats markedly reduced poly(GP) production without affecting antisense RNA levels, demonstrating that this sequence is required for efficient poly(GP) synthesis. We further found that unspliced sense transcripts containing the repeat expansion likely serve as templates for poly(GA) and poly(GR) production in motor neurons. Finally, ASOs targeting the antisense AUG-containing region reduced poly(PR) and poly(GP) levels without altering repeat RNA abundance, supporting a role for AUG-dependent translation of the antisense repeat RNA. These findings provide new insights into the mechanisms of DPR production and suggest that translation-blocking ASOs may represent a therapeutic strategy for C9ORF72-associated ALS/FTD.

Keywords:  ALS; ASO; FTD; dipeptide repeat protein; iPSC-derived neurons; repeat RNA translation

DOI:  https://doi.org/10.3390/cells15181701
bioRxiv. 2026 Sep 20. pii: 2026.09.17.752169. [Epub ahead of print]

Loss of TDP-43 function drives cryptic circular RNAs in neurodegenerative diseases.

Chengzhang Zhu, Zhiyan Zhao, Bojun Song, Yan-Ming Chen, Yu Xiao, Zhe Zhang, Yingzhi Ye, Niannian Xu, Ruijia Zhang, Yongxin Huang, Juan C Troncoso, Chuan He, Shuying Sun.

TAR DNA-binding protein 43 (TDP-43) is a key pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) and a critical regulator of RNA splicing. While loss of TDP-43 induces aberrant splicing in linear transcripts, its impact on circular RNA (circRNA) biogenesis remains unexplored. Here, we show that TDP-43 depletion in human neurons induces widespread circRNA changes, especially upregulation of a distinct class of cryptic circRNAs that arise specifically upon loss of TDP-43. Some of these cryptic circRNAs incorporate cryptic exons derived from intronic sequences. Notably, these cryptic circRNAs exhibit greater stability than their corresponding linear RNA isoforms and accumulate progressively in neurons. Moreover, cryptic circRNAs are elevated in postmortem brain tissues from ALS, FTD and Alzheimer's disease (AD) patients. These findings reveal a previously unrecognized role for TDP-43 in repressing cryptic circRNA formation and establish these circRNAs as stable molecular signatures of TDP-43 dysfunction.

DOI: https://doi.org/10.64898/2026.09.17.752169
Transl Neurodegener. 2026 Sep 20. pii: 49. [Epub ahead of print]15(1):

What can we learn on ALS pathophysiology from iPSC-derived motor neurons harbouring TARBDP mutations: a systematic review.

Elena Pasho, Alberto Catanese, Edor Kabashi, Sorana Ciura.

  The degenerating motor neurons of amyotrophic lateral sclerosis (ALS) patients are characterized by the accumulation of cytoplasmic aggregates, specifically enriched in ubiquitinated TDP-43. Expressed mainly in the nucleus and partially in the cytoplasm to execute its role in RNA metabolism, the exact mechanisms that bring TDP-43 to aggregate in disease have yet to be described. Unfolding these processes could bring us closer to effective drug development that is drastically lacking in the ALS research field. Induced pluripotent stem cells (iPSCs) offer a promising platform for studying ALS pathogenesis directly in the relevant human genetic backgrounds of ALS patients. Since 2012, more than 30 published studies have investigated altered cellular and subcellular features in iPSC-derived motor neurons from ALS patients harbouring mutations in the TARDBP gene (encoding for TDP-43 protein). However, there are discrepancies in the obtained results, which call into question the relevance of this model for ALS disease modelling and its use for drug development efforts. Thus, there is a need in the field for a clear and detailed layout and summary of all the published data on ALS modelling using TARDBP mutant iPSC-derived motor neurons. In this systematic review, we analyse all phenotypic assessments that have been done on iPSC-derived motor neurons derived from TARDBP ALS patients on the morphology, functionality and the viability of these cells. We also analyse if this model recapitulates ALS pathology in vitro by comparing studies that looked at TDP-43 and neurofilament aggregation, as well as stress granule dynamics. Importantly, we compare the technical details of all the discussed studies, such as differentiation protocol, age and purity of the used motor neurons and quantification method, in order to discuss how all of these parameters affect the observed phenotypes. Lastly, we also expand our review to all other different cell types that have been differentiated from TARDBP iPSCs, highlighting non-cell autonomous mechanisms of TDP-43-ALS pathophysiology. By integrating findings across studies, this review identifies commonalities and discrepancies, discusses methodological advancements and limitations, and highlights potential therapeutic targets revealed through TARDBP iPSC-derived models.

Keywords:  ALS; Disease modelling; Drug screening; Motor neuron; Phenotype; TARDBP; TDP-43; iPSC

DOI:  https://doi.org/10.1186/s40035-026-00580-2
Acta Neuropathol. 2026 Sep 25. pii: 41. [Epub ahead of print]152(1):

Integrative synaptosome multi-omics reveals disrupted synapse organization and localized cryptic transcripts in C9ORF72-frontotemporal dementia.

Ashton M Spillman, Eric B Alsop, Lauren M Gittings, Krystine Garcia-Mansfield, Ignazio Piras, Anna Bonfitto, Melissa N Martinez, Ritin Sharma, Kim R Preller, Matthew Huentelman, Patrick Pirrotte, Kendall Van Keuren-Jensen, Rita Sattler.

  Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are linked neurodegenerative diseases characterized by both synaptic dysfunction and TDP-43 pathology. A hexanucleotide repeat expansion (HRE) in the C9ORF72 (C9) gene represents the most common genetic cause of FTD and ALS, yet the synapse-specific mechanisms underlying disease pathogenesis remain poorly understood. Here, we performed integrated multi-omic profiling of synaptosomes enriched from postmortem frontal cortex and patient-derived induced pluripotent stem cell (iPSC)-cortical neurons to define molecular alterations associated with C9-FTD-mediated synaptic dysfunction. Proteomic profiling of frontal cortex-derived synaptosomes identified 1,324 differentially abundant proteins (p<0.05) enriched in pathways regulating synaptic vesicle transport and synapse organization, while synaptosomal RNA sequencing revealed 2,835 differentially expressed protein-coding genes. C9-FTD iPSC-cortical neurons exhibited reductions in excitatory and inhibitory postsynaptic markers, accompanied by progressive impairment of neuronal network activity, supporting both structural and functional deficits. iPSC-derived synaptosomes recapitulated key molecular pathways observed in patient brain, revealing convergent dysregulation of synaptic signaling pathways. Comparative analyses revealed divergence between protein and RNA alterations, consistent with the disruption of regulatory processes that link RNA and protein abundance in diseased synapses. Consistent with TDP-43 loss-of-function pathology we identified cryptic exon (CE)-containing transcripts within C9-FTD frontal cortex-derived synaptosomes, including KALRN and STMN2, providing evidence that aberrantly spliced RNAs localize to synaptic compartments. Together, these findings define convergent molecular pathways underlying synapse vulnerability in both C9-FTD model systems and identify synaptic localization of CE-containing transcripts as a previously unrecognized feature of TDP-43 proteinopathy.

Keywords:  Cryptic Exon (CE); Frontotemporal Dementia (FTD); Proteomics; Synaptosome; TAR DNA-binding protein 43 (TDP-43); Transcriptomics

DOI:  https://doi.org/10.1007/s00401-026-03084-5
bioRxiv. 2026 Sep 18. pii: 2026.09.11.751082. [Epub ahead of print]

Restoring multiple TDP-43 cryptic targets, but not solely Unc13a , rescues motor neuron disease.

Aswathy Peethambaran Mallika, Meghraj Singh Baghel, Opal Sitzman, Jessica G Yu, Tianyu Cao, Shruti Renganathan, Irika R Sinha, Tatiana Melnikova, Jonathan P Ling, Philip C Wong.

Dysfunction of TAR DNA-binding protein 43kDa (TDP-43) underlies amyotrophic lateral sclerosis (ALS), a neurodegenerative disorder with limited therapeutic options. While current therapeutic approaches are designed to individually target unique cryptic exons of TDP-43 such as UNC13A , the sufficiency of such a strategy to mitigate motor neuron disease remains unclear. Using a mouse model lacking TDP-43 in spinal motor neurons which mimics early stages of ALS, we show that the exclusion of Unc13a cryptic exon fails to mitigate motor neuron disease. In contrast, the restoration of multiple TDP-43 cryptic targets, including Unc13a , attenuated motor neuron loss, and rescued motor neuron disease. Additionally, compared to brain neurons, spinal motor neurons accumulate markedly lower amounts of Unc13a cryptic exons in mice and humans, suggesting that the contribution of this TDP-43 cryptic target to spinal motor neuron loss may be limited. Together, these results strongly support ALS therapeutic strategies designed to simultaneously restore multiple TDP-43 cryptic targets to attenuate spinal motor neuron loss.

DOI: https://doi.org/10.64898/2026.09.11.751082
J Huntingtons Dis. 2026 Sep 24. 18796397261490952

Defective membrane dynamics in Huntington's disease: A role of Rab11 dysfunction.

An Wu, Xueyi Li.

  Huntington's disease (HD) is a life-threatening neurodegenerative disease caused by a mutation in Huntingtin (HTT). Neuropathology in HD is marked by a progressive loss of neurons in the caudate putamen and the deposition of mutant HTT aggregates as cytoplasmic and nuclear inclusions. At present, no disease-modifying treatment for HD exists. Vesicular trafficking is a fundamental way to target proteins to their functional sites and thus essential for the maintenance of cellular homeostasis. HTT associates with vesicles and interacts with molecular motor proteins driving vesicle movement along cytoskeletons and with the molecular machines orchestrating vesicle formation, tethering and fusion. Rab proteins play key roles in each of the four major steps in vesicular trafficking. Here we provide an overview of Rab proteins perturbed in HD, how their disturbance contributes to disease progression, and ways to target them for therapy in Huntington's disease (HD).

Keywords:  GTPase-activating protein; Huntington's disease; Rab11; guanine nucleotide exchange factor; recycling endosome; vesicular trafficking

DOI:  https://doi.org/10.1177/18796397261490952
bioRxiv. 2026 Sep 20. pii: 2026.09.17.752342. [Epub ahead of print]

TDP-43 dysfunction induces cryptic circular RNAs in ALS/FTD.

NYGC ALS Consortium

Nuclear depletion of TDP-43 is a defining pathological feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), leading to widespread RNA misprocessing, including the formation of cryptic exons. Here, we identified TDP-43 as a regulator of circular RNA (circRNA) biogenesis in multiple human neuronal cell models, and showed that its dysfunction induces the de novo formation of cryptic circular RNAs (c-circRNAs). Analysis of post-mortem brain transcriptomic data identified a subset of c-circRNAs which are specific for ALS and FTD cases with TDP-43 pathology. Further, we developed highly sensitive rolling-circle amplification-based circRNA detection assays that allow to distinguish TDP-43 pathology in human CNS tissues with a 0.99 AUC. We found that c-circRNAs can co-occur with cryptic linear splicing events, uncovering complex RNA misprocessing hotspots that induce loss of disease-relevant proteins, including RPTOR and EHMT1. Notably, one of these c-circRNAs originates from UNC13A, a gene whose cryptic exon has previously been linked to one of the major GWAS hits in ALS/FTD and that is being pursued as a therapeutic target through splice-switching ASOs. We showed that c-circUNC13A is co-regulated with the linear cryptic transcript and suppression of UNC13A cryptic exon results in c-circUNC13A reduction in cultured neurons and in vivo, highlighting its potential as a target engagement biomarker for emerging UNC13A-directed therapies. Overall, this work identifies a novel molecular mechanism for TDP-43 dysfunction, opening novel avenues for understanding disease pathogenesis and developing much needed pathology biomarkers.

DOI: https://doi.org/10.64898/2026.09.17.752342
Antioxidants (Basel). 2026 Aug 22. pii: 1051. [Epub ahead of print]15(9):

Mitochondrial Quality Control Impairment Is a Hallmark of TDP-43G376D ALS Patient-Derived Fibroblasts.

Giuseppe Petito, Maria Ventriglia, Victoria Stefania Del Fiore, Arianna Cuomo, Federica Cioffi, Francesco Manfrevola, Flora Guerra, Lucia Bertuccini, Giulia Ricci, Gilda Cobellis, Antonia Lanni, Cecilia Bucci, Roberta Romano, Rosalba Senese.

  Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disorder strongly associated with mitochondrial dysfunction and impaired proteostasis. Mutations in TARDBP, encoding TAR DNA-binding protein 43 (TDP-43), contribute to disease pathogenesis through cytoplasmic mislocalization and aggregation. Among these, the ALS-linked TDP-43G376D mutation has been previously associated with oxidative stress, mitochondrial fragmentation, and impaired oxidative phosphorylation. Here, we investigated the impact of TDP-43G376D on Mitochondrial Quality Control (MQC) pathways using patient-derived dermal fibroblasts carrying the mutation at early and advanced disease stages, complemented by HEK293T and Neuro2a cellular models expressing mutant TDP-43. We show that TDP-43G376D impairs mitophagic flux, as evidenced by reduced delivery of damaged mitochondria to lysosomes. This was accompanied by pronounced disruption of mitochondrial cristae architecture and accumulation of mitochondrial DNA damage, indicating compromised mitochondrial genome integrity. Furthermore, TDP-43G376D induces sustained activation of the mitochondrial Unfolded Protein Response (UPRmt), consistent with persistent mitochondrial stress, while selectively impairing the sirtuin-dependent antioxidant branch. In parallel, activation of the Endoplasmic Reticulum UPR (UPRER) was observed, indicating a coordinated engagement of cellular stress pathways. Collectively, our findings identify coordinated alterations in multiple MQC pathways associated with TDP-43G376D rather than isolated mitochondrial defects, supporting further investigation of these pathways in larger and disease-relevant ALS models.

Keywords:  Amyotrophic Lateral Sclerosis (ALS); Endoplasmic Reticulum stress; Mitochondrial Quality Control (MQC); TDP-43; mitochondrial Unfolded Protein Response (UPRmt); mitochondrial dysfunction; mitophagy; oxidative stress

DOI:  https://doi.org/10.3390/antiox15091051
Cell Death Differ. 2026 Sep 21.

Convergent TBK1 loss of function drives gasdermin E dependent pyroptosis and motor neuron degeneration across ALS genotypes.

Zihan Qi, Xuehe Liu, Lu Geng, Xiaoni Liu, Siqi Dong, Jiatong Li, Wenyi Hua, Yifan Deng, Suomei Lai, Yuhao Wang, Yuhong Xia, Yuyun Han, Dongqing Zhu, Xiang Zhang, Jinsong Li, Jixi Li, Xiangjun Chen.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive loss of upper and lower motor neurons, yet it is unknown whether diverse genetic risks converge on a shared pathogenic pathway. Here we identify a common mechanism linking TBK1 insufficiency to inflammatory cell death across distinct ALS genotypes. Whole-exome sequencing of 8 familial ALS patients revealed that 3 of them carried pathogenic variants, namely TBK1 (R573H), TDP-43 (G298V)/GARS (I44M)/SETX (L1304W), or SOD1 (G94R)/SQSTM1 (G262R). Motor neurons differentiated from patient iPSCs, despite their different mutations, consistently exhibited axonal and neurite shortening, reduced TBK1 activity, increased phosphorylated TDP-43 with cytoplasmic aggregation, and co-assembly of caspase-8/7 with the pyroptosis executor gasdermin E (GSDME). Neuroinflammatory stimulation further enhanced caspase-8/7 and GSDME cleavage, increased RIPK1 phosphorylation and suppressed TBK1 activation in patient-derived motor neurons, whereas TBK1 knockdown in SH-SY5Y cells recapitulated these phenotypes. Furthermore, a generated humanized TBK1 R573H knock-in mouse developed progressive motor deficits accompanied by spinal motor-neuron loss, neuroinflammation, and TDP-43 pathology. In these mice, LPS challenge produced increased cleavage of GSDME and caspase-7, along with a marked upregulation of p-TDP-43 expression in vulnerable neurons. Together, these data identify TBK1 loss of function as an upstream driver of a caspase-8/7-GSDME pyroptotic program in motor neurons and provide a mechanistic bridge between neuroinflammation, TDP-43 proteinopathy, and neurodegeneration. Targeting the TBK1-caspase-GSDME axis may offer a tractable strategy for disease modification in ALS.

DOI: https://doi.org/10.1038/s41418-026-01874-8
Neurol Sci. 2026 Sep 24. pii: 807. [Epub ahead of print]47(10):

Dysregulation of mitochondrial bioenergetics, RNA processing, and cellular stress responses in FUS-ALS: a multi-dataset transcriptomic perspective.

Francisca Walden, Yasna Reihani, Austin Peters, Masood Sepehrimanesh.

   BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by motor neuron (MN) degeneration. Mutations in the FUS gene, particularly the aggressive P525L variant, disrupt nuclear localization of the FUS RNA-binding protein, resulting in cytoplasmic accumulation, altered RNA metabolism, and toxic aggregation. Astrocytes also contribute to disease progression through non-cell-autonomous mechanisms.
OBJECTIVE: To identify shared and cell-type-specific molecular mechanisms underlying FUS-associated ALS through integrated transcriptomic analysis of human induced pluripotent stem cell (hiPSC)-derived motor neurons and astrocytes.
METHODS: Five publicly available RNA-sequencing datasets from hiPSC-derived motor neurons and astrocytes carrying FUS mutations were analyzed. Differential gene expression and Gene Ontology enrichment analyses were performed to identify dysregulated genes and pathways.
RESULTS: Both cell types showed convergent dysregulation of mitochondrial/bioenergetic pathways (oxidative phosphorylation, ATP metabolism, translation, membrane potential). Pathway analysis across motor neuron datasets identified largely MN-specific enrichment in cell-cycle regulation, intracellular trafficking, and stimulus response, while only four pathways were shared with astrocytes, most notably NADH dehydrogenase complex assembly, enriched across all datasets and both cell types, marking mitochondrial complex I as the strongest convergent signature. RNA processing/splicing pathways were also enriched, consistent with FUS function. Shared DEGs included upregulated COMT, TXNRD2, and PCDHGB4, and downregulated PCDH17 and C11orf87, implicating neurotransmitter metabolism, antioxidant defenses, and adhesion. Stress pathways (oxidative stress, autophagy, heat-shock, ER stress/UPR) were broadly altered, with UPR genes mainly upregulated and inflammatory genes downregulated.
CONCLUSION: FUS-ALS involves shared mitochondrial complex I and stress-response disturbances layered onto MN-intrinsic cell-cycle and trafficking dysregulation, reflecting convergent and cell-type-specific mechanisms.

Keywords:  Amyotrophic lateral sclerosis; Astrocytes; ER stress/UPR; FUS; Motor neurons; iPSC

DOI:  https://doi.org/10.1007/s10072-026-09412-7
bioRxiv. 2026 Sep 18. pii: 2026.09.13.751248. [Epub ahead of print]

CDKL5 deficiency impairs TBK1-mediated autophagy and clearance of neuronal protein aggregates.

Zhongju Zou, Bilal Kahn, Salwa Sebti, Jason Lucavs, Giomar Rivera-Cancel, Josephine Thinwa.

The clearance of unwanted protein aggregates is essential for maintaining proteostasis and cellular function, particularly in long-lived cells such as neurons, yet the signaling pathways that activate selective autophagy of protein aggregates remain incompletely understood. Here, we identify the neurodevelopmental kinase CDKL5 as an upstream regulator of a signaling pathway involving the TBK1 adaptor SINTBAD and the selective autophagy receptors p62 and TAX1BP1. CDKL5-deficient mice show age-dependent accumulation of detergent-insoluble protein aggregates in the brain, accompanied by impaired TAX1BP1 recruitment and reduced p62 Ser405 phosphorylation. In cultured cells and primary cortical neurons, loss of CDKL5 delays clearance of puromycin- and proteasome-inhibitor-induced aggregates in a manner dependent on CDKL5 kinase activity. Mechanistically, CDKL5 kinase activity is required for SINTBAD Ser504 phosphorylation, a SINTBAD modification that promotes TBK1 activation, resulting in p62 Ser403/405 phosphorylation and TAX1BP1-dependent aggregate clearance. Phosphomimetic SINTBAD rescues these responses in CDKL5-deficient cells. These findings define a CDKL5/SINTBAD/TBK1 signaling axis that couples proteotoxic stress to activation of selective autophagy receptors and identify impaired proteostasis as a previously unrecognized consequence of CDKL5 deficiency.

DOI: https://doi.org/10.64898/2026.09.13.751248
Int Rev Cell Mol Biol. 2026 ;pii: S1937-6448(26)00034-1. [Epub ahead of print]406 1-42

Lysosomal function and homeostasis: From cellular adaptation to disease pathogenesis.

Satoshi Minami, Maho Hamasaki.

  Lysosomes were once considered terminal degradative organelles responsible for disposing of cellular waste. However, recent studies have revealed that lysosomes serve as dynamic signalling and metabolic hubs at the center of diverse biological processes, including nutrient sensing, metabolic regulation, membrane trafficking, autophagy, inflammation, and cell death. To support this broad functional repertoire, lysosomes must possess robust mechanisms to maintain their integrity in the face of damage or stress. In response to lysosomal membrane damage, cells engage multilayered adaptive mechanisms that act in coordination-membrane repair (Repair), selective removal of damaged organelles (Removal), and de novo biogenesis of lysosomes (Regeneration). These processes are mediated by a range of molecular pathways, including the ESCRT complex, the PITT pathway, lysophagy, and TFEB-dependent lysosomal regeneration. Notably, recent findings highlight the noncanonical autophagy-like pathway known as ATG8ylation (conjugation of ATG8s on single membranes), which is activated via the STING-V-ATPase-ATG16L1 axis and functions as a critical hub connecting multiple arms of the lysosomal damage response. In this review, we systematically outline the molecular basis of lysosomal damage responses, including ATG8ylation, and explore how these networks are implicated in a broad spectrum of pathological conditions such as aging, neurodegeneration, cancer, obesity-related disorders, and immune dysfunction. Understanding these lysosomal quality control mechanisms not only sheds light on the fundamental principles of organelle homeostasis but also opens new avenues for therapeutic innovation.

Keywords:  Lysophagy; Lysosome; Organelle damage; Selective autophagy

DOI:  https://doi.org/10.1016/bs.ircmb.2026.04.002
Neurobiol Dis. 2026 Sep 19. pii: S0969-9961(26)00356-6. [Epub ahead of print]229 107611

TBK1 overexpression reduces neuronal loss in the P497S UBQLN2 mouse model of ALS/FTD.

Shaoteng Wang, Melanie A Dolce, Angelina M Burkett, Victoria L Collis, Peyton E Fuller, Grace A Blanco, Nhat T T Le, John H Fingert, Mervyn J Monteiro.

  Missense mutations in UBQLN2 are linked to dominant inheritance of amyotrophic lateral sclerosis (ALS) which is frequently accompanied by frontotemporal dementia (FTD). The encoded UBQLN2 protein functions to maintain proteostasis, collapse of which is increasingly being realized as the cause of many neurodegenerative diseases. During investigations of our UBQLN2 mouse models of ALS/FTD, we observed a significant decline in Tank-binding kinase 1 (TBK1) protein in end-stage mice. The decline could be significant, as haploinsufficiency of TBK1 expression is linked to ALS/FTD. To determine whether the reduction in TBK1 levels is responsible for driving pathogenesis, we crossed P497S UBQLN2 transgenic (Tg) mice with BacTBK1 Tg mice that overexpress TBK1 and analyzed the progeny for signs of pathology. The analysis revealed that double transgenic mice had a significant reduction in neurodegeneration in both the brain and spinal cord (SC) compared to P497S single-Tg mice. Double immunofluorescence staining of P497S mouse tissue revealed TBK1 colocalizes with UBQLN2 aggregates in spinal motor neurons. Biochemical extraction studies of mouse brain tissue revealed increased enrichment of TBK1 along with wild type and mutant UBQLN2 proteins in detergent-insoluble material, suggesting TBK1 gets sequestered by UBQLN2 aggregates. GST-pulldown assays revealed that UBQLN2 binds directly with TBK1, but paradoxically the P497S mutation was found to reduce TBK1 binding. Turnover studies indicated that loss of UBQLN2 destabilizes TBK1, providing another route for its reduction. These findings lead us to propose that efforts directed toward increasing TBK1 expression may provide a therapeutic approach for treating ALS/FTD caused by UBQLN2 mutations.

Keywords:  Amyotrophic lateral sclerosis; Motor neuron disease; Proteostasis; TBK1; UBQLN2

DOI:  https://doi.org/10.1016/j.nbd.2026.107611
Brain Commun. 2026 ;8(5): fcag339

Ciliary neurotrophic factor slows axonal transport of signalling endosomes.

Elena R Rhymes, Cassandra Fetkowitz, Giampietro Schiavo, James N Sleigh, Andrew P Tosolini.

  Efficient axonal transport is essential for maintaining neuronal function, enabling the bidirectional delivery of diverse cargoes between the cell body and distal compartments. In the neuromuscular system, neurotrophic factors regulate motor neuron survival, function and synaptic connectivity, in part, through retrograde trafficking of activated neurotrophic factor-receptor complexes from the neuromuscular junction to the cell body. We recently demonstrated that brain-derived neurotrophic factor stimulation to muscles selectively enhances retrograde transport of signalling endosomes in fast, but not slow, motor neurons in vivo. Moreover, both axonal endosome transport and its brain-derived neurotrophic factor-mediated regulation are disrupted in mouse models of diseases impacting motor neurons, including amyotrophic lateral sclerosis and Charcot-Marie-Tooth disease. Here, we examined whether additional neurotrophic factors, when applied to distal axon terminals, share this transport-modulating property. Through imaging sciatic nerves in anaesthetised mice, we tracked the in vivo dynamics of signalling endosomes in fast and slow motor neurons via intramuscular injections of a fluorescent atoxic fragment of tetanus neurotoxin. These injections were co-administered with ciliary neurotrophic factor, hepatocyte growth factor, neurturin, or cleavage-resistant pro-brain-derived neurotrophic factor-four growth factors with known effects on motor neurons. Compared to vehicle-treated controls, pro-brain-derived neurotrophic factor, hepatocyte growth factor and neurturin produced no detectable change in transport dynamics. In contrast, ciliary neurotrophic factor markedly reduced endosome speeds in both fast and slow motor neurons, indicating remarkable selectivity of specific neurotrophic factors in the regulation of signalling endosome transport in motor neurons. Understanding this selectivity may aid the development of muscle-targeted neurotrophic factor-based therapeutic strategies aimed at restoring axonal transport in neurodegenerative disease, peripheral neuropathy and nerve injury.

Keywords:  intravital imaging; motor neurons; neurotrophic factors; neurotrophins; signalling endosomes

DOI:  https://doi.org/10.1093/braincomms/fcag339
Nat Commun. 2026 Aug 20. pii: 9980. [Epub ahead of print]17(1):

Drug screen and machine learning predict neuroprotective agents in a preclinical human model of childhood dementia.

Zarina Greenberg, Ella McDonald, Alejandra Noreña Puerta, Manam Inushi De Silva, Cade Christensen, Robert Adams, Jenne Tran, Paris Mazzachi, Sebastian Loskarn, Siti N Mubarokah, Leanne Winner, Drew Neavin, Megan Maack, Kristina L Elvidge, Lisa Melton, Mark R Hutchinson, Kim M Hemsley, Nicholas Smith, Cedric Bardy.

Childhood dementias are a group of paediatric neurodegenerative disorders characterised by neurocognitive decline, and in many cases underpinned by pathophysiological mechanisms similar to adult-onset dementias. In this study, we use patient-derived induced pluripotent stem cells (iPSCs) from children with one of the most prevalent childhood dementias, Mucopolysaccharidosis Type IIIA (MPS IIIA), also known as Sanfilippo syndrome. The derived cortical cultures exhibit lysosomal dysfunction, heparan sulfate accumulation, progressive neurodegeneration and astrocytic reactivity, recapitulating prototypical in-vivo phenotypes. Using a multimodal drug screening platform that integrates machine learning, high-content confocal imaging, single-nuclei transcriptomics and electrophysiology, we identify at least nine repurposed compounds that significantly mitigate these adverse effects within two weeks of treatment in vitro, demonstrating potential for rapid clinical translation. This human preclinical model for MPS IIIA, coupled with a robust multimodal therapeutic interrogation platform, serves as an exemplar for advancing drug discovery for childhood dementias and the broader neurodegenerative disease spectrum.

DOI: https://doi.org/10.1038/s41467-026-76837-1
J Cell Physiol. 2026 Sep;241(9): e70231

Chronic Contractile Activity Remediates Lysosomal Dysfunction in a Muscle Cell Lysosomal Disease Model.

Debasmita Bhattacharya, Neushaw Moradi, David A Hood.

  Lysosomes are important organelles for the degradation of unwanted biomolecules via autophagy. Lysosomal dysfunction is apparent in ageing tissues, and can cause various neurodegenerative diseases. It is imperative to understand the mechanisms and implications of lysosomal malfunction and to find strategies to ameliorate diseases. To investigate this, we induced lysosomal dysfunction with Bafilomycin A1 (BAF), a drug that hinders lysosomal acidification by blocking vATPase-mediated proton pumping, in myoblasts and myotubes. Myotubes were subjected to chronic contractile activity (CCA) to mimic "exercise" to evaluate any therapeutic potential and reversal of pathophysiology. Induced lysosomal dysfunction was evident from impaired processing of the protease cathepsin B, enhanced lysosomal accumulation and increased autophagic markers Lamp1, p62, and the LC3II/I ratio. BAF attenuated lysosomal protease degradation measured using the substrate DQ-BSA in both myoblasts and myotubes. Many of the adverse effects generated by BAF in myotubes were reversed by CCA, manifested by a decrease in immature cathepsin B, a down-regulation of Lamp1, p62, LC3II/I and partial restoration of lysosomal protein degradation enzymatic capacity. To investigate further improvements in lysosomal function in a healthy cellular model, we treated myotubes with C1, a curcumin analogue. C1 decreased Lamp1, p62 and the LC3II/I ratio, all of which indicate improved lysosomal function and autophagosome clearance with a greater lysosomal ability to degrade substrates. Additionally, C1 was able to mimic the improved mitochondrial content induced by CCA. Thus, contractile activity and curcumin analogues may provide useful therapeutic potential to resuscitate lysosomal function, improve muscle health and ameliorate lysosome-mediated diseases. NEW AND NOTEWORTHY: This study explores the potential of chronic contractile activity (CCA) in reversing the compromised lysosomes caused by the disruption of lysosomal acidification in myotubes. Remarkably, CCA mitigated the effects of Bafilomycin A1-induced lysosomal dysfunction, enhancing protease activity and increased mitochondrial content. Treatment of myotubes with the curcumin analogue C1 reduced lysosomal accumulation and enhanced mitochondrial content suggesting that contractile activity-based interventions and small-molecule modulators of lysosomal pathways may represent promising complementary strategies for treating lysosome-related diseases and enhancing muscle health.

Keywords:  autophagy; bafilomycin A; exercise; lysosomes; mitochondria

DOI:  https://doi.org/10.1002/jcp.70231
J Clin Invest. 2026 Sep 15. pii: e188241. [Epub ahead of print]

Microtubule-associated protein 1S phosphorylation regulates dynein-driven transport in the brain and synaptogenesis in neurons.

André T Lopes, Ondine Janiv, Suzanne Claxton, Sila K Ultanir.

  Cyclin-dependent kinase like 5 (CDKL5) is a serine-threonine kinase enriched in the mammalian brain whose loss of function causes a severe developmental and epileptic encephalopathy named CDKL5 Deficiency Disorder. We previously showed that CDKL5 phosphorylates the microtubule-associated protein MAP1S, but how this regulates microtubule-dependent functions is not well understood. To address this question, we generated MAP1S phosphomutant mice in which the CDKL5 phosphorylation sites S786 and S812 were mutated to alanine (MAP1S S786/812A; MAP1S SA). Using a microtubule cosedimentation assay, we found that dynein binding to microtubules was reduced in MAP1S SA and CDKL5 knockout (KO) brain lysates, and time-lapse imaging showed impaired dynein motility in dendrites from both genotypes. MAP1S SA mice also exhibited reduced AMPA receptor transport, dendritic spine density, and excitatory synapses, accompanied by anxiety-like behavior and motor, social, and memory deficits relevant to CDD. Mechanistically, MAP1S SA and CDKL5 KO neurons showed increased microtubule stability and reduced tubulin tyrosination, consistent with excessive MAP1S-mediated stabilization. Restoring tubulin tyrosination by expressing tubulin-tyrosine ligase rescued dynein transport defects. Together, these findings identify MAP1S phosphorylation as a critical regulator of microtubule dynamics and dynein-dependent transport.

Keywords:  Cytoskeleton; Development; Neurodevelopment; Neuroscience; Synapses

DOI:  https://doi.org/10.1172/JCI188241
JCI Insight. 2026 Sep 22. pii: e200761. [Epub ahead of print]11(18):

Mitochonic acid 5 alleviates amyotrophic lateral sclerosis phenotypes via mitochondrial augmentation.

Yoshitsugu Oikawa, Yuhan Luo, Naoki Suzuki, Tomoko Kasahara, Yoshiyasu Tongu, Yuki Yoshida, Tsukasa Tominari, Shogo Tanabe, Yoshiko Suto, Hitomi Kashiwagi, Saki Saito, Kensuke Ikeda, Chitose Suzuki, Arata Kuranaga, Tetsuya Akiyama, Satoru Morimoto, Yoshitsugu Aoki, Rieko Muramatsu, Tomoyoshi Soga, Masashi Aoki, Hideyuki Okano, Tetsuhiro Tanaka, Takaaki Abe, Erina Kuranaga, Takafumi Toyohara.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that urgently requires effective treatment. Mitochondrial dysfunction underlies ALS pathology and represents a potential therapeutic target. Here, we demonstrated the therapeutic potential of mitochonic acid 5 (MA-5), a novel mitochondria-targeted compound that ameliorated ALS phenotypes by enhancing mitochondrial function. In a Drosophila ALS model expressing a mutant human SOD1 (G85R), MA-5 significantly improved locomotor activity, with a trend toward restoration of mitochondrial integrity. In skin fibroblasts derived from ALS patients and motor neurons derived from induced pluripotent stem cells, MA-5 restored ATP production and increased mitochondrial motility. Multiomics analyses suggested that MA-5 modulated mitochondria-linked gene expression and downregulated the glycerophosphate shuttle, contributing to mitochondrial reactive oxygen species production. Transcriptomic analysis identified C7orf31 as a potential marker for monitoring the therapeutic effects of MA-5 and diagnosing ALS subtypes. These findings support MA-5 as a promising therapeutic candidate for ALS and propose C7orf31 as a potential biomarker for treatment monitoring and for disease subtyping.

Keywords:  ALS; Biomarkers; Drug therapy; Metabolism; Neuroscience

DOI:  https://doi.org/10.1172/jci.insight.200761
Curr Neurol Neurosci Rep. 2026 Sep 24. pii: 51. [Epub ahead of print]26(1):

Fluid and Imaging Biomarkers in Huntington's Disease.

Elizabeth L Broom, Joelle Hanson-Baiden, Lauren M Byrne.

   PURPOSE OF REVIEW: Growing interest in earlier and mechanism-targeted interventions for Huntington's disease (HD) has brought focus on biomarkers capable of informing trial design and regulatory pathways. This review discusses recent advances in fluid and neuroimaging biomarkers since the introduction of the HD-Integrated Staging System (HD-ISS), focusing on candidates with the most relevant evidence for regulatory applications.
RECENT FINDINGS: Neurofilament light (NfL) has become a versatile biomarker for prognostic enrichment and safety monitoring. Mutant huntingtin (mHTT) and somatic expansion ratio (SER) provide mechanistic evidence of target engagement. HD-YAS showed SER predicts striatal atrophy over multi-year intervals, but its short-term sensitivity remains uncertain. Structural MRI remains the most sensitive imaging marker of progression, whilst PET and diffusion imaging offer mechanistic insight but limited temporal sensitivity. The most advanced biomarkers now span neuronal injury, target engagement and neurodegeneration. Moving toward qualified endpoints will require harmonised methods, improved analytical validation and stronger evidence linking biomarker change to clinical benefit.

Keywords:  Biomarkers; Huntington’s disease; Mutant huntingtin; Neurofilament light; Regulatory qualification; Structural MRI

DOI:  https://doi.org/10.1007/s11910-026-01522-1
Nat Commun. 2026 Aug 22. pii: 10159. [Epub ahead of print]17(1):

Enhanced axonal mitochondrial motility and neural activity-induced energy deficits destabilize synaptic transmission in models of bipolar disorder.

Sunan Li, Gui-Jing Xiong, Zezhi Li, Zu-Hang Sheng.

Synaptic communication requires mitochondria to supply ATP and buffer calcium at presynaptic terminals. In bipolar disorder, manic episodes are associated with elevated mood and neural activity, but the underlying cellular mechanisms remain unclear. Here we show that hiPSC-derived cortical neurons from donors with bipolar disorder exhibit increased axonal mitochondrial motility and frequent mitochondrial entry-exit transitions, reducing stable mitochondrial retention at presynaptic terminals. This destabilizes local ATP maintenance and calcium buffering, increasing synaptic variability without altering mean synaptic strength. Knockdown of the bipolar disorder risk gene AKAP11 in mouse neurons reproduced these synaptoenergetic deficits. HiPSC-derived neurons from donors with bipolar disorder exhibited reduced expression of the mitochondrial anchor protein syntaphilin(SNPH), and snph knockout mice displayed manic-like behavioral phenotypes. Lithium restored presynaptic mitochondrial retention, improved ATP maintenance, rescued synaptic variability, and reversed behavioral phenotypes. These findings support impaired presynaptic mitochondrial retention and activity-induced synaptoenergetic deficits as cellular mechanisms contributing to bipolar disorder.

DOI: https://doi.org/10.1038/s41467-026-76722-x
Biol Open. 2026 Sep 21. pii: bio.062632. [Epub ahead of print]

Dynamin-2 promotes Atg9A retrieval from phagophores during autophagy.

Andrew Caliri, Alejandro Martorell Riera, Panagiota Kolitsida, Akash Saha, Cinta Iriondo Martinez, Samuel Itskanov, Janos Steffen, Carla M Koehler, Alexander M van der Bliek.

  Autophagy involves the rapid growth of phagophores through membrane addition. This growth is triggered by vesicles containing the Atg9A protein. However, Atg9A is not incorporated into mature autophagosomes. We now demonstrate that Dynamin-2 (Dnm2) colocalizes with the BAR domain protein Endophilin-B1 (EndoB1/Bif-1/SH3GLB1) and other autophagy proteins when autophagy is induced. Our data suggest that Atg9A is retrieved from phagophores via fission, with help from Dnm2. Blocking Atg9A recycling, either by mutating Dnm2, using RNA interference, or applying chemical inhibitors, results in Atg9A remaining in autophagosomes and being degraded during autophagy. Overall, these findings provide new insights into the roles of Dnm2 in autophagy.

Keywords:  Atg9A; Autophagy; Dynamin; Endophilin; LC3

DOI:  https://doi.org/10.1242/bio.062632
J Biomed Sci. 2026 Sep 24. pii: 90. [Epub ahead of print]33(1):

miR-196a reduces mutant Huntingtin aggregates by Rad23b-mediated degradation in Huntington's disease.

Chih-Wei Tung, Siew Chin Chan, Yi-Ching Chen, Po-Ming Wu, Pei-Hsun Cheng, Chuan-Mu Chen, Shang-Hsun Yang.

   BACKGROUND: Huntington's disease (HD) is a neurodegenerative disorder caused by abnormal expansions of poly-glutamine repeats in the mutant Huntingtin (mHTT). The expanded proteins form pathological aggregates to disrupt neuronal functions during disease progression, suggesting the clearance of the aggregates is considered as a potential direction for development of therapy. Our previous studies have demonstrated one specific microRNA, miR-196a, downregulates mHTT aggregates and improves the pathological phenotypes in HD. However, the detailed mechanism remains unclear.
METHODS: Proteomic analyses and bioinformatic tools were used to identify putative miR-196a targets in HD, and the expression of one target gene, Rad23b, was manipulated in different HD models to assess its role in mHTT aggregates.
RESULTS: Here, we identify Rad23b as a key target, and miR-196a directly downregulates Rad23b to reduce mHTT aggregates and toxicity. We also show Rad23b overexpression worsens mHTT pathology, while its knockdown or knockout diminishes aggregates, primarily through the ubiquitin-proteasome system (UPS). Moreover, Rad23b interacts with mHTT aggregates via its ubiquitin-binding domains, and promotes their ubiquitination; however, Rad23b disrupts the chymotrypsin-like activity of UPS, and contributes to mHTT accumulation. In transgenic mouse models in vivo, Rad23b increases mHTT aggregates and cell death in brains, and also worsens motor dysfunction in HD transgenic mice.
CONCLUSIONS: These findings demonstrate that critical role of Rad23b in miR-196a-reduced pathological aggregates, and highly suggest downregulating Rad23b or disrupting its interaction with mHTT may offer novel strategies to mitigate mHTT aggregates to delay disease progression.

Keywords:  Huntington's disease; Protein aggregation; Proteomic analysis; Rad23b; Ubiquitin-proteasome system; miR-196a

DOI:  https://doi.org/10.1186/s12929-026-01292-5
bioRxiv. 2026 Sep 20. pii: 2026.09.18.752796. [Epub ahead of print]

A lifespan single-cell atlas of the human developing hippocampus benchmarks familial Alzheimer's disease brain organoids.

Ekaterina Ivleva, Emil Kruikov, Joseph F Arboleda-Velasquez, Petr Baranov.

Familial Alzheimer's disease (fAD) is an early-onset form of AD caused by autosomal-dominant variants in APP, PSEN1, or PSEN2, with PSEN1 accounting for most genetically defined cases [1]. The hippocampus is among the earliest and most severely affected brain regions in AD [2,3]. Human induced pluripotent stem cell (iPSC)-derived brain organoids recapitulate key features of early human brain development and provide a tractable model for studying how fAD mutations perturb neurodevelopmental processes [4]. However, their interpretation is complicated by heterogeneous regional identity, variable maturation state, and cell-type composition across protocols [5,6]. Existing single-cell studies of human hippocampus cover prenatal [7] and postnatal [8-10] stages but do not provide a continuous developmental reference. By elevating the atlas approach in utilizing single-cell RNA-sequencing data, we obtain standardized information on the organoid cell class and type composition and maturation states. Here, we constructed the Human Developing Hippocampus Atlas (HuDeHA), an integrated single-cell reference comprising 658,059 cells spanning post-conceptional week 3 to 15.3 years, and used it to benchmark iPSC-derived brain organoids carrying PSEN1 E280A which is associated with fAD in a large Colombian population. Reference-based mapping revealed altered cellular composition in PSEN1 E280A organoids, including reduced radial glia and increased neural crest-derived neurons. These changes were accompanied by cross-lineage transcriptional alterations, including broad upregulation of the ventral patterning factor MEIS2 and reduced expression of the αβ-binding protein transthyretin (TTR) in choroid-plexus and ependymal-associated populations. Reconstructed neuronal-lineage trajectories showed a shift toward mature states in PSEN1 E280A organoids. Together, these findings establish HuDeHA as a resource for developmental benchmarking of hippocampus-relevant organoid systems and describe cell-lineage-specific developmental changes in PSEN1 E280A organoids that may inform interpretation of early cellular alterations in fAD.

DOI: https://doi.org/10.64898/2026.09.18.752796
iScience. 2026 Oct 16. 29(10): 117518

PP2A and CDK16 antagonistically regulate WIPI2B phosphorylation and neuronal autophagosome biogenesis.

Heather Tsong, M Neal Waxham, Andrea K H Stavoe.

  Autophagy is a homeostatic mechanism for recycling cellular constituents. In primary murine neurons, autophagosome biogenesis declines during aging. Importantly, this decline can be restored by the ectopic expression of key autophagy component WIPI2B. The phosphorylation state of WIPI2B serine 395 is critical for this restoration, suggesting that WIPI2B S395 phosphorylation regulates autophagosome biogenesis. Here, we identified protein phosphatase 2A (PP2A) and CDK16 as regulators of WIPI2B S395 phosphorylation and neuronal autophagy. Using Caenorhabditis elegans, we showed that PP2A and CDK16 regulate neuronal autophagy through the same genetic pathway as WIPI2B. Further, purified mammalian PP2A and CDK16 directly modified WIPI2B S395 phosphorylation in vitro. In primary murine neurons, PP2A and CDK16 colocalized with WIPI2B at autophagosomes, and manipulation of PP2A and CDK16 expression altered WIPI2B puncta formation and rates of autophagosome biogenesis. Altogether, our data support the conclusion that PP2A and CDK16 regulate WIPI2B S395 phosphorylation, modulating autophagosome biogenesis in neurons.

Keywords:  C. elegans; autophagy; neuron; phosphorylation

DOI:  https://doi.org/10.1016/j.isci.2026.117518
Methods Mol Biol. 2027 ;3059 273-282

Deep Insights Without Clearing: An Optimized Protocol for Patient-Derived Whole-Organoid Immunofluorescence Imaging.

Laura Gull, Mireia Sueca-Comes, Ana Pires.

  Here, we present an optimized whole-organoid immunofluorescence staining protocol that preserves morphology, achieves uniform antibody penetration, and enables deep-tissue imaging without the need for tissue clearing. Organoids are three-dimensional, self-organizing structures that replicate many of the cellular and architectural features of their tissue of origin, making them powerful preclinical models for studying development, disease, and therapeutic responses. Most organoid research is performed on formalin-fixed, paraffin-embedded (FFPE) samples, in which organoids are sectioned across the Z-planes. While this method facilitates conventional histological analysis, it limits spatially resolved visualization of protein expression and tissue organization. The inherent density and abundant extracellular matrix of organoids present substantial barriers to reagent penetration and imaging depth, further constraining the ability to capture their three-dimensional architecture in full. Our new workflow includes steps for fixation, permeabilization, and antibody incubation and has been validated on intestinal organoids using markers for different membrane-associated proteins. By providing high-quality staining through the full organoid depth without clearing, this protocol streamlines sample preparation, reduces processing artifacts, and allows more rapid, accurate 3D mapping of cell types and structures in organoid-based research.

Keywords:  3D cell culture; Antibody penetration; Confocal microscopy; Deep-tissue imaging; No tissue clearing; Organoids; Whole-mount immunostaining

DOI:  https://doi.org/10.1007/978-1-0716-5412-5_20