bims-axbals 2025-02-16 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2025–02–16
thirty-two papers selected by
TJ Krzystek, ALS Therapy Development Institute

A guide to selecting high-performing antibodies for ADNP (UniProt ID: Q9H2P0) for use in western blot, immunoprecipitation, and immunofluorescence.
A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence.
The dynamic axon initial segment: From neuronal polarity to network homeostasis.
Antisense oligonucleotide-mediated MSH3 suppression reduces somatic CAG repeat expansion in Huntington's disease iPSC-derived striatal neurons.
Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.
Development of selective deconjugases for membrane-anchored LC3A/B in post-mitotic neurons.
Small molecule treatment alleviates photoreceptor cilia defects in LCA5-deficient human retinal organoids.
Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.
Dysregulation of Retinal and Photoreceptor Structural Integrity Genes in ATF6-/- Retinal Organoids.
Live Imaging Microscopy of Human Retina Organoids: Photoreceptor Pathology.
Oxidative Stress Suppresses Trk Signaling While Stimulating JNK-Mediated Endocytosis and Cleavage of p75NTR: A Targetable Pathway for Neuroprotection in a Parkinson's Disease Model.
Bioelectric stimulation outperforms brain derived neurotrophic factor in promoting neuronal maturation.
Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9.
The proteomic landscape of stool-derived extracellular vesicles in patients with pre-cancerous lesions and colorectal cancer.
Protein misfolding: understanding biology to classify and treat synucleinopathies.
Protocols for the application of human embryonic stem cell-derived neurons for aging modeling and gene manipulation.
Polyamine metabolism dysregulation contributes to muscle fiber vulnerability in ALS.
The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.
The Fragile X Messenger Ribonucleoprotein 1 Regulates the Morphology and Maturation of Human and Rat Oligodendrocytes.
Disruption of mitochondrial homeostasis and permeability transition pore opening in OPA1 iPSC-derived retinal ganglion cells.
Prime Editing Strategy to Install the RPE65 c.1430A>G Dominant Mutation.
Bafilomycin A1 induces colon cancer cell death through impairment of the endolysosome system dependent on iron.
Heat-shock chaperone HSPB1 mitigates poly-glycine-induced neurodegeneration via restoration of autophagic flux.
The Importance of Differentiated RPE Cultures for studying Cell Biological Processes.
Reprogramming astrocytes into dopaminergic neurons to restore motor dysfunction in Parkinson's disease model rats.
NF2 is Essential for Human Endoderm Development.
Generation of Retinal Organoids Using Human-Induced Pluripotent Stem Cells.
A microscopy-based screen identifies cellular kinases modulating mitochondrial translation.
AXL signaling in cancer: from molecular insights to targeted therapies.
Xenopus laevis neural stem progenitor cells exhibit a transient metabolic shift toward glycolysis during spinal cord regeneration.
Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network.
The fundamental role of mitochondria-endoplasmic reticulum contacts in ageing and declining healthspan.

F1000Res. 2024 ;13 1545

A guide to selecting high-performing antibodies for ADNP (UniProt ID: Q9H2P0) for use in western blot, immunoprecipitation, and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  ADNP is a multifunctional protein involved in chromatin remodeling, transcription, and microtubule interaction, playing a critical role in brain development, with mutations linked to ADNP-Related Disorder. Here we have characterized seven ADNP 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:  ADNP; Activity-Dependent Neuroprotective Protein; Activity-Dependent Neuroprotector homeobox Protein; Q9H2P0; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation; western blot

DOI:  https://doi.org/10.12688/f1000research.160121.1
F1000Res. 2024 ;13 1

A guide to selecting high-performing antibodies for Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform (PPP2R5D) for use in Western Blot, immunoprecipitation and immunofluorescence.

NeuroSGC/YCharOS/EDDU collaborative group

  Protein phosphatase 2A is a serine/threonine phosphatase with activity dependent on an associated regulatory subunit, serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta (δ) isoform (PPP2R5D). PPP2R5D is the δ isoform in the B56 family of regulatory subunits. Abundantly expressed in the brain and involved in a broad range of cellular processes, PPP2R5D plays an essential role in modulating key neuronal pathways and signalling. Pathogenic mutations in the PPP2R5D gene are linked to clinical symptoms characterized by neurodevelopmental delay, intellectual disability, and autism spectrum disorders. The etiology of these genetic disorders remains unknown, which can partly be due to the lack of independently characterized antibodies. Here we have characterized six PPP2R5D 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 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:  PPP2R5D; Serine/threonine-protein phosphatase 2A 56 kDa regulatory subunit delta isoform; Uniprot ID Q14738; Western Blot; antibody characterization; antibody validation; immunofluorescence; immunoprecipitation

DOI:  https://doi.org/10.12688/f1000research.145146.2
Neuron. 2025 Feb 04. pii: S0896-6273(25)00008-X. [Epub ahead of print]

The dynamic axon initial segment: From neuronal polarity to network homeostasis.

Amélie Fréal, Casper C Hoogenraad.

  The axon initial segment (AIS) is a highly specialized compartment in neurons that resides in between axonal and somatodendritic domains. The localization of the AIS in the proximal part of the axon is essential for its two major functions: generating and modulating action potentials and maintaining neuron polarity. Recent findings revealed that the incredibly stable AIS is generated from highly dynamic components and can undergo extensive structural and functional changes in response to alterations in activity levels. These activity-dependent alterations of AIS structure and function have profound consequences for neuronal functioning, and AIS plasticity has emerged as a key regulator of network homeostasis. This review highlights the functions of the AIS, its architecture, and how its organization and remodeling are influenced by developmental plasticity and both acute and chronic adaptations. It also discusses the mechanisms underlying these processes and explores how dysregulated AIS plasticity may contribute to brain disorders.

Keywords:  AIS; actin; ankyrin; axon initial segment; microtubule; neurological disease; neuron; plasticity; self-organization; synapse; transport

DOI:  https://doi.org/10.1016/j.neuron.2025.01.004
Sci Transl Med. 2025 Feb 12. 17(785): eadn4600

Antisense oligonucleotide-mediated MSH3 suppression reduces somatic CAG repeat expansion in Huntington's disease iPSC-derived striatal neurons.

Emma L Bunting, Jasmine Donaldson, Sarah A Cumming, Jessica Olive, Elizabeth Broom, Mihai Miclăuș, Joseph Hamilton, Matthew Tegtmeyer, Hien T Zhao, Jonathan Brenton, Won-Seok Lee, Robert E Handsaker, Susan Li, Brittany Ford, Mina Ryten, Steven A McCarroll, Holly B Kordasiewicz, Darren G Monckton, Gabriel Balmus, Michael Flower, Sarah J Tabrizi.

Expanded CAG alleles in the huntingtin (HTT) gene that cause the neurodegenerative disorder Huntington's disease (HD) are genetically unstable and continue to expand somatically throughout life, driving HD onset and progression. MSH3, a DNA mismatch repair protein, modifies HD onset and progression by driving this somatic CAG repeat expansion process. MSH3 is relatively tolerant of loss-of-function variation in humans, making it a potential therapeutic target. Here, we show that an MSH3-targeting antisense oligonucleotide (ASO) effectively engaged with its RNA target in induced pluripotent stem cell (iPSC)-derived striatal neurons obtained from a patient with HD carrying 125 HTT CAG repeats (the 125 CAG iPSC line). ASO treatment led to a dose-dependent reduction of MSH3 and subsequent stalling of CAG repeat expansion in these striatal neurons. Bulk RNA sequencing revealed a safe profile for MSH3 reduction, even when reduced by >95%. Maximal knockdown of MSH3 also effectively slowed CAG repeat expansion in striatal neurons with an otherwise accelerated expansion rate, derived from the 125 CAG iPSC line where FAN1 was knocked out by CRISPR-Cas9 editing. Last, we created a knock-in mouse model expressing the human MSH3 gene and demonstrated effective in vivo reduction in human MSH3 after ASO treatment. Our study shows that ASO-mediated MSH3 reduction can prevent HTT CAG repeat expansion in HD 125 CAG iPSC-derived striatal neurons, highlighting the therapeutic potential of this approach.

DOI: https://doi.org/10.1126/scitranslmed.adn4600
Cell Mol Life Sci. 2025 Feb 13. 82(1): 76

Enhanced secretion of the amyotrophic lateral sclerosis ALS-associated misfolded TDP-43 mediated by the ER-ubiquitin specific peptidase USP19.

Flavien Picard, Takashi Nonaka, Edwige Belotti, Alexis Osseni, Elisabeth Errazuriz-Cerda, Coline Jost-Mousseau, Emilien Bernard, Agnès Conjard-Duplany, Delphine Bohl, Masato Hasegawa, Cédric Raoul, Thierry Galli, Laurent Schaeffer, Pascal Leblanc.

  Proteinopathies, such as amyotrophic lateral sclerosis (ALS), are marked by the accumulation of misfolded proteins that disrupt cellular processes. Eukaryotic cells have developed protein quality control systems to eliminate these aberrant proteins, but these systems often fail to differentiate between normal and misfolded proteins. In ALS, pathological inclusions primarily composed of misfolded TDP-43 are a hallmark of the disease. Recently, a novel unconventional secretion process called misfolding-associated protein secretion (MAPS) has been discovered to selectively export misfolded proteins. USP19, an Endoplasmic Reticulum-associated ubiquitin peptidase, plays a crucial role in this process. In this study, we investigated the impact of ER-anchored USP19 on the secretion of misfolded TDP-43. Here we found that USP19 overexpression significantly promotes the secretion of soluble and aggregated misfolded TDP-43, requiring both ER anchoring and ubiquitin peptidase activity. Characterization of the cellular and molecular mechanisms involved in this process highlighted the importance of early autophagosomal and late endosomal/amphisomal compartments, while lysosomes did not play a key role. By using dominant-negative mutants and small interfering RNAs, we identified that USP19-mediated secretion of misfolded TDP-43 is modulated by key factors involved in cellular trafficking and secretion pathways, such as ATG7, the ESCRT-O HGS/HRS, the Rab GTPases RAB11A, RAB8A, and RAB27A, and the v-SNARE VAMP7. We also confirmed the crucial role of the DNAJC5/CSPα cochaperone. Overall, this study provides new insights into how cells manage the secretion of misfolded TDP-43 proteins and potentially opens new avenues for therapeutic interventions in ALS and related disorders.

Keywords:  ALS; Aggregates; Autophagosomes; Autophagy; Endosomes; Misfolding; Release/secretion; TDP-43; USP19; Ubiquitin peptidase

DOI:  https://doi.org/10.1007/s00018-025-05589-w
Mol Brain. 2025 Feb 12. 18(1): 11

Development of selective deconjugases for membrane-anchored LC3A/B in post-mitotic neurons.

Haneul Choi, Sang-Won Park, Deok-Jin Jang, Jin-A Lee.

  Neuronal autophagy is essential for maintaining protein and organelle turnover, thereby safeguarding neuronal health. LC3, a central autophagy protein, exists in lipidated (LC3-II) and non-lipidated (LC3-I) forms, both critical for neurons due to their sensitivity to metabolic and proteostatic stress. To elucidate the specific roles of membrane-anchored LC3A/B in post-mitotic neurons, we engineered deconjugases with enhanced selectivity for lipidated LC3. By modifying LC3-interacting regions (LIRs) at the deconjugase termini, we significantly improved targeting specificity toward LC3A/B. Deconjugases with N-terminal LIR modifications reduced LC3A/B-associated autophagosomes, highlighting the importance of LIR positioning for specificity. Sequential N-terminal LIR arrangements further refined LC3A/B targeting without affecting GABARAP-associated autophagosomes. Moreover, reducing the hydrophobicity of the α3 helix to limit membrane residence time further improved selectivity. These targeted modifications demonstrate the potential of customized deconjugases to dissect and modulate specific autophagic pathways in neurons, paving the way for novel therapeutic strategies against neurodegenerative diseases associated with autophagy dysregulation.

Keywords:  Deconjugases; LC3/GABARAP; Neuronal autophagy; RavZ

DOI:  https://doi.org/10.1186/s13041-025-01184-z
Acta Neuropathol Commun. 2025 Feb 11. 13(1): 26

Small molecule treatment alleviates photoreceptor cilia defects in LCA5-deficient human retinal organoids.

Dimitra Athanasiou, Tess A V Afanasyeva, Niuzheng Chai, Kalliopi Ziaka, Katarina Jovanovic, Rosellina Guarascio, Karsten Boldt, Julio C Corral-Serrano, Naheed Kanuga, Ronald Roepman, Rob W J Collin, Michael E Cheetham.

  Bialleleic pathogenic variants in LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report the use of gene editing to generate isogenic LCA5 knock-out (LCA5 KO) induced pluripotent stem cells (iPSC) and their differentiation to retinal organoids. The molecular and cellular phenotype of the LCA5 KO retinal organoids was studied in detail and compared to isogenic controls as well as patient-derived retinal organoids. The absence of LCA5 was confirmed in retinal organoids by immunohistochemistry and western blotting. There were no major changes in retinal organoid differentiation or ciliation, however, the localisation of CEP290 and IFT88 was significantly altered in LCA5 KO and patient photoreceptor cilia with extension along the axoneme. The LCA5-deficient organoids also had shorter outer segments and rhodopsin was mislocalised to the outer nuclear layer. We also identified transcriptomic and proteomic changes associated with the loss of LCA5. Importantly, treatment with the small molecules eupatilin, fasudil or a combination of both drugs reduced CEP290 and IFT88 accumulation along the cilia. The treatments also improved rhodopsin traffic to the outer segment and reduced mislocalisation of rhodopsin in the outer nuclear layer (ONL). The improvements in cilia-associated protein localisation and traffic were accompanied by significant changes in the transcriptome towards control gene expression levels in many of the differentially expressed genes. In summary, iPSC-derived retinal organoids are a powerful model for investigating the molecular and cellular changes associated with loss of LCA5 function and highlight the therapeutic potential of small molecules to treat retinal ciliopathies.

Keywords:  Cilia; Gene editing; LCA; LCA5; Organoid; Photoreceptor; Retina; Retinal dystrophy; Stem cell; Therapy

DOI:  https://doi.org/10.1186/s40478-025-01943-y
Elife. 2025 Feb 12. pii: RP100541. [Epub ahead of print]13

Pharmacologic activation of integrated stress response kinases inhibits pathologic mitochondrial fragmentation.

Kelsey R Baron, Samantha Oviedo, Sophia Krasny, Mashiat Zaman, Rama Aldakhlallah, Prerona Bora, Prakhyat Mathur, Gerald Pfeffer, Michael J Bollong, Timothy E Shutt, Danielle A Grotjahn, R Luke Wiseman.

  Excessive mitochondrial fragmentation is associated with the pathologic mitochondrial dysfunction implicated in the pathogenesis of etiologically diverse diseases, including many neurodegenerative disorders. The integrated stress response (ISR) - comprising the four eIF2α kinases PERK, GCN2, PKR, and HRI - is a prominent stress-responsive signaling pathway that regulates mitochondrial morphology and function in response to diverse types of pathologic insult. This suggests that pharmacologic activation of the ISR represents a potential strategy to mitigate pathologic mitochondrial fragmentation associated with human disease. Here, we show that pharmacologic activation of the ISR kinases HRI or GCN2 promotes adaptive mitochondrial elongation and prevents mitochondrial fragmentation induced by the calcium ionophore ionomycin. Further, we show that pharmacologic activation of the ISR reduces mitochondrial fragmentation and restores basal mitochondrial morphology in patient fibroblasts expressing the pathogenic D414V variant of the pro-fusion mitochondrial GTPase MFN2 associated with neurological dysfunctions, including ataxia, optic atrophy, and sensorineural hearing loss. These results identify pharmacologic activation of ISR kinases as a potential strategy to prevent pathologic mitochondrial fragmentation induced by disease-relevant chemical and genetic insults, further motivating the pursuit of highly selective ISR kinase-activating compounds as a therapeutic strategy to mitigate mitochondrial dysfunction implicated in diverse human diseases.

Keywords:  cell biology; human; integrated stress response; mitochondrial fragmentation; mitochondrial morphology; mouse; stress signaling

DOI:  https://doi.org/10.7554/eLife.100541
Adv Exp Med Biol. 2025 ;1468 401-407

Dysregulation of Retinal and Photoreceptor Structural Integrity Genes in ATF6-/- Retinal Organoids.

Allyssa Bradley, Masako Le, Soyeon Park, Heike Kroeger, R Luke Wiseman, Eun-Jin Lee, Jonathan H Lin.

  ATF6 is a key regulator of the unfolded protein response (UPR) pathway that maintains cellular homeostasis during ER stress. In people, loss of ATF6 function causes cone dysfunction, manifesting as achromatopsia (ACHM). Previously, we generated ACHM retinal organoids (ROs) from patient induced pluripotent stem cells (iPSCs) carrying mutant ATF6 variants and gene-edited ATF6-knockout (KO) human embryonic stem cells (hESCs). ACHM and ATF6-KO ROs both showed severe stunting of cone inner and outer segments. RNA-Seq analysis of ACHM 290-day-old ROs showed downregulated cone gene expression and dysregulated mitochondria and ER stress gene expression. Here, we analyzed RNA-Seq analysis of 203-day-old ATF6-KO ROs. In younger ROs, we found dysregulation of genes involved in retinal and photoreceptor structural integrity, including CRB1, EGFLAM, and VTN. In addition, we found dysregulation of ATF6 and UPR-regulated transcriptional signatures. Dysregulation of retinal and photoreceptor structural integrity genes may underlie the observed stunting of cone inner/outer segments in ATF6-achromatopsia patients.

Keywords:  ATF6; Achromatopsia; Brush border (inner segment/outer segment); Cones; Outer limiting membrane (external limiting membrane); Photoreceptor structure; Retinal organoid; UPR

DOI:  https://doi.org/10.1007/978-3-031-76550-6_66
Adv Exp Med Biol. 2025 ;1468 383-387

Live Imaging Microscopy of Human Retina Organoids: Photoreceptor Pathology.

Felix Wagner, Mike O Karl.

  Neurodegenerative diseases involve many different (sub)cellular pathologic processes like mitochondria damage, which are still incompletely understood and present future targets for therapy development. Live imaging microscopy of dynamic pathologic changes at single cell resolution might advance studies of pathomechanisms and biomarkers. Organoid technologies may complement and facilitate animal and patient studies: We recently reported a novel pathomechanism of photoreceptor degeneration via cell extrusion, identified and validated by live imaging in human retina organoids. Here, we describe the related workflow for live imaging organoid studies. As another use-case, we describe mitochondrial alterations in photoreceptors acutely damaged by the ionophore CCCP. Our data further support a CCCP-induced experimental model for photoreceptor pathology, which can be monitored by JC-1 live dye-assisted live imaging. Thus, we demonstrate that live imaging advances studies of early subcellular pathologic events in human organoids. Such approaches might facilitate future therapeutic target identification and development for early intervention.

Keywords:  Human; Live imaging; Mitochondria; Neurodegeneration; Organoid; Retina

DOI:  https://doi.org/10.1007/978-3-031-76550-6_63
J Neurochem. 2025 Feb;169(2): e70010

Oxidative Stress Suppresses Trk Signaling While Stimulating JNK-Mediated Endocytosis and Cleavage of p75NTR: A Targetable Pathway for Neuroprotection in a Parkinson's Disease Model.

Poshan V Pokharel, Aaron M Newchurch, Sunny C Overby, Cassidy A Spease, Isaac T Perkins, Lorelei G Darzi, Nabin Ghimire, Ahmed Lawan, Bradley R Kraemer.

The p75 Neurotrophin Receptor (p75NTR) is a multifunctional transmembrane protein that mediates neuronal responses to pathological conditions in specific regions of the nervous system. In many biological contexts, p75NTR signaling is initiated through sequential cleavage of the receptor by α- and γ-secretases, which releases receptor fragments for downstream signaling. Our previous research demonstrated that proteolytic processing of p75NTR in this manner is stimulated by oxidative stress in Lund Human Mesencephalic (LUHMES) cells, a dopaminergic neuronal cell line derived from human mesencephalic tissue. Considering the vulnerability of dopaminergic neurons in the ventral mesencephalon to oxidative stress and neurodegeneration associated with Parkinson's disease (PD), we investigated the role of this signaling cascade in neurodegeneration and explored cellular processes that govern oxidative stress-induced p75NTR signaling. In the present study, we provide evidence that oxidative stress induces cleavage of p75NTR by promoting c-Jun N-terminal Kinase (JNK)-dependent internalization of p75NTR from the cell surface. This activation of p75NTR signaling is counteracted by tropomyosin-related kinase (Trk) receptor signaling; however, oxidative stress leads to Trk receptor downregulation, thereby enhancing p75NTR processing. Importantly, we demonstrate that this pathway can be inhibited by LM11a-31, a small molecule modulator of p75NTR, thereby conferring protection against neurodegeneration. Treatment with LM11a-31 significantly reduced p75NTR cleavage and neuronal death associated with oxidative stress. These findings reveal novel mechanisms underlying activation of p75NTR in response to oxidative stress, underscore a key role for p75NTR in dopaminergic neurodegeneration, and highlight p75NTR as a potential therapeutic target for reducing neurodegeneration in PD.

DOI: https://doi.org/10.1111/jnc.70010
Sci Rep. 2025 Feb 08. 15(1): 4772

Bioelectric stimulation outperforms brain derived neurotrophic factor in promoting neuronal maturation.

María Del Pilar Diego-Santiago, María Ujué González, Esther María Zamora Sánchez, Nuria Cortes-Carrillo, Carlos Dotti, Francesc Xavier Guix, Sahba Mobini.

  Neuronal differentiation and maturation are crucial for developing research models and therapeutic applications. Brain-derived neurotrophic factor (BDNF) is a widely used biochemical stimulus for promoting neuronal maturation. However, the broad effects of biochemical stimuli on multiple cellular functions limit their applicability in both in vitro models and clinical settings. Electrical stimulation (ES) offers a promising physical method to control cell fate and function, but it is hampered by lack of standard and optimised protocols. In this study, we demonstrate that ES outperforms BDNF in promoting neuronal maturation in human neuroblastoma SH-SY5Y. Additionally, we address the question regarding which ES parameters regulate biological responses. The neuronal differentiation and maturation of SH-SY5Y cells were tested under several pulsed ES regimes. We identified accumulated charge and effective electric field time as novel criteria for determining optimal ES regimes. ES parameters were obtained using electrochemical characterisation and equivalent circuit modelling. Our findings show that neuronal maturation in SH-SY5Y cells correlates with the amount of accumulated charge during ES. Higher charge accumulation (~ 50 mC/h) significantly promotes extensive neurite outgrowth and ramification, and enhances the expression of synaptophysin, yielding effects exceeding those of BDNF. In contrast, fewer charge injection to the culture (~ 0.1 mC/h) minimally induces maturation but significantly increases cell proliferation. Moreover, ES altered the concentration and protein cargo of secreted extracellular vesicles (EV). ES with large enough accumulated charge significantly enriched EV proteome associated with neural development and function. These results demonstrate that each ES regime induces distinct cellular responses. Increased accumulated charge facilitates the development of complex neuronal morphologies and axonal ramification, outperforming exogenous neurotrophic factors. Controlled ES methods are immediately applicable in creating mature neuronal cultures in vitro with minimal chemical intervention.

Keywords:  Biochemical-free stimulation; Charge injection; Electrical stimulation; Neural extracellular vesicles; Neuronal differentiation and maturation

DOI:  https://doi.org/10.1038/s41598-025-89330-4
Adv Exp Med Biol. 2025 ;1468 107-111

Megabase Deletion of the Human EYS Locus Using CRISPR/Cas9.

Bruna Lopes da Costa, Anders Steen Knudsen, C Henrique Alves, Stephen H Tsang, Peter M J Quinn.

  Mutations in the Eyes Shut Homolog (EYS) gene are associated with autosomal recessive retinitis pigmentosa (arRP). To date, four retinal isoforms of EYS have been identified. However, the precise retinal function of EYS is not fully understood, but it has apparent roles in retinal morphogenesis, architecture, and ciliary transport. Clustered-regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) nuclease-mediated approaches are powerful tools for genome engineering in mammalian cells. The use of paired CRISPR/Cas9-induced double-strand breaks (DSBs) using dual single guide RNAs (sgRNA) can lead to precise genomic deletions. In this study, we developed a dual sgRNA strategy to facilitate CRISPR/Cas9-mediated deletion of 1,988,210 bp of the EYS locus, removing the four currently identified human retinal EYS isoforms. This approach can be used to produce EYSdel induced pluripotent stem cell (iPSC) lines to explore the function of EYS in human iPSC-derived retinal organoids.

Keywords:  CRISPR/Cas9; Eyes Shut Homolog (EYS); Megabase-scale deletion; Retinitis Pigmentosa type 25 (RP25)

DOI:  https://doi.org/10.1007/978-3-031-76550-6_18
Commun Biol. 2025 Feb 13. 8(1): 228

The proteomic landscape of stool-derived extracellular vesicles in patients with pre-cancerous lesions and colorectal cancer.

Emmalee J Northrop-Albrecht, Yohan Kim, William R Taylor, Shounak Majumder, John B Kisiel, Fabrice Lucien.

Colorectal cancer (CRC) is the 2nd most fatal cancer in the United States, but when detected early it is highly curable. Stool-derived extracellular vesicles (EVs) are a novel biomarker source that could augment the sensitivity for detection of CRC precursors. However, standardization of isolation methods for stool-derived EVs remains underexplored. We previously reported that size-exclusion chromatography (SEC) followed by ultrafiltration (UF-100kDa) was suitable for human stool supernatant EV isolation. In this study, we first assess alternative EV concentration methods (ultrafiltration [UF]; 10 kDa, 30 kDa, 50 kDa, 100 kDa and speed vacuum [SV]). Second, we investigate the host/bacterial EV proteomes by mass spectrometry. We report no difference in recovery, RNA and soluble protein contamination among concentration methods. Proteomic analysis reveals a diverse bacterial proteome, while human-derived proteins are more abundant. Specifically, pancreatic enzymes are among the most abundant proteins, further exploration revealed that zymogen granules are likely co-isolated in stool EV preparations. To enable discovery of EV-based molecular signatures of CRC precursors with high sensitivity, immunocapture strategies will likely be needed. Notably, we identified 10 surface proteins that may serve as candidates for the purification of colon-derived EVs. This work serves as framework for the future discovery and validation of EV-based biomarkers for CRC.

DOI: https://doi.org/10.1038/s42003-025-07652-5
J Neural Transm (Vienna). 2025 Feb 11.

Protein misfolding: understanding biology to classify and treat synucleinopathies.

Tiago Fleming Outeiro, Günter Höglinger, Anthony E Lang, Tuane C R G Vieira.

  Protein misfolding and aggregation is a major pathological hallmark in a variety of human conditions, including cancer, diabetes, and neurodegeneration. However, we still do not fully understand the role of protein accumulation in disease. Interestingly, recent breakthroughs in artificial intelligence (AI) are having a tremendous impact on our ability to predict three-dimensional protein structures and understand the molecular rules governing protein folding/misfolding. This progress will enable us to understand how intrinsic and extrinsic factors trigger protein misfolding, thereby changing protein function. These changes, in some cases, are related to normal biological responses and, in other cases, associated with pathological alterations, such as those found in many neurodegenerative disorders. Here, we provide a brief historical perspective of how findings in the field of prion diseases and prion biology have enabled tremendous advances that are now forming the basis for our understanding of disease processes and discuss how this knowledge is now emerging as central for our ability to classify, diagnose, and treat devastating neurodegenerative disorders such as Parkinson's and Alzheimer's diseases.

Keywords:  Alpha-synuclein; Biomarkers; Disease classification; Neurodegeneration; Parkinson’s disease; Protein aggregation

DOI:  https://doi.org/10.1007/s00702-025-02889-0
STAR Protoc. 2025 Feb 10. pii: S2666-1667(25)00039-5. [Epub ahead of print]6(1): 103633

Protocols for the application of human embryonic stem cell-derived neurons for aging modeling and gene manipulation.

Hui Zhang, Shuhui Sun, Juan Carlos Izpisua Belmonte, Guang-Hui Liu, Si Wang, Weiqi Zhang, Jing Qu.

  In vitro models of neuronal aging and gene manipulation in human neurons (hNeurons) are valuable tools for investigating human brain aging and diseases. Here, we present a protocol for applying human embryonic stem cell (hESC)-derived neurons to model aging and the further application of small interfering RNA (siRNA)-mediated gene silencing for functional investigations. We describe steps for neuronal differentiation and culture, siRNA transfection, and technical considerations to ensure reproducibility. Our protocol enables investigations of the molecular mechanism underlying neuronal aging and facilitates drug evaluation. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

Keywords:  Cell Biology; Cell Differentiation; Developmental biology; Molecular Biology; Neuroscience; Stem Cells

DOI:  https://doi.org/10.1016/j.xpro.2025.103633
Cell Rep. 2025 Jan 28. pii: S2211-1247(24)01474-8. [Epub ahead of print]44(1): 115123

Polyamine metabolism dysregulation contributes to muscle fiber vulnerability in ALS.

Veronica Ruggieri, Silvia Scaricamazza, Andrea Bracaglia, Chiara D'Ercole, Cristina Parisi, Paolo D'Angelo, Daisy Proietti, Chiara Cappelletti, Alberto Macone, Biliana Lozanoska-Ochser, Marina Bouchè, Lucia Latella, Cristiana Valle, Alberto Ferri, Lorenzo Giordani, Luca Madaro.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease causing progressive paralysis due to motor neuron degeneration with no curative therapy despite extensive biomedical research. One of the primary targets of ALS is skeletal muscle, which undergoes profound functional changes as the disease progresses. To better understand how altered innervation interferes with muscle homeostasis during disease progression, we generated a spatial transcriptomics dataset of skeletal muscle in the SOD1G93A mouse model of ALS. Using this strategy, we identified polyamine metabolism as one of the main altered pathways in affected muscle fibers. By establishing a correlation between the vulnerability of muscle fibers and the dysregulation of this metabolic pathway, we show that disrupting polyamine homeostasis causes impairments similar to those seen in ALS muscle. Finally, we show that restoration of polyamine homeostasis rescues the muscle phenotype in SOD1G93A mice, opening new perspectives for the treatment of ALS.

Keywords:  CP: Metabolism; CP: Neuroscience; amyotrophic lateral sclerosis; muscular atrophy; neuromuscular junction; polyamines; skeletal muscle; spatial transcriptomics

DOI:  https://doi.org/10.1016/j.celrep.2024.115123
J Cell Biol. 2025 Apr 07. pii: e202407105. [Epub ahead of print]224(4):

The LC3-interacting region of NBR1 is a protein interaction hub enabling optimal flux.

Brian J North, Amelia E Ohnstad, Michael J Ragusa, Christopher J Shoemaker.

During autophagy, toxic cargo is encapsulated by autophagosomes and trafficked to lysosomes for degradation. NBR1, an autophagy receptor targeting ubiquitinated aggregates, serves as a model for studying the multivalent, heterotypic interactions of cargo-bound receptors. Here, we find that three critical NBR1 partners-ATG8-family proteins, FIP200, and TAX1BP1-each bind to distinct, overlapping determinants within a short linear interaction motif (SLiM). To explore whether overlapping SLiMs extend beyond NBR1, we analyzed >100 LC3-interacting regions (LIRs), revealing that FIP200 and/or TAX1BP1 binding to LIRs is a common phenomenon and suggesting LIRs as protein interaction hotspots. Phosphomimetic peptides demonstrate that phosphorylation generally enhances FIP200 and ATG8-family binding but not TAX1BP1, indicating differential regulation. In vivo, LIR-mediated interactions with TAX1BP1 promote optimal NBR1 flux by leveraging additional functionalities from TAX1BP1. These findings reveal a one-to-many binding modality in the LIR motif of NBR1, illustrating the cooperative mechanisms of autophagy receptors and the regulatory potential of multifunctional SLiMs.

DOI: https://doi.org/10.1083/jcb.202407105
Glia. 2025 Feb 10.

The Fragile X Messenger Ribonucleoprotein 1 Regulates the Morphology and Maturation of Human and Rat Oligodendrocytes.

Vidya Ramesh, Eleni Tsoukala, Ioanna Kougianou, Zrinko Kozic, Karen Burr, Biju Viswanath, David Hampton, David Story, Bharath Kumar Reddy, Rakhi Pal, Owen Dando, Peter C Kind, Sumantra Chattarji, Bhuvaneish T Selvaraj, Siddharthan Chandran, Lida Zoupi.

  The Fragile X Messenger Ribonucleoprotein (FMRP) is an RNA binding protein that regulates the translation of multiple mRNAs and is expressed by neurons and glia in the mammalian brain. Loss of FMRP leads to fragile X syndrome (FXS), a common inherited form of intellectual disability and autism. While most research has been focusing on the neuronal contribution to FXS pathophysiology, the role of glia, particularly oligodendrocytes, is largely unknown. FXS individuals are characterized by white matter changes, which imply impairments in oligodendrocyte differentiation and myelination. We hypothesized that FMRP regulates oligodendrocyte maturation and myelination during postnatal development. Using a combination of human pluripotent stem cell-derived oligodendrocytes and an Fmr1 knockout rat model, we studied the role of FMRP on mammalian oligodendrocyte development. We found that the loss of FMRP leads to shared defects in oligodendrocyte morphology in both rat and human systems in vitro, which persist in the presence of FMRP-expressing axons in chimeric engraftment models. Our findings point to species-conserved, cell-autonomous defects during oligodendrocyte maturation in FXS.

Keywords:  fragile X syndrome; myelin; neurodevelopmental disorder; oligodendrocytes

DOI:  https://doi.org/10.1002/glia.24680
Acta Neuropathol Commun. 2025 Feb 13. 13(1): 28

Disruption of mitochondrial homeostasis and permeability transition pore opening in OPA1 iPSC-derived retinal ganglion cells.

Michael Whitehead, Joshua P Harvey, Paul E Sladen, Giada Becchi, Kritarth Singh, Yujiao Jennifer Sun, Thomas Burgoyne, Michael R Duchen, Patrick Yu-Wai-Man, Michael E Cheetham.

  Dominant optic atrophy (DOA) is the most common inherited optic neuropathy, characterised by the selective loss of retinal ganglion cells (RGCs). Over 60% of DOA cases are caused by pathogenic variants in the OPA1 gene, which encodes a dynamin-related GTPase protein. OPA1 plays a key role in the maintenance of the mitochondrial network, mitochondrial DNA integrity and bioenergetic function. However, our current understanding of how OPA1 dysfunction contributes to vision loss in DOA patients has been limited by access to patient-derived RGCs. Here, we used induced pluripotent stem cell (iPSC)-RGCs to study how OPA1 dysfunction affects cellular homeostasis in human RGCs. iPSCs derived from a DOA+ patient with the OPA1 R445H variant and isogenic CRISPR-Cas9-corrected iPSCs were differentiated to iPSC-RGCs. Defects in mitochondrial networks and increased levels of reactive oxygen species were observed in iPSC-RGCs carrying OPA1 R445H. Ultrastructural analyses also revealed changes in mitochondrial shape and cristae structure, with decreased endoplasmic reticulum (ER): mitochondrial contact length in DOA iPSC-RGCs. Mitochondrial membrane potential was reduced and its maintenance was also impaired following inhibition of the F1Fo-ATP synthase with oligomycin, suggesting that mitochondrial membrane potential is maintained in DOA iPSC-RGCs through reversal of the ATP synthase and ATP hydrolysis. These impairments in mitochondrial structure and function were associated with defects in cytosolic calcium buffering following ER calcium release and store-operated calcium entry, and following stimulation with the excitatory neurotransmitter glutamate. In response to mitochondrial calcium overload, DOA iPSC-RGCs exhibited increased sensitivity to opening of the mitochondrial permeability transition pore. These data reveal novel aspects of DOA pathogenesis in R445H patient-derived RGCs. The findings suggest a mechanism in which primary defects in mitochondrial network dynamics disrupt core mitochondrial functions, including bioenergetics, calcium homeostasis, and opening of the permeability transition pore, which may contribute to vision loss in DOA patients.

Keywords:  Calcium homeostasis; Dominant optic atrophy; Mitochondrial networks; Neurodegeneration; OPA1; Retinal ganglion cells; iPSCs

DOI:  https://doi.org/10.1186/s40478-025-01942-z
Adv Exp Med Biol. 2025 ;1468 101-106

Prime Editing Strategy to Install the RPE65 c.1430A>G Dominant Mutation.

Bruna Lopes da Costa, Salvatore Marco Caruso, Yi-Ting Tsai, David S Castillejos, Mohamed Sylla, Stephen H Tsang, Peter M J Quinn.

  The retinal pigment epithelium 65-kDa protein (RPE65) is a retinal isomerase that is an essential component of the visual cycle. Mutations in RPE65 are typically associated with autosomal recessive retinitis pigmentosa and Leber congenital amaurosis. Here, we report on a patient with RPE65-mediated autosomal dominant retinitis pigmentosa (adRP) who has widespread chorioretinal atrophy with significant macular involvement and only small areas of retinal preservation. In future studies, we plan to model the pathobiology of RPE65-mediated adRP using induced pluripotent stem cell (iPSC)-derived RPE. To effectively model rare mutations using iPSC-derived RPE and screen gene editing correction approaches, we require a strategy to install the desired mutation in wild-type iPSC and HEK293T. In this study, we developed a prime editing strategy for the installation of the pathogenic RPE65 c.1430A>G mutation underlying our patient's disease.

Keywords:  Autosomal dominant retinitis pigmentosa (adRP); Prime editing; RPE65

DOI:  https://doi.org/10.1007/978-3-031-76550-6_17
Sci Rep. 2025 Feb 12. 15(1): 5148

Bafilomycin A1 induces colon cancer cell death through impairment of the endolysosome system dependent on iron.

Dong Hwa Min, Dasom Kim, Seung Taek Hong, Joohee Kim, Min Jung Kim, Seung-Hae Kwon, Aeree Kim, Ji-Yun Lee.

  The late endolysosomal compartment plays a crucial role in cancer cell metabolism by regulating lysosomal activity, essential for cell proliferation, and the degradation of cellular components during the final stages of autophagy. Modulating late endolysosomal function represents a new target for cancer therapy. In this study, we investigated the effects of bafilomycin A1 (BA1), a vacuolar H+-ATPase inhibitor, on colon cancer and normal colon fibroblasts (CCD-18Co) cells. We found that very low concentrations (~ 2 nM) of BA1 selectively induced cell death in colon cancer cells. This cytotoxicity was associated with lysosomal stress response and dysregulation of iron homeostasis. BA1 treatment resulted in significant alterations to the endolysosomal system, including an increased number and size of lysosomes, lysosomal membrane permeabilization, and autophagy flux blockade. These changes were accompanied by endoplasmic reticulum stress and lipid droplet accumulation. Furthermore, BA1 decreased intracellular Fe2+ levels, as measured using FerroOrange. Notably, iron (III)-citrate supplementation rescued cells from BA1-induced death. These findings suggest that BA1-induced endolysosomal dysfunction impairs iron homeostasis, ultimately leading to colon cancer cell death. Our results highlight the potential of targeting endolysosomal function and iron homeostasis as novel therapeutic strategies for colon cancer, paving the way for more selective and effective treatments.

Keywords:  Autophagy; Bafilomycin A1; Colorectal cancer; Endolysosome; Iron

DOI:  https://doi.org/10.1038/s41598-025-89127-5
Autophagy. 2025 Feb 12.

Heat-shock chaperone HSPB1 mitigates poly-glycine-induced neurodegeneration via restoration of autophagic flux.

Ning Ding, Yijie Song, Yuhang Zhang, Wei Yu, Xinnan Li, Wei Li, Lei Li.

  The CGG repeat expansions in the 5"-UTR regions of certain genes have been implicated in various neurodegenerative and muscular disorders. However, the underlying pathogenic mechanisms are not well understood. In this study, we explore the role of the small molecular chaperone HSPB1 in counteracting neurodegeneration induced by poly-glycine (poly-G) aggregates. Employing a reporter system, we demonstrate that CGG repeat expansions within the 5"-UTR of the GIPC1 gene produce poly-G proteins, by repeat-associated non-AUG (RAN) translation. Through proximity labeling and subsequent mass spectrometry analysis, we characterize the composition of poly-G insoluble aggregates and reveal that these aggregates sequester key macroautophagy/autophagy receptors, SQSTM1/p62 and TOLLIP. This sequestration disrupts MAP1LC3/LC3 recruitment and impairs autophagosome formation, thereby compromising the autophagic pathway. Importantly, we show that HSPB1 facilitates the dissociation of these receptors from poly-G aggregates and consequently restores autophagic function. Overexpressing HSPB1 alleviates poly-G-induced neurodegeneration in mouse models. Taken together, these findings highlight a mechanistic basis for the neuroprotective effects of HSPB1 and suggest its potential as a therapeutic target in treating poly-G-associated neurodegenerative diseases.

Keywords:  Autophagy receptors; CGG repeat expansions; HSPB1; RAN translation; p62; poly-glycine

DOI:  https://doi.org/10.1080/15548627.2025.2466144
Adv Exp Med Biol. 2025 ;1468 351-355

The Importance of Differentiated RPE Cultures for studying Cell Biological Processes.

Roni A Hazim, David S Williams.

  The retinal pigment epithelium (RPE) is a polarized monolayer of cells that provides essential functions to the light-sensitive photoreceptors in the retina. Many of the cell biological processes involving the RPE, including those underlying disease mechanisms, can be studied using in vitro culture systems. For such models to be informative, the RPE cultures must be well-differentiated and fully mature, exhibiting the key characteristics of their native counterparts. In this mini review, we emphasize this requirement to use fully differentiated RPE cultures by discussing structural, functional, and metabolic aspects of the RPE.

Keywords:  Actin filaments; Cytoskeleton; Metabolism; Microtubules; Phagocytosis; RPE

DOI:  https://doi.org/10.1007/978-3-031-76550-6_58
Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2024 Sept 28;49(9):pii: 1672-7347(2024)09-1377-13. [Epub ahead of print]49(9): 1377-1389

Reprogramming astrocytes into dopaminergic neurons to restore motor dysfunction in Parkinson's disease model rats.

Chunbo Liu, Mengjiao Ying, Ao Wang, Yumeng Liu, Ying Chen, Wenhao Ye, Hebao Wen, Caiyun Ma, Changqing Liu, Yu Guo.

   OBJECTIVES: Parkinson's disease (PD) is a neurodegenerative disorder primarily caused by the loss of dopaminergic neurons (DA) in the brain. Since DA neurons are non-renewable, conventional therapies only alleviate symptoms without addressing the root cause. This study aims to reprogram astrocyte (AS) into DA neurons for transplantation into the brain to reconstruct damaged neural circuits and treat PD.
METHODS: Astrocytes were isolated from neonatal rat brain tissues. A lentiviral vector carrying the transcription factors nuclear receptor-related factor 1 (UNRR1) and achaete-scute family bHLH transcription factor 1 (ASCL1), named LV-NURR1-ASCL1, was constructed and used to infect cultured rat AS in vitro. Immunofluorescence, Western blotting, and reverse transcription polymerase chain reaction (RT-PCR) were employed to detect and compare the expression levels of NURR1 and ASCL1 in lentivirus-infected AS (LV group) and AS cultured in complete medium without LV-NURR1-ASCL1 (Con group). The virus-infected AS was then cultured in neuronal induction medium for 18 days. Immunofluorescence was used to detect the expression of DA markers, including tyrosine hydroxylase (TH) and forkhead box A2 (FOXA2), as well as the neuronal marker class III beta tubulin (TUJ1). To establish the PD rat model, 6-hydroxydopamine (6-OHDA) was injected into 2 sites in the medial forebrain bundle (MFB) region of the right brain in rats. The reprogrammed cells (AS-iDA) were quantified and transplanted into the right MFB region of the PD model rats using a stereotaxic instrument. Four weeks after transplantation, immunofluorescence was used to assess the survival, differentiation, and migration of AS-iDA in the brain and the expression of TH, TUJ1, and FOXA2 in the brain tissue of PD rats. Eight weeks post-transplantation, the recovery of motor function in PD rats was evaluated using the apomorphine (APO)-induced rotation test, rotarod fatigue test, and open-field test.
RESULTS: Immunofluorescence analysis showed positive expression of NURR1 and ASCL1 in AS after lentiviral infection. RT-PCR results demonstrated that the mRNA expression levels of NURR1 and ASCL1 in the LV group were significantly higher than those in the Con group, with increases of (7.483±0.706)-fold and (10.830±1.940)-fold, respectively. Western blotting analysis further confirmed that the protein expression levels of NURR1 and ASCL1 in the LV group were (2.403±0.511)-fold and (4.423±0.603)-fold higher, respectively, compared to the control group. After 18 days of directed induction culture lentivirus-infected AS (AS-iDA) displayed significant morphological changes, developing neuron-like long neurites. At this stage, AS-iDA highly expressed the neuronal marker TUJ1 as well as the DA markers TH and FOXA2. Four weeks post-transplantation, immunofluorescence on brain slices from PD rats revealed that AS-iDA survived in the transplant region, migrated to surrounding areas, and expressed TUJ1, TH, and FOXA2. At 8 weeks post-transplantation, compared to untreated PD rats, PD rats transplanted with AS-iDA exhibited significantly reduced rotational behavior in the APO-induced rotation test, increased mobility in the open-field test, and extended time on the rotarod in the fatigue test (all P<0.05).
CONCLUSIONS: Lentiviral overexpression of NURR1 and ASCL1 efficiently reprograms AS into DA neurons. Transplantation of reprogrammed DA neurons significantly improves motor function in PD rats, highlighting their potential as donor cells for the treatment of neurodegenerative diseases.

Keywords:  astrocytes; cell transdifferentiation; dopaminergic neurons; parkinson’s disease; transcription factors

DOI:  https://doi.org/10.11817/j.issn.1672-7347.2024.240078
Adv Sci (Weinh). 2025 Feb 08. e2410909

NF2 is Essential for Human Endoderm Development.

Minjin Jeong, Dongjun Han, Preetida Bhetariya, D Bradley Welling, Miodrag Stojkovic, Konstantina M Stankovic.

  Vertebrate embryogenesis requires the precisely timed specification of 3 germ cell layers- ectoderm, mesoderm, and endoderm- which give rise to tissues and organs in the developing organism. The tumor suppressor gene NF2, moesin-ezrin-radixin like (MERLIN) tumor suppressor (Nf2) is expressed in all 3 germ layers during mouse development and its homozygous deletion causes embryonic lethality. People with heterozygous NF2 mutations typically develop Schwann cell tumors, especially vestibular schwannoma, but the specific role of NF2 in human embryonic development is unclear. Here, human induced pluripotent stem cells (hiPSCs) are used to demonstrate that NF2 is essential for endoderm specification and formation in humans. Although endoderm differentiation is not impaired in hiPSCs with heterozygous NF2 mutation, NF2 knockout (NF2-/-) abolished the capacity to form endoderm in vitro, confirmed by loss of expression of endoderm-related genes and proteins, or teratomas in vivo. This defect is mediated by the nuclear translocation of yes-associated protein 1 (YAP1), a transcription co-activator regulating lineage fate via the Hippo pathway and subsequent YAP1-mediated shutdown of Activin/Nodal signaling. Endoderm formation can be rescued via YAP1 knockdown or forced re-expression of NF2 in NF2-/- cells. Taken together, the essential role of NF2 during endoderm specification in human embryogenesis as a regulator of YAP1 is reported.

Keywords:  NF2; YAP1; endoderm; hippo pathway; human induced pluripotent stem cells

DOI:  https://doi.org/10.1002/advs.202410909
Methods Mol Biol. 2025 Feb 11.

Generation of Retinal Organoids Using Human-Induced Pluripotent Stem Cells.

Keith Theodore, Joel Alan Imventarza, Saleha Tahir, Bruna Lopes da Costa, Peter M J Quinn.

  Retinal organoids (ROs) derived from human-induced pluripotent stem cells (hiPSCs) serve as relevant models for studying retinal disease pathogenesis, as well as furthering gene therapy efforts. These complex, three-dimensional (3D), multicellular structures recapitulate the development and functionality of the maturing human retina. Here, we describe an in-depth method for the generation of ROs from hiPSCs and evaluate the morphology of these multilayered structures.

Keywords:  Human-induced pluripotent stem cells (hiPSCs); Retinal organoids

DOI:  https://doi.org/10.1007/7651_2024_600
Cell Rep. 2025 Jan 28. pii: S2211-1247(24)01494-3. [Epub ahead of print]44(1): 115143

A microscopy-based screen identifies cellular kinases modulating mitochondrial translation.

Roya Yousefi, Luis Daniel Cruz-Zaragoza, Anusha Valpadashi, Carina Hansohn, Drishan Dahal, Ricarda Richter-Dennerlein, Silvio Rizzoli, Henning Urlaub, Peter Rehling, David Pacheu-Grau.

  Mitochondrial DNA encodes 13 subunits of the oxidative phosphorylation (OXPHOS) system, which are synthesized inside the organelle and essential for cellular energy supply. How mitochondrial gene expression is regulated and integrated into cellular physiology is little understood. Here, we perform a high-throughput screen combining fluorescent labeling of mitochondrial translation products with small interfering RNA (siRNA)-mediated knockdown to identify cellular kinases regulating translation. As proof of principle, the screen identifies known kinases that affect mitochondrial translation, and it also reveals several kinases not yet linked to this process. Among the latter, we focus on the primarily cytosolic kinase, fructosamine 3 kinase (FN3K), which localizes partially to the mitochondria to support translation. FN3K interacts with the mitochondrial ribosome and modulates its assembly, thereby affecting translation. Overall, our work provides a reliable approach to identify protein functions for mitochondrial gene expression in a high-throughput manner.

Keywords:  CP: Metabolism; CP: Molecular biology; cellular kinases; click chemistry; mito-FUNCAT; mitochondrial translation; siRNA library

DOI:  https://doi.org/10.1016/j.celrep.2024.115143
Signal Transduct Target Ther. 2025 Feb 10. 10(1): 37

AXL signaling in cancer: from molecular insights to targeted therapies.

Monika Yadav, Akansha Sharma, Ketki Patne, Saba Tabasum, Jyoti Suryavanshi, Laxminarayan Rawat, Marc Machaalani, Marc Eid, Rana P Singh, Toni K Choueiri, Soumitro Pal, Akash Sabarwal.

AXL, a member of the TAM receptor family, has emerged as a potential target for advanced-stage human malignancies. It is frequently overexpressed in different cancers and plays a significant role in various tumor-promoting pathways, including cancer cell proliferation, invasion, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, stemness, DNA damage response, acquired therapeutic resistance, immunosuppression, and inflammatory responses. Beyond oncology, AXL also facilitates viral infections, including SARS-CoV-2 and Zika highlighting its importance in both cancer and virology. In preclinical models, small-molecule kinase inhibitors targeting AXL have shown promising anti-tumorigenic potential. This review primarily focuses on the induction, regulation and biological functions of AXL in mediating these tumor-promoting pathways. We discuss a range of therapeutic strategies, including recently developed small-molecule tyrosine kinase inhibitors (TKIs), monoclonal antibodies, and antibody-drug conjugates (ADCs), anti-AXL-CAR, and combination therapies. These interventions are being examined in both preclinical and clinical studies, offering the potential for improved drug sensitivity and therapeutic efficacy. We further discuss the mechanisms of acquired therapeutic resistance, particularly the crosstalk between AXL and other critical receptor tyrosine kinases (RTKs) such as c-MET, EGFR, HER2/HER3, VEGFR, PDGFR, and FLT3. Finally, we highlight key research areas that require further exploration to enhance AXL-mediated therapeutic approaches for improved clinical outcomes.

DOI: https://doi.org/10.1038/s41392-024-02121-7
Front Cell Dev Biol. 2025 ;13 1529093

Xenopus laevis neural stem progenitor cells exhibit a transient metabolic shift toward glycolysis during spinal cord regeneration.

Paula G Slater, Miguel E Domínguez-Romero, Guillermo Campos, Vania Aravena, Javier Cavieres-Lepe, Verónica Eisner.

  Spinal cord injury (SCI) results in severe disruption of communication between the brain and body, causing motor, sensory, and autonomic dysfunctions. While SCI in mammals leads to permanent impairment due to limited regenerative capacity, certain non-mammalian species, such as Xenopus laevis larval stages, exhibit remarkable regenerative abilities. During Xenopus laevis spinal cord regeneration, neural stem precursor cells (NSPCs) surrounding the central canal rapidly proliferate in response to SCI, compensating for cellular loss, restoring canal continuity, and generating new neurons to reestablish lost connections. It has been described that mitochondria and cellular metabolism play essential roles in stem cell proliferation, self-renewal, and differentiation. However, the mitochondrial and cellular metabolic response during spinal cord regeneration remains unexplored. This study uses electron and confocal microscopy to investigate the NSPCs mitochondrial response in Xenopus laevis following SCI. We observed that mitochondria exhibit a rapid and transient response after SCI, characterized by a disruption of the mitochondrial localization, a decrease in mitochondrial number per cell section, and an increase in mitochondrial area and circularity. Furthermore, mitochondria adopted a swollen phenotype, which did not impair mitochondrial function or cellular energy balance. This morphological shift was accompanied by a transient decrease in the mitochondrial membrane potential and a metabolic switch favoring glycolysis. Therefore, these findings demonstrate that a transient metabolic shift toward glycolysis occurs during spinal cord regeneration.

Keywords:  Xenopus laevis; glycolitic shift; metabolic regulation; mitochondria; neural stem progenitor cells (NSPCs); regeneration

DOI:  https://doi.org/10.3389/fcell.2025.1529093
Am J Hum Genet. 2025 Feb 04. pii: S0002-9297(25)00013-8. [Epub ahead of print]

Isogenic hiPSC models of Turner syndrome development reveal shared roles of inactive X and Y in the human cranial neural crest network.

Darcy T Ahern, Prakhar Bansal, Isaac V Faustino, Owen M Chambers, Erin C Banda, Heather R Glatt-Deeley, Rachael E Massey, Yuvabharath Kondaveeti, Stefan F Pinter.

  Viable human aneuploidy can be challenging to model in rodents due to syntenic boundaries or primate-specific biology. Human monosomy-X (45,X) causes Turner syndrome (TS), altering craniofacial, skeletal, endocrine, and cardiovascular development, which in contrast remain unaffected in X-monosomic mice. To learn how monosomy-X may impact embryonic development, we turned to 45,X and isogenic euploid human induced pluripotent stem cells (hiPSCs) from male and female mosaic donors. Because the neural crest (NC) is hypothesized to give rise to craniofacial and cardiovascular changes in TS, we assessed differential expression of hiPSC-derived anterior NC cells (NCCs). Across three independent isogenic panels, 45,X NCCs show impaired acquisition of PAX7+SOX10+ markers and disrupted expression of other NCC-specific genes relative to isogenic euploid controls. Additionally, 45,X NCCs increase cholesterol biosynthesis genes while reducing transcripts with 5' terminal oligopyrimidine (TOP) motifs, including those of ribosomal and nuclear-encoded mitochondrial proteins. Such metabolic pathways are also over-represented in weighted co-expression modules that are preserved in monogenic neurocristopathy and reflect 28% of all TS-associated terms of the human phenotype ontology. We demonstrate that 45,X NCCs reduce protein synthesis despite activation of mammalian target of rapamycin (mTOR) but are partially rescued by mild mTOR suppression. Our analysis identifies specific sex-linked genes that are expressed from two copies in euploid males and females alike and qualify as candidate haploinsufficient drivers of TS phenotypes in NC-derived lineages. This study demonstrates that isogenic hiPSC-derived NCC panels representing monosomy-X can serve as powerful models of early NC development in TS and inform new hypotheses toward its etiology.

Keywords:  Turner syndrome; X chromosome inactivation; Y chromosome; gene dosage; haploinsufficiency; human iPSC; neural crest; protein synthesis; ribosome biogenesis; sex chromosome evolution

DOI:  https://doi.org/10.1016/j.ajhg.2025.01.013
Open Biol. 2025 Feb;15(2): 240287

The fundamental role of mitochondria-endoplasmic reticulum contacts in ageing and declining healthspan.

Richard M Monaghan.

  This open question research article highlights mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), which have emerged as crucial cellular structures that challenge our traditional understanding of organelle function. This review highlights the critical importance of MAMs as a frontier in cell biology with far-reaching implications for health, disease and ageing. MAMs serve as dynamic communication hubs between the ER and mitochondria, orchestrating essential processes such as calcium signalling, lipid metabolism and cellular stress responses. Recent research has implicated MAM dysfunction in a wide array of conditions, including neurodegenerative diseases, metabolic disorders, cardiovascular diseases and cancer. The significant lack of biological knowledge behind MAM function emphasizes the need to study these enigmatic subcellular sites in greater detail. Key open questions include the mechanisms controlling MAM formation and disassembly, the full complement of MAM-associated proteins and how MAMs contribute to cellular decision-making and ageing processes. Advancing our understanding of MAMs through interdisciplinary approaches and cutting-edge technologies promises to reveal new insights into fundamental cellular signalling pathways and potentially lead to innovative therapeutic strategies for a range of diseases. As such, MAM research represents a critical open question in biology with the potential to transform our understanding of cellular life and human health.

Keywords:  ageing; endoplasmic reticulum; healthspan; membrane contact sites; metabolism; mitochondria

DOI:  https://doi.org/10.1098/rsob.240287