bims-axbals 2024-12-01 papers

bims-axbals

Biomed News

on Axonal biology and ALS

Issue of 2024–12–01
forty-one papers selected by
TJ Krzystek, ALS Therapy Development Institute

Epigenetic age acceleration as a biomarker of amyotrophic lateral sclerosis severity?
Seeding-competent TDP-43 persists in human patient and mouse muscle.
Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUS P525L spinal motor neurons.
Conserved region of human TDP-43 is structurally similar to membrane binding protein FARP1 and protein chaperons BAG6 and CYP33.
Excitatory Cortical Neurons from CDKL5 Deficiency Disorder Patient-Derived Organoids Show Early Hyperexcitability Not Identified in Neurogenin2 Induced Neurons.
A rare genetic variant in APEX1 is associated with familial amyotrophic lateral sclerosis with slow progression.
Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model.
14-3-3 binding maintains the Parkinson's associated kinase LRRK2 in an inactive state.
Small molecule modulation of p75NTR engages the autophagy-lysosomal pathway and reduces huntingtin aggregates in cellular and mouse models of Huntington's disease.
Early disruption of the CREB pathway drives dendritic morphological alterations in FTD/ALS cortical neurons.
Upregulated miR-10b-5p as a potential miRNA signature in amyotrophic lateral sclerosis patients.
Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Mechanistic Insights into RNA Oligonucleotide-Mediated Inhibition of TDP-43 Aggregation.
Human VCP mutant ALS/FTD microglia display immune and lysosomal phenotypes independently of GPNMB.
G2019S Mutation of Leucine-Rich Repeat Kinase 2 Is a Cause of Lewy Body Dementia in Patients With North African Ancestors.
Co-Aggregation of TDP-43 with Other Pathogenic Proteins and Their Co-Pathologies in Neurodegenerative Diseases.
ALS-linked mutant TDP-43 in oligodendrocytes induces oligodendrocyte damage and exacerbates motor dysfunction in mice.
MicroRNAs as Biomarkers in Spinal Muscular Atrophy.
Rapidly Progressing and Early-Onset Forms of Amyotrophic Lateral Sclerosis Caused by a Novel SOD1 Variant in a Lithuanian Family.
Measurements of neurite extension and nucleokinesis in an iPSC-derived model system following microtubule perturbation.
Navigating the pathways: TAR-DNA-binding-protein-43 aggregation, axonal transport, and local synthesis in amyotrophic lateral sclerosis pathology.
Analysis of dopaminergic neuron-specific mitochondrial morphology and function using tyrosine hydroxylase reporter iPSC lines.
Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection.
Development of a Novel Microphysiological System for Peripheral Neurotoxicity Prediction Using Human iPSC-Derived Neurons with Morphological Deep Learning.
Impairments of inhibitory neurons in amyotrophic lateral sclerosis and frontotemporal dementia.
Myelinated Glial Cells: Their Proposed Role in Waste Clearance and Neurodegeneration in Arachnid and Human Brain.
Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids.
Investigating Inherited Heart Diseases Using Human Induced Pluripotent Stem Cell-Based Models.
Tracing ALS Degeneration: Insights from Spinal Cord and Cortex Transcriptomes.
Loss of neuronal Imp induces seizure behavior through Syndecan function.
Protein translocation through α-helical channels and insertases.
Development of MAPT S305 mutation human iPSC lines exhibiting elevated 4R tau expression and functional alterations in neurons and astrocytes.
Protective Effects of Ambroxol on Aβ and α-Synuclein-Induced Neurotoxicity Through Glucocerebrosidase Activation in HT-22 Hippocampal Neuronal Cells.
Microglial APOE3 Christchurch protects neurons from Tau pathology in a human iPSC-based model of Alzheimer's disease.
Epileptiform activity in brain organoids derived from patient with Glucose Transporter 1 Deficiency Syndrome.
Generating Skin-Derived Precursor-Like Cells From Human-Induced Pluripotent Stem Cell-Derived Skin Organoids.
Modeling early phenotypes of Parkinson's disease by age-induced midbrain-striatum assembloids.
Single-cell transcriptomic landscape of the neuroimmune compartment in amyotrophic lateral sclerosis brain and spinal cord.
Loss of Dnah5 Downregulates Dync1h1 Expression, Causing Cortical Development Disorders and Congenital Hydrocephalus.
Molecular architecture of synaptic vesicles.
RNA-containing extracellular vesicles in infection.
Profiling Human iPSC-Derived Sensory Neurons for Analgesic Drug Screening Using a Multi-Electrode Array.

EBioMedicine. 2024 Nov 23. pii: S2352-3964(24)00506-1. [Epub ahead of print]110 105470

Epigenetic age acceleration as a biomarker of amyotrophic lateral sclerosis severity?

Sebastian A Lewandowski, Lara Kular, Maja Jagodic.

DOI: https://doi.org/10.1016/j.ebiom.2024.105470
Sci Transl Med. 2024 Nov 27. 16(775): eadp5730

Seeding-competent TDP-43 persists in human patient and mouse muscle.

Eileen M Lynch, Sara Pittman, Jil Daw, Chiseko Ikenaga, Sheng Chen, Dhruva D Dhavale, Meredith E Jackrel, Yuna M Ayala, Paul Kotzbauer, Cindy V Ly, Alan Pestronk, Thomas E Lloyd, Conrad C Weihl.

TAR DNA binding protein 43 (TDP-43) is an RNA binding protein that accumulates as aggregates in the central nervous systems of some patients with neurodegenerative diseases. However, TDP-43 aggregation is also a sensitive and specific pathologic feature found in a family of degenerative muscle diseases termed inclusion body myopathy. TDP-43 aggregates from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia brain lysates may serve as self-templating aggregate seeds in vitro and in vivo, supporting a prion-like spread from cell to cell. Whether a similar process occurs in patient muscle is not clear. We developed a mouse model of inducible, muscle-specific cytoplasmic localized TDP-43. These mice develop muscle weakness with robust accumulation of insoluble and phosphorylated sarcoplasmic TDP-43, leading to eosinophilic inclusions, altered proteostasis, and changes in TDP-43-related RNA processing that resolve with the removal of doxycycline. Skeletal muscle lysates from these mice also have seeding-competent TDP-43, as determined by a FRET-based biosensor, that persists for weeks upon resolution of TDP-43 aggregate pathology. Human muscle biopsies with TDP-43 pathology also contain TDP-43 aggregate seeds. Using lysates from muscle biopsies of patients with sporadic inclusion body myositis (IBM), immune-mediated necrotizing myopathy (IMNM), and ALS, we found that TDP-43 seeding capacity was specific to IBM. TDP-43 seeding capacity anticorrelated with TDP-43 aggregate and vacuole abundance. These data support that TDP-43 aggregate seeds are present in IBM skeletal muscle and represent a unique TDP-43 pathogenic species not previously appreciated in human muscle disease.

DOI: https://doi.org/10.1126/scitranslmed.adp5730
bioRxiv. 2024 Nov 21. pii: 2024.11.20.624439. [Epub ahead of print]

Axonal transcriptome reveals upregulation of PLK1 as a protective mechanism in response to increased DNA damage in FUS P525L spinal motor neurons.

Vitaly Zimyanin, Banaja P Dash, Dajana Großmann, Theresa Simolka, Hannes Glaß, Riya Verma, Vivek Khatri, Christopher Deppmann, Eli Zunder, Stefanie Redemann, Andreas Hermann.

Mutations in the gene FUSED IN SARCOMA ( FUS ) are among the most frequently occurring genetic forms of amyotrophic lateral sclerosis (ALS). Early pathogenesis of FUS -ALS involves impaired DNA damage response and axonal degeneration. However, it is still poorly understood how these gene mutations lead to selective spinal motor neuron (MN) degeneration and how nuclear and axonal phenotypes are linked. To specifically address this, we applied a compartment specific RNA-sequencing approach using microfluidic chambers to generate axonal as well as somatodendritic compartment-specific profiles from isogenic induced pluripotent stem cells (iPSCs)-derived MNs. We demonstrate high purity of axonal and soma fractions and show that the axonal transcriptome is unique and distinct from that of somas including significantly fewer number of transcripts. Functional enrichment analysis revealed that differentially expressed genes (DEGs) in axons were mainly enriched in key pathways like RNA metabolism and DNA damage, complementing our knowledge of early phenotypes in ALS pathogenesis and known functions of FUS. In addition, we demonstrate a strong enrichment for cell cycle associated genes including significant upregulation of polo-like kinase 1 (PLK1) in FUS P525L mutant MNs. PLK1 was increased upon DNA damage induction and PLK1 inhibition further increased the number of DNA damage foci in etoposide-treated cells, an effect that was diminished in case of FUS mutant MNs. In contrast, inhibition of PLK1 increased late apoptotic or necrosis-induced neuronal cell death in mutant neurons. Taken together, our findings provide insights into compartment-specific transcriptomics in human FUS -ALS MNs and we propose that specific upregulation of PLK1 might represent an early event in the pathogenesis of ALS, possibly modulating DNA damage response and other associated pathways.

DOI: https://doi.org/10.1101/2024.11.20.624439
MicroPubl Biol. 2024 ;2024

Conserved region of human TDP-43 is structurally similar to membrane binding protein FARP1 and protein chaperons BAG6 and CYP33.

Ljiljana Sjekloća, Emanuele Buratti.

Transactive response DNA-binding protein of 43 KDa (TDP-43) is important for RNA metabolism in all animals and in humans is involved in neuromuscular diseases. Full-length TDP-43 is prone to oligomerization and misfolding what renders difficult its characterization. We report that TDP-43 domains are structurally similar to lipid binding protein FARP1 and protein chaperons BAG6 and CYP33. Sequence analysis suggests putative lipid binding sites throughout TDP-43 and in vitro thioflavin T fluorescence assays show that cholesterol and phosphatidylcholine affect fibrillation of recombinant TDP-43 fragments. Our findings suggest that TDP-43 can bind lipids directly and it may contribute to its own chaperoning.

DOI: https://doi.org/10.17912/micropub.biology.001388
bioRxiv. 2024 Nov 11. pii: 2024.11.11.622878. [Epub ahead of print]

Excitatory Cortical Neurons from CDKL5 Deficiency Disorder Patient-Derived Organoids Show Early Hyperexcitability Not Identified in Neurogenin2 Induced Neurons.

Madison R Glass, Dosh Whye, Nickesha C Anderson, Delaney Wood, Nina R Makhortova, Taryn Polanco, Kristina H Kim, Kathleen E Donovan, Lorenzo Vaccaro, Ashish Jain, Davide Cacchiarelli, Liang Sun, Heather Olson, Elizabeth D Buttermore, Mustafa Sahin.

CDKL5 deficiency disorder (CDD) is a rare developmental and epileptic encephalopathy resulting from variants in cyclin-dependent kinase-like 5 (CDKL5) that lead to impaired kinase activity or loss of function. CDD is one of the most common genetic etiologies identified in epilepsy cohorts. To study how CDKL5 variants impact human neuronal activity, gene expression and morphology, CDD patient-derived induced pluripotent stem cells and their isogenic controls were differentiated into excitatory neurons using either an NGN2 induction protocol or a guided cortical organoid differentiation. Patient-derived neurons from both differentiation paradigms had decreased phosphorylated EB2, a known molecular target of CDKL5. Induced neurons showed no detectable differences between cases and isogenic controls in network activity using a multielectrode array, or in MAP2+ neurite length, and only two genes were differentially expressed. However, patient-derived neurons from the organoid differentiation showed increased synchrony and weighted mean firing rate on the multielectrode array within the first month of network maturation. CDD patient-derived cortical neurons had lower expression of CDKL5 and HS3ST1, which may change the extracellular matrix around the synapse and contribute to hyperexcitability. Similar to the induced neurons, there were no differences in neurite length across or within patient-control cell lines. Induced neurons have poor cortical specification while the organoid derived neurons expressed cortical markers, suggesting that the changes in neuronal excitability and gene expression are specific to cortical excitatory neurons. Examining molecular mechanisms of early hyperexcitability in cortical neurons is a promising avenue for identification of CDD therapeutics.

DOI: https://doi.org/10.1101/2024.11.11.622878
Acta Neurol Belg. 2024 Nov 28.

A rare genetic variant in APEX1 is associated with familial amyotrophic lateral sclerosis with slow progression.

Yuxin Mi, Peipei Zhang, Xiaotong Hou, Yuqi Ding, Yiying Wang, Hongwu Du, Min Deng.

  Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder characterized by loss of motor neurons and progressive muscle weakness. We aimed to identify the pathogenic genetic variants in familial ALS (fALS) pedigrees and to elucidate their impact on the disease phenotype. Through the analysis of whole-genome sequencing data of 34 fALS probands that screened negative for mutations in the most common ALS-causing genes, we identified a rare missense variant in APEX1 (NM_001641.4: c.22G > A, p.Gly8Arg) associated with ALS in one pedigree. Fluorescence microscopy images using green fluorescent protein (GFP)-fusion proteins suggested that this amino acid substitution could cause an impairment in nuclear localization of the protein. We described the clinical characteristics of this cohort analyzed and found that patients carrying this variant exhibit lower motor neuron onset and prolonged survival. The relation between APEX1 and ALS occurrence has been elusive despite evidence of a neuroprotective role for the gene. This study provides evidence linking an APEX1 variant with fALS and information on the distinct clinical manifestation. This study contributes to the understanding of the genetic basis of ALS, as well as a potential mechanism leading to loss of neurons, highlighting possible opportunities of targeted treatment harnessing the DNA repair process or ameliorating the oxidative stress.

Keywords:  APEX1; Amyotrophic lateral sclerosis; DNA repair; Single nucleotide variant; Whole genome sequencing

DOI:  https://doi.org/10.1007/s13760-024-02692-w
Sci Adv. 2024 Nov 29. 10(48): eadq3350

Inflammatory cytokines disrupt astrocyte exosomal HepaCAM-mediated protection against neuronal excitotoxicity in the SOD1G93A ALS model.

Shijie Jin, Yang Tian, Jonathan Hacker, Xuan Chen, Marcela Bertolio, Caroline Reynolds, Rachel Jarvis, Jingwen Hu, Vanessa Promes, Dilara Halim, Fen-Biao Gao, Yongjie Yang.

Astrocyte secreted signals substantially affect disease pathology in neurodegenerative diseases. It remains little understood about how proinflammatory cytokines, such as interleukin-1α/tumor necrosis factor-α/C1q (ITC), often elevated in neurodegenerative diseases, alter astrocyte-secreted signals and their effects in disease pathogenesis. By selectively isolating astrocyte exosomes (A-Exo.) and employing cell type-specific exosome reporter mice, our current study showed that ITC cytokines significantly reduced A-Exo. secretion and decreased spreading of focally labeled A-Exo. in diseased SOD1G93A mice. Our results also found that A-Exo. were minimally associated with misfolded SOD1 and elicited no toxicity to mouse spinal and human iPSC-derived motor neurons. In contrast, A-Exo. were neuroprotective against excitotoxicity, which was completely diminished by ITC cytokines and partially abolished by SOD1G93A expression. Subsequent proteomic characterization of A-Exo. and genetic analysis identified that surface expression of glial-specific HepaCAM preferentially mediates A-Exo's axon protection effect. Together, our study defines a cytokine-induced loss-of-function mechanism of A-Exo. in protecting neurons from excitotoxicity in amyotrophic lateral sclerosis.

DOI: https://doi.org/10.1126/sciadv.adq3350
bioRxiv. 2024 Nov 22. pii: 2024.11.22.624879. [Epub ahead of print]

14-3-3 binding maintains the Parkinson's associated kinase LRRK2 in an inactive state.

Juliana A Martinez Fiesco, Ning Li, Astrid Alvarez de la Cruz, Riley D Metcalfe, Alexandra Beilina, Mark R Cookson, Ping Zhang.

Leucine-rich repeat kinase 2 (LRRK2) is a central player in cellular signaling and a significant contributor to Parkinson's disease (PD) pathogenesis. 14-3-3 proteins are essential regulators of LRRK2, modulating its activity. Here, we present the cryo- electron microscopy structure of the LRRK2:14-3-3 2 autoinhibitory complex, showing that a 14-3-3 dimer stabilizes an autoinhibited LRRK2 monomer by binding to key phosphorylation sites and the COR-A and COR-B subdomains within the Roc-COR GTPase domain of LRRK2. This interaction locks LRRK2 in an inactive conformation, restricting LRR domain mobility and preventing dimerization and oligomer formation. Our mutagenesis studies reveal that PD-associated mutations at the COR:14-3-3 interface and within the GTPase domain reduce 14-3-3 binding, diminishing its inhibitory effect on LRRK2. These findings provide a structural basis for understanding how LRRK2 likely remains dormant within cells, illuminate aspects of critical PD biomarkers, and suggest therapeutic strategies to enhance LRRK2-14-3-3 interactions to treat PD and related disorders.

DOI: https://doi.org/10.1101/2024.11.22.624879
Neurotherapeutics. 2024 Nov 25. pii: S1878-7479(24)00182-X. [Epub ahead of print] e00495

Small molecule modulation of p75NTR engages the autophagy-lysosomal pathway and reduces huntingtin aggregates in cellular and mouse models of Huntington's disease.

Danielle A Simmons, Namitha Alexander, Gloria Cao, Ido Rippin, Yarine Lugassy, Hagit Eldar-Finkelman, Frank M Longo.

  Huntington's disease (HD) is a neurodegenerative disorder caused by a CAG repeat expansion in the HTT gene encoding a mutant huntingtin (mHtt) protein. mHtt aggregates within neurons causing degeneration primarily in the striatum. There is currently a need for disease-modifying treatments for HD. Many therapeutic studies have focused on lowering mHtt levels by reducing its production or enhancing its clearance. One way to clear mHtt aggregates is to promote autophagy, which is disrupted in HD. Our previous studies showed that the small molecule p75 neurotrophin receptor (p75NTR) ligand, LM11A-31, prevented HD-related neuropathologies and behavioral deficits in multiple HD mouse models. This study investigated whether modulating p75NTR with LM11A-31, would reduce mHtt aggregates via autophagic/lysosomal mechanisms in HD models. LM11A-31 decreased mHtt aggregates in human neuroblastoma SH-SY5Y cells expressing mHtt (exon 1 with 74 CAG repeats) and in the striatum of R6/2 and zQ175dn mouse models of HD. The LM11A-31 associated decrease in mHtt aggregates in vitro was accompanied by increased autophagic/lysosomal activity as indicated by altered levels of relevant markers including p62/SQSTM1 and the lysosomal protease, mature cathepsin D, and increased autophagy flux. In R6/2 and/or zQ175dn striatum, LM11A-31 increased AMPK activation, normalized p62/SQSTM1 and LC3II levels, and enhanced LAMP1 and decreased LC3B association with mHtt. Thus, LM11A-31 reduces mHtt aggregates and may do so via engaging autophagy/lysosomal systems. LM11A-31 has successfully completed a Phase 2a clinical trial for mild-to-moderate Alzheimer's disease and our results here strengthen its potential as a candidate for HD clinical testing.

Keywords:  Autophagy; Huntingtin inclusions; Neurodegeneration; Neurotrophin; p75(NTR)

DOI:  https://doi.org/10.1016/j.neurot.2024.e00495
Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2406998121

Early disruption of the CREB pathway drives dendritic morphological alterations in FTD/ALS cortical neurons.

Michelle Jean Gregoire, Riccardo Sirtori, Liviana Donatelli, Emily Morgan Potts, Alicia Collins, Danielo Zamor, Natallia Katenka, Claudia Fallini.

  Synaptic loss and dendritic degeneration are common pathologies in several neurodegenerative diseases characterized by progressive cognitive and/or motor decline, such as Alzheimer's disease (AD) and frontotemporal dementia/amyotrophic lateral sclerosis (FTD/ALS). An essential regulator of neuronal health, the cAMP-dependent transcription factor CREB positively regulates synaptic growth, learning, and memory. Phosphorylation of CREB by protein kinase A (PKA) and other cellular kinases promotes neuronal survival and maturation via transcriptional activation of a wide range of downstream target genes. CREB pathway dysfunction has been strongly implicated in AD pathogenesis, and recent data suggest that impaired CREB activation may contribute to disease phenotypes in FTD/ALS as well. However, the mechanisms behind reduced CREB activity in FTD/ALS pathology are not clear. In this study, we found that cortical-like neurons derived from iPSC lines carrying the hexanucleotide repeat expansion in the C9ORF72 gene, a common genetic cause of FTD/ALS, displayed a diminished activation of CREB, resulting in decreased dendritic and synaptic health. Importantly, we determined such impairments to be mechanistically linked to an imbalance in the ratio of regulatory and catalytic subunits of the CREB activator PKA and to be conserved in C9-ALS patient's postmortem tissue. Modulation of cAMP upstream of this impairment allowed for a rescue of CREB activity and an amelioration of dendritic morphology and synaptic protein levels. Our data elucidate the mechanism behind early CREB pathway dysfunction and discern a feasible therapeutic target for the treatment of FTD/ALS and possibly other neurodegenerative diseases.

Keywords:  ALS; CREB; FTD; PKA; dendrites

DOI:  https://doi.org/10.1073/pnas.2406998121
Front Cell Neurosci. 2024 ;18 1457704

Upregulated miR-10b-5p as a potential miRNA signature in amyotrophic lateral sclerosis patients.

Banaja P Dash, Axel Freischmidt, Anika M Helferich, Albert C Ludolph, Peter M Andersen, Jochen H Weishaupt, Andreas Hermann.

  Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset disease marked by a progressive degeneration of motor neurons (MNs) present in the spinal cord, brain stem and motor cortex. Death in most patients usually occurs within 2-4 years after symptoms onset. Despite promising progress in delineating underlying mechanisms, such as disturbed proteostasis, DNA/RNA metabolism, splicing or proper nucleocytoplasmic shuttling, there are no effective therapies for the vast majority of cases. A reason for this might be the disease heterogeneity and lack of substantial clinical and molecular biomarkers. The identification and validation of such pathophysiology driven biomarkers could be useful for early diagnosis and treatment stratification. Recent advances in next generation RNA-sequencing approaches have provided important insights to identify key changes of non-coding RNAs (ncRNAs) implicated with ALS disease. Especially, microRNAs (miRNAs) have emerged as key post-transcriptional regulators of gene expression to target several genes/pathways by degrading messenger RNAs (mRNAs) or repressing levels of gene expression. In this study, we expand our previous work to identify top-regulated differentially expressed (DE)-miRNAs by combining different normalizations to search for important and generalisable pathomechanistic dysregulations in ALS as putative novel biomarkers of the disease. For this we performed a consensus pipeline of existing datasets to investigate the transcriptomic profile (mRNAs and miRNAs) of MN cell lines from iPSC-derived SOD1- and TARDBP (TDP-43 protein)-mutant-ALS patients and healthy controls to identify potential signatures and their related pathways associated with neurodegeneration. Transcriptional profiling of miRNA-mRNA interactions from MN cell lines in ALS patients revealed differential expression of genes showed greater vulnerability to KEAP1-NRF2 stress response pathway, sharing a common molecular denominator linked to both disease conditions. We also reported that mutations in above genes led to significant upregulation of the top candidate miR-10b-5p, which we could validate in immortalized lymphoblast cell lines (LCLs) derived from sporadic and familial ALS patients and postmortem tissues of familial ALS patients. Collectively, our findings suggest that miRNA analysis simultaneously performed in various human biological samples may reveal shared miRNA profiles potentially useful as a biomarker of the disease.

Keywords:  amyotrophic lateral sclerosis; differentially expressed; human induced pluripotent stem cells; microRNA; motor neurons; next generation RNA sequencing

DOI:  https://doi.org/10.3389/fncel.2024.1457704
J Am Chem Soc. 2024 Nov 29.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Mechanistic Insights into RNA Oligonucleotide-Mediated Inhibition of TDP-43 Aggregation.

Thomas C Minshull, Emily J Byrd, Monika Olejnik, Antonio N Calabrese.

Deposits of aggregated TAR DNA-binding protein 43 (TDP-43) in the brain are associated with several neurodegenerative diseases. It is well established that binding of RNA/DNA to TDP-43 can prevent TDP-43 aggregation, but an understanding of the structure(s) and conformational dynamics of TDP-43, and TDP-43-RNA complexes, is lacking, including knowledge of how the solution environment modulates these properties. Here, we address this challenge using hydrogen-deuterium exchange-mass spectrometry. In the presence of RNA olignoucleotides, we observe protection from exchange in the RNA recognition motif (RRM) domains of TDP-43 and the linker region between the RRM domains, consistent with nucleic acid binding modulating interdomain interactions. Intriguingly, at elevated salt concentrations, the extent of protection from exchange is reduced in the RRM domains when bound to an RNA sequence derived from the 3' UTR of the TDP-43 mRNA (CLIP34NT) compared to when bound to a (UG)6 repeat sequence. Under these conditions, CLIP34NT is no longer able to prevent TDP-43 aggregation. This suggests that a salt-induced structural rearrangement occurs when bound to this RNA, which may play a role in facilitating aggregation. Additionally, upon RNA binding, we identify differences in exchange within the short α-helical region located in the C-terminal domain (CTD) of TDP-43. These allosterically altered regions may influence the ability of TDP-43 to aggregate and fine-tune its RNA binding repertoire. Combined, these data provide additional insights into the intricate interplay between TDP-43 aggregation and RNA binding, an understanding of which is crucial for unraveling the molecular mechanisms underlying TDP-43-associated neurodegeneration.

DOI: https://doi.org/10.1021/jacs.4c11229
Mol Neurodegener. 2024 Nov 26. 19(1): 90

Human VCP mutant ALS/FTD microglia display immune and lysosomal phenotypes independently of GPNMB.

Benjamin E Clarke, Oliver J Ziff, Giulia Tyzack, Marija Petrić Howe, Yiran Wang, Pierre Klein, Claudia A Smith, Cameron A Hall, Adel Helmy, Michael Howell, Gavin Kelly, Rickie Patani.

BACKGROUND: Microglia play crucial roles in maintaining neuronal homeostasis but have been implicated in contributing to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the role of microglia in ALS/FTD remains incompletely understood.
METHODS: Here, we generated highly enriched cultures of VCP mutant microglia derived from human induced pluripotent stem cells (hiPSCs) to investigate their cell autonomous and non-cell autonomous roles in ALS pathogenesis. We used RNA-sequencing, proteomics and functional assays to study hiPSC derived VCP mutant microglia and their effects on hiPSC derived motor neurons and astrocytes.
RESULTS: Transcriptomic, proteomic and functional analyses revealed immune and lysosomal dysfunction in VCP mutant microglia. Stimulating healthy microglia with the inflammatory inducer lipopolysaccharide (LPS) showed partial overlap with VCP mutant microglia in their reactive transformation. LPS-stimulated VCP mutant microglia displayed differential activation of inflammatory pathways compared with LPS-stimulated healthy microglia. Conserved gene expression changes were identified between VCP mutant microglia, SOD1 mutant mice microglia, and postmortem ALS spinal cord microglial signatures, including increased expression of the transmembrane glycoprotein GPNMB. While knockdown of GPNMB affected inflammatory and phagocytosis processes in microglia, this was not sufficient to ameliorate cell autonomous phenotypes in VCP mutant microglia. Secreted factors from VCP mutant microglia were sufficient to activate the JAK-STAT pathway in hiPSC derived motor neurons and astrocytes.
CONCLUSIONS: VCP mutant microglia undergo cell autonomous reactive transformation involving immune and lysosomal dysfunction that partially recapitulate key phenotypes of microglia from other ALS models and post mortem tissue. These phenotypes occur independently of GPNMB. Additionally, VCP mutant microglia elicit non cell autonomous responses in motor neurons and astrocytes involving the JAK-STAT pathway.

DOI: https://doi.org/10.1186/s13024-024-00773-1
Alzheimer Dis Assoc Disord. 2024 Oct-Dec 01;38(4):38(4): 328-331

G2019S Mutation of Leucine-Rich Repeat Kinase 2 Is a Cause of Lewy Body Dementia in Patients With North African Ancestors.

Kurt Segers, Florence Benoit, Sophie Levy, Valérie Martinet, Joachim G Schulz, Frédéric Bertrand, Gabrielle De Bourgoing, Chiara Tatillo, Jean-Philippe Praet, Isabelle Vandernoot, Laurence Desmyter, Xavier Peyrassol, Pashalina Kehagias, Guillaume Smits, Baptiste Dumoulin, Tatiana Besse-Hammer, Bernard Dachy, Murielle Surquin.

BACKGROUND: Mutations in the LRRK2 gene are the most common genetic cause of Parkinson disease but are believed to play no significant role in Lewy body disease (LBD).
OBJECTIVES: As the frequency of G2019S LRRK2 mutation is extremely high in North African patients with Parkinson disease, we postulate that the high prevalence of LBD in North Africa might be due to the same mutation because LBD and Parkinson disease share many clinical, pathological, and genetic features.
METHODS: We screened patients with LBD or prodromal LBD for the G2019S mutation of LRRK2.
RESULTS: A total of 162 patients were tested for the mutation, which was present in 5 of the 47 patients with North African ancestors. This is a much higher prevalence (10.6%) than in healthy North African subjects (1.45%) but lower than in North African patients with Parkinson disease (36% to 39%). Carriers tended to develop more often orthostatic hypotension and swallowing problems.
CONCLUSIONS: Where previous studies in European and North American patients found no link between LRRK2 mutations and LBD, we found an LRRK2 mutation associated with Lewy body disease, namely the G2019S mutation that might be restricted to patients with North African ancestors. Our study illustrates the need to introduce ethnic diversity as stratifying factor in the analysis of genetic causes of neurodegenerative disorders. The current development of disease-modifying drugs modulating LRRK2 kinase activity could justify to screen North African patients with LBD for the G2019S LRRK2 mutation.

DOI: https://doi.org/10.1097/WAD.0000000000000643
Int J Mol Sci. 2024 Nov 18. pii: 12380. [Epub ahead of print]25(22):

Co-Aggregation of TDP-43 with Other Pathogenic Proteins and Their Co-Pathologies in Neurodegenerative Diseases.

Lei-Lei Jiang, Xiang-Le Zhang, Hong-Yu Hu.

  Pathological aggregation of a specific protein into insoluble aggregates is a common hallmark of various neurodegenerative diseases (NDDs). In the earlier literature, each NDD is characterized by the aggregation of one or two pathogenic proteins, which can serve as disease-specific biomarkers. The aggregation of these specific proteins is thought to be a major cause of or deleterious result in most NDDs. However, accumulating evidence shows that a pathogenic protein can interact and co-aggregate with other pathogenic proteins in different NDDs, thereby contributing to disease onset and progression synergistically. During the past years, more than one type of NDD has been found to co-exist in some individuals, which may increase the complexity and pathogenicity of these diseases. This article reviews and discusses the biochemical characteristics and molecular mechanisms underlying the co-aggregation and co-pathologies associated with TDP-43 pathology. The TDP-43 aggregates, as a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), can often be detected in other NDDs, such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD) and spinocerebellar ataxia type 2 (SCA2). In many cases, TDP-43 is shown to interact and co-aggregate with multiple pathogenic proteins in vitro and in vivo. Furthermore, the co-occurrence and co-aggregation of TDP-43 with other pathogenic proteins have important consequences that may aggravate the diseases. Thus, the current viewpoint that the co-aggregation of TDP-43 with other pathogenic proteins in NDDs and their relevance to disease progression may gain insights into the patho-mechanisms and therapeutic potential of various NDDs.

Keywords:  TDP-43 pathology; co-aggregation; co-pathology; neurodegenerative disease; pathogenic protein

DOI:  https://doi.org/10.3390/ijms252212380
Acta Neuropathol Commun. 2024 Nov 27. 12(1): 184

ALS-linked mutant TDP-43 in oligodendrocytes induces oligodendrocyte damage and exacerbates motor dysfunction in mice.

Mai Horiuchi, Seiji Watanabe, Okiru Komine, Eiki Takahashi, Kumi Kaneko, Shigeyoshi Itohara, Mayuko Shimada, Tomoo Ogi, Koji Yamanaka.

  Nuclear clearance and cytoplasmic aggregation of TAR DNA-binding protein of 43 kDa (TDP-43) are pathological hallmarks of amyotrophic lateral sclerosis (ALS) and its pathogenic mechanism is mediated by both loss-of-function and gain-of-toxicity of TDP-43. However, the role of TDP-43 gain-of-toxicity in oligodendrocytes remains unclear. To investigate the impact of excess TDP-43 on oligodendrocytes, we established transgenic mice overexpressing the ALS-linked mutant TDP-43M337V in oligodendrocytes through crossbreeding with Mbp-Cre mice. Two-step crossbreeding of floxed TDP-43M337V and Mbp-Cre mice resulted in the heterozygous low-level systemic expression of TDP-43M337V with (Cre-positive) or without (Cre-negative) oligodendrocyte-specific overexpression of TDP-43M337V. Although Cre-negative mice also exhibit subtle motor dysfunction, TDP-43M337V overexpression in oligodendrocytes aggravated clasping signs and gait disturbance accompanied by myelin pallor in the corpus callosum and white matter of the lumbar spinal cord in Cre-positive mice. RNA sequencing analysis of oligodendrocyte lineage cells isolated from whole brains of 12-month-old transgenic mice revealed downregulation of myelinating oligodendrocyte marker genes and cholesterol-related genes crucial for myelination, along with marked upregulation of apoptotic pathway genes. Immunofluorescence staining showed cleaved caspase 3-positive apoptotic oligodendrocytes surrounded by activated microglia and astrocytes in aged transgenic mice. Collectively, our findings demonstrate that an excess amount of ALS-linked mutant TDP-43 expression in oligodendrocytes exacerbates motor dysfunction in mice, likely through oligodendrocyte dysfunction and neuroinflammation. Therefore, targeting oligodendrocyte protection, particularly through ameliorating TDP-43 pathology, could represent a potential therapeutic approach for ALS.

Keywords:  Amyotrophic lateral sclerosis; Animal model; Apoptosis; Behavioral test; Myelin; Neuroinflammation; Oligodendrocyte; RNA-sequencing; TDP-43

DOI:  https://doi.org/10.1186/s40478-024-01893-x
Biomedicines. 2024 Oct 23. pii: 2428. [Epub ahead of print]12(11):

MicroRNAs as Biomarkers in Spinal Muscular Atrophy.

Maruša Barbo, Damjan Glavač, Gregor Jezernik, Metka Ravnik-Glavač.

  Spinal muscular atrophy (SMA) is a severe neurodegenerative disease caused by the loss of the survival motor neuron (SMN) protein, leading to degeneration of anterior motor neurons and resulting in progressive muscle weakness and atrophy. Given that SMA has a single, well-defined genetic cause, gene-targeted therapies have been developed, aiming to increase SMN production in SMA patients. The SMN protein is likely involved in the synthesis of microRNAs (miRNAs), and dysregulated miRNA expression is increasingly associated with the pathophysiology of SMA. Currently, there is a lack of reliable biomarkers to monitor SMA; therefore, the search for novel SMA biomarkers, including miRNAs, is crucial as reliable tools are needed to track disease progression, predict the response to therapy and understand the different clinical outcomes of available treatments. In this review, we compile data on miRNAs associated with SMA pathogenesis and their potential use as biomarkers. Based on current knowledge, the most frequently deregulated miRNAs between SMA patients and controls, as well as pre- and post-treatment in SMA patients, include miR-1-3p, miR-133a-3p, miR-133b, and miR-206. These findings offer promising possibilities for improving patient classification and monitoring disease progression and response to treatment. Additionally, these findings provide insights into the broader molecular mechanisms and networks of SMA that could inform the development of future therapeutic strategies.

Keywords:  SMA; biomarkers; circulating miRNA; molecular networks; spinal muscular atrophy

DOI:  https://doi.org/10.3390/biomedicines12112428
Neurol Genet. 2024 Dec;10(6): e200217

Rapidly Progressing and Early-Onset Forms of Amyotrophic Lateral Sclerosis Caused by a Novel SOD1 Variant in a Lithuanian Family.

Domantas Valančius, Birutė Burnytė, Raminta Masaitienė, Aušra Morkūnienė, Aušra Klimašauskienė.

Objectives: To describe a novel familial variant of superoxide dismutase 1 (SOD1)-associated amyotrophic lateral sclerosis (ALS) in a Lithuanian family, highlighting its variable progression and implications for treatment inclusion criteria.
Methods: This study presents the clinical and genetic findings of a family with the novel SOD1 variant, including one member diagnosed with early-onset ALS (onset <40 years) and one with a particularly rapidly progressing course of ALS.
Results: The SOD1 variant NM_000454.5:c.446T>C, NP_000445.1:p.(Val149Ala) was identified in affected family members and 4 asymptomatic members aged 32-56 years. We present detailed disease course of the affected family members obtained during follow-up. Clinically, this variant is associated with variable disease progression, with the time from symptom onset to death ranging from 5 to 77 months.
Discussion: The novel SOD1 variant p.Val149Ala in this Lithuanian family causes ALS of variable onset and course, including a case of early-onset ALS and one case of rapidly progressing ALS, necessitating recognition by the scientific community and development of tailored therapeutic approaches.

DOI: https://doi.org/10.1212/NXG.0000000000200217
Mol Biol Cell. 2024 Nov 27. mbcE24020061

Measurements of neurite extension and nucleokinesis in an iPSC-derived model system following microtubule perturbation.

Muriel Sébastien, Alexandra L Paquette, Lilian Ferotin, Adam G Hendricks, Gary J Brouhard.

In neurons, patterns of different microtubule types are essential for neurite extension and nucleokinesis. Cellular model systems such as rodent primary cultures and induced pluripotent stem cells (iPSC)-derived neurons have provided key insights into how these patterns are created and maintained through the action of microtubule-associated proteins (MAPs), motor proteins, and regulatory enzymes. iPSC-derived models show tremendous promise but lack benchmarking and validation relative to rodent primary cultures. Here we have characterized a recent iPSC-derived model, in which doxycycline-induced expression of Neurogenin-2 drives consistent trans-differentiation into the neuronal state (EBiSC-NEUR1 neurons, referred to as NGN2 neurons below). We developed a suite of open-access, semi-automated methods to measure neurite extension and nucleokinesis of NGN2 neurons, which compare favorably to published data from other models. Then, we challenged NGN2 neurons with a panel of drugs that perturb microtubule physiology. NGN2 neurons extension and nucleokinesis were significantly perturbed by two microtubule-targeting drugs, namely a taxane (paclitaxel) and a vinca alkaloid (DZ-2384). In contrast, inhibition of microtubule severing (spastazoline) or of deacetylation (trichostatin A) had a limited effect on nucleokinesis only. Our results support the primary importance of microtubule dynamics in neuronal development and demonstrate the power of NGN2 neurons as a model system. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

DOI: https://doi.org/10.1091/mbc.E24-02-0061
Neural Regen Res. 2025 Oct 01. 20(10): 2921-2922

Navigating the pathways: TAR-DNA-binding-protein-43 aggregation, axonal transport, and local synthesis in amyotrophic lateral sclerosis pathology.

Ori Bar Avi, Eran Perlson.

DOI: https://doi.org/10.4103/NRR.NRR-D-24-00726
Anat Sci Int. 2024 Nov 29.

Analysis of dopaminergic neuron-specific mitochondrial morphology and function using tyrosine hydroxylase reporter iPSC lines.

Mutsumi Yokota.

  Changes in mitochondrial function and morphology contribute to the development of many neurological diseases. Parkinson's disease is one of the neurodegenerative diseases suspected to be associated with defects in mitochondrial function and quality control. The loss of dopaminergic neurons in the substantia nigra pars compacta is a well-known pathological feature of Parkinson's disease. It is important for elucidating the pathogenesis of Parkinson's disease to analyze mitochondrial function and morphology specific to dopaminergic neurons using live-cell imaging or electron microscopy. However, the cells differentiated into dopaminergic neurons from induced pluripotent stem cells generally comprise heterogeneous populations. We generated tyrosine hydroxylase (TH) reporter iPSC lines to distinguish dopaminergic neurons from other cells for live-cell imaging and electron microscopy. This review summarizes previous studies utilizing the TH reporter iPSC lines and discusses the importance of studying mitochondria specific to dopaminergic neurons. Additionally, it provides overviews of recent studies reporting changes in endoplasmic reticulum-mitochondrial contact sites in Parkinson's disease models.

Keywords:  Dopaminergic neurons; Electron microscopy; Mitochondria; Parkinson’s disease; Tyrosine hydroxylase reporter iPSC

DOI:  https://doi.org/10.1007/s12565-024-00816-z
Anal Biochem. 2024 Nov 22. pii: S0003-2697(24)00264-1. [Epub ahead of print]697 115720

Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection.

Bashkim Kokona, Nicole R Cunningham, Jeanne M Quinn, Danielle R Jacobsen, F Jay Garcia, Sierra M Galindo, Leonard Petrucelli, Walter F Stafford, Thomas M Laue, Robert Fairman.

  Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large G4C2 hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)47, (Gly-Ala)50, and (Gly-Arg)50 fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala)50 in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP)47 is largely monomeric. In contrast, (GA)50 forms both intermediate and large-scale aggregates. (GR)50 is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.

Keywords:  Amyotrophic lateral sclerosis; Analytical ultracentrifugation; Drosophila melanogaster; Sedimentation velocity; Semi-denaturing detergent agarose gel electrophoresis

DOI:  https://doi.org/10.1016/j.ab.2024.115720
Toxics. 2024 Nov 11. pii: 809. [Epub ahead of print]12(11):

Development of a Novel Microphysiological System for Peripheral Neurotoxicity Prediction Using Human iPSC-Derived Neurons with Morphological Deep Learning.

Xiaobo Han, Naoki Matsuda, Makoto Yamanaka, Ikuro Suzuki.

  A microphysiological system (MPS) is an in vitro culture technology that reproduces the physiological microenvironment and functionality of humans and is expected to be applied for drug screening. In this study, we developed an MPS for the structured culture of human iPSC-derived sensory neurons and then predicted drug-induced neurotoxicity by morphological deep learning. Using human iPSC-derived sensory neurons, after the administration of representative anti-cancer drugs, the toxic effects on soma and axons were evaluated by an AI model with neurite images. Significant toxicity was detected in positive drugs and could be classified by different effects on soma or axons, suggesting that the current method provides an effective evaluation of chemotherapy-induced peripheral neuropathy. The results of neurofilament light chain expression changes in the MPS device also agreed with clinical reports. Therefore, the present MPS combined with morphological deep learning is a useful platform for in vitro peripheral neurotoxicity assessment.

Keywords:  human iPSC-derived sensory neuron; microphysiological system; morphological deep learning; peripheral neuropathy

DOI:  https://doi.org/10.3390/toxics12110809
Neurobiol Dis. 2024 Nov 26. pii: S0969-9961(24)00350-4. [Epub ahead of print]203 106748

Impairments of inhibitory neurons in amyotrophic lateral sclerosis and frontotemporal dementia.

Félicie Lorenc, Luc Dupuis, Raphaelle Cassel.

  Amyotrophic lateral sclerosis and frontotemporal dementia are two fatal neurodegenerative disorders. They are part of a pathophysiological continuum, displaying clinical, neuropathological, and genetic overlaps. There is compelling evidence that neuronal circuit dysfunction is an early feature of both diseases. Impaired neuronal excitability, imbalanced excitatory and inhibitory influences, and altered functional connectivity have been reported. These phenomena are likely due to combined alterations in the various cellular components involved in the functioning of neuronal networks. This review focuses on one of these cellular components: inhibitory neurons. We assess the evidence for inhibitory neuron impairments in amyotrophic lateral sclerosis and frontotemporal dementia, as well as the mechanisms leading to the loss of inhibition. We also discuss the contributions of these alterations to symptoms, and the potential therapeutic strategies for targeting inhibitory neuron deficits.

Keywords:  Amyotrophic lateral sclerosis; Frontotemporal dementia; GABA; Glycine; Inhibition; Inhibitory neurons; Interneurons

DOI:  https://doi.org/10.1016/j.nbd.2024.106748
J Comp Neurol. 2024 Nov;532(11): e70000

Myelinated Glial Cells: Their Proposed Role in Waste Clearance and Neurodegeneration in Arachnid and Human Brain.

Ruth Fabian-Fine, Adam L Weaver, Abigail G Roman, Melanie J Winters, John C DeWitt.

  One of the most important goals in biomedical sciences is understanding the causal mechanisms of neurodegeneration. A prevalent hypothesis relates to impaired waste clearance mechanisms from the brain due to reported waste aggregation in the brains of Alzheimer patients, including amyloid-β plaques and neurofibrillary tau tangles. Currently, our understanding of the mechanisms by which waste is removed from the brain is only fragmentary. Here we provide compelling evidence that waste clearance from brain tissue is highly conserved in arachnids and humans. Utilizing RNAscope in situ hybridization, immunohistochemical, ultrastructural, and histological approaches, we demonstrate that cellular debris in spider neurons is engulfed by myelin-forming ependymal glial cells that transect into neuronal somata and form myelin-derived waste-internalizing receptacles. These canal systems channel this debris into the lymphatic system likely in an aquaporin-4 (AQP4) water channel-dependent manner. We provide robust evidence that a similar process may be true in human hippocampus where vast numbers of myelinated AQP4-immunoreactive ependymal glial cells send cellular projections into the somata of neurons and glial cells where they differentiate into waste internalizing receptacles. In the brains of Alzheimer decedents, hypertrophic impairment of these myelinated glial cells leads to the catastrophic obstruction and depletion of neuronal cytoplasm into the ependymal glial cells. At the cellular level, the structural impairment of macroglia leads to swelling myelin protrusions that appear as electron-lucent circular profiles, explaining spongiform abnormalities associated with the neurodegenerative diseases described here. We propose to term this novel type of macroglia-mediated cell death "gliaptosis."

Keywords:  Alzheimer disease; Cupiennius salei; glymphatic system; tanycyte; waste clearance

DOI:  https://doi.org/10.1002/cne.70000
Front Bioeng Biotechnol. 2024 ;12 1467412

Morphogen-driven differentiation is precluded by physical confinement in human iPSCs spheroids.

Haneen S Alsehli, Errin Roy, Thomas Williams, Alicja Kuziola, Yunzhe Guo, Cecile A Dreiss, Jeremy B A Green, Eileen Gentleman, Davide Danovi.

   Introduction: Cell lineage specification is tightly associated with profound morphological changes in the developing human embryo, particularly during gastrulation. The interplay between mechanical forces and biochemical signals is poorly understood.
Methods: Here, we dissect the effects of biochemical cues and physical confinement on a 3D in vitro model based on spheroids formed from human induced pluripotent stem cells (hiPSCs).
Results: First, we compare self-renewing versus differentiating media conditions in free-floating cultures and observe the emergence of tri-germ layers. In these unconfined conditions, BMP4 exposure induces polarised expression of SOX17 in conjunction with spheroid elongation. We then physically confine spheroids using PEG-peptide hydrogels and observe dramatically reduced SOX17 expression, albeit rescued if gels that soften over time are used instead.
Discussion: Our study combines high-content imaging, synthetic hydrogels, and hiPSCs-derived models of early development to define the drivers that cause changes in the shape and the emergence of germ layers.

Keywords:  PEG-based hydrogels; germ layer differentiation; high content image analysis; morphogenesis; pluripotent stem cells

DOI:  https://doi.org/10.3389/fbioe.2024.1467412
Life (Basel). 2024 Oct 25. pii: 1370. [Epub ahead of print]14(11):

Investigating Inherited Heart Diseases Using Human Induced Pluripotent Stem Cell-Based Models.

Brian Xiangzhi Wang.

  Inherited heart diseases (IHDs) are caused by genetic mutations that disrupt the physiological structure and function of the heart. Understanding the mechanisms behind these diseases is crucial for developing personalised interventions in cardiovascular medicine. Development of induced pluripotent stem cells, which can then be differentiated to any nucleated adult cell type, has enabled the creation of personalised single-cell and multicellular models, providing unprecedented insights into the pathophysiology of IHDs. This review provides a comprehensive overview of recent advancements in human iPSC models used to dissect the molecular and genetic underpinnings of common IHDs. We examine multicellular models and tissue engineering approaches, such as cardiac organoids, engineered heart tissue, and multicellular co-culture systems, which simulate complex intercellular interactions within heart tissue. Recent advancements in stem cell models offer a more physiologically relevant platform to study disease mechanisms, enabling researchers to observe cellular interactions, study disease progression, and identify therapeutic strategies. By leveraging these innovative models, we can gain deeper insights into the molecular and cellular mechanisms underlying IHDs, ultimately paving the way for more effective diagnostic and therapeutic strategies.

Keywords:  arrhythmia; disease modelling; inherited heart disease; stem cell; tissue engineering

DOI:  https://doi.org/10.3390/life14111370
Genes (Basel). 2024 Nov 02. pii: 1431. [Epub ahead of print]15(11):

Tracing ALS Degeneration: Insights from Spinal Cord and Cortex Transcriptomes.

Nela Pragathi Sneha, S Akila Parvathy Dharshini, Y-H Taguchi, M Michael Gromiha.

   BACKGROUND/OBJECTIVES: Amyotrophic Lateral Sclerosis is a progressive neurodegenerative disorder characterized by the loss of upper and lower motor neurons. Key factors contributing to neuronal death include mitochondrial energy damage, oxidative stress, and excitotoxicity. The frontal cortex is crucial for action initiation, planning, and voluntary movements whereas the spinal cord facilitates communication with the brain, walking, and reflexes. By investigating transcriptome data from the frontal cortex and spinal cord, we aim to elucidate common pathological mechanisms and pathways involved in ALS for understanding the disease progression and identifying potential therapeutic targets.
METHODS: In this study, we quantified gene and transcript expression patterns, predicted variants, and assessed their functional effects using computational tools. It also includes predicting variant-associated regulatory effects, constructing functional interaction networks, and performing a gene enrichment analysis.
RESULTS: We found novel genes for the upregulation of immune response, and the downregulation of metabolic-related and defective degradation processes in both the spinal cord and frontal cortex. Additionally, we observed the dysregulation of histone regulation and blood pressure-related genes specifically in the frontal cortex.
CONCLUSIONS: These results highlight the distinct and shared molecular disruptions in ALS, emphasizing the critical roles of immune response and metabolic dysfunction in neuronal degeneration. Targeting these pathways may provide new therapeutic avenues to combat neurodegeneration and preserve neuronal health.

Keywords:  ALS; gene expression; network; neurodegenerative diseases; transcriptome analysis

DOI:  https://doi.org/10.3390/genes15111431
bioRxiv. 2024 Nov 22. pii: 2024.11.21.624719. [Epub ahead of print]

Loss of neuronal Imp induces seizure behavior through Syndecan function.

Paula R Roy, Nichole Link.

Seizures affect a large proportion of the global population and occur due to abnormal neuronal activity in the brain. Unfortunately, widespread genetic and phenotypic heterogeneity contribute to insufficient treatment options. It is critical to identify the genetic underpinnings of how seizures occur to better understand seizure disorders and improve therapeutic development. We used the Drosophila model to identify that IGF-II mRNA Binding Protein (Imp) is linked to the onset of this phenotype. Specific reduction of Imp in neurons causes seizures after mechanical stimulation. Importantly, gross motor behavior is unaffected, showing Imp loss does not affect general neuronal activity. Developmental loss of Imp is sufficient to cause seizures in adults, thus Imp-modulated neuron development affects mature neuronal function. Since Imp is an RNA-binding protein, we sought to identify the mRNA target that Imp regulates in neurons to ensure proper neuronal activity after mechanical stress. We find that Imp protein binds Syndecan ( Sdc ) mRNA, and reduction of Sdc also causes mechanically-induced seizures. Expression of Sdc in Imp deficient neurons rescues seizure defects, showing that Sdc is sufficient to restore normal behavior after mechanical stress. We suggest that Imp protein binds Sdc mRNA in neurons, and this functional interaction is important for normal neuronal biology and animal behavior in a mechanically-induced seizure model. Since Imp and Sdc are conserved, our work highlights a neuronal specific pathway that might contribute to seizure disorder when mutated in humans.

DOI: https://doi.org/10.1101/2024.11.21.624719
Structure. 2024 Nov 19. pii: S0969-2126(24)00487-8. [Epub ahead of print]

Protein translocation through α-helical channels and insertases.

Jingxia Chen, Xueyin Zhou, Yuqi Yang, Long Li.

Protein translocation systems are essential for distributing proteins across various lipid membranes in cells. Cellular membranes, such as the endoplasmic reticulum (ER) membrane and mitochondrial inner membrane, require highly regulated protein translocation machineries that specifically allow the passage of protein polypeptides while blocking smaller molecules like ions and water. Key translocation systems include the Sec translocation channel, the protein insertases of the Oxa1 superfamily, and the translocases of the mitochondrial inner membrane (TIM). These machineries utilize different mechanisms to create pathways for proteins to move across membranes while preventing ion leakage during the dynamic translocation processes. In this review, we highlight recent advances in our understanding of these α-helical translocation machineries and examine their structures, mechanisms, and regulation. We also discuss the therapeutic potential of these translocation pathways and summarize the progress in drug development targeting these systems for treating diseases.

DOI: https://doi.org/10.1016/j.str.2024.10.032
Cell Rep. 2024 Nov 26. pii: S2211-1247(24)01364-0. [Epub ahead of print]43(12): 115013

Development of MAPT S305 mutation human iPSC lines exhibiting elevated 4R tau expression and functional alterations in neurons and astrocytes.

Kathryn R Bowles, Chiara Pedicone, Derian A Pugh, Laura-Maria Oja, Filipa H Sousa, Lois K Keavey, Brian Fulton-Howard, Sarah A Weitzman, Yiyuan Liu, Jonathan L Chen, Matthew D Disney, Alison M Goate.

  Due to the importance of 4R tau (with four microtubule-binding-repeat domains) in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in induced pluripotent stem cell (iPSC)-derived neurons, which express very low levels of 4R tau. To address this, we have developed a panel of isogenic iPSC lines carrying MAPT splice-site mutations, S305S, S305I, or S305N, derived from four different donors. All mutations significantly increase 4R tau expression in iPSC neurons and astrocytes. Functional analyses of S305 mutant neurons reveal shared disruption in synaptic signaling and maturity but divergent effects on mitochondrial bioenergetics. In iPSC astrocytes, S305 mutations promote internalization of exogenous tau that may be a precursor to glial pathology. These lines recapitulate previously characterized tauopathy-relevant phenotypes and highlight functional differences between the wild-type 4R and the mutant 4R proteins in both neurons and astrocytes. As such, these lines enable a more complete understanding of pathogenic mechanisms underlying 4R tauopathies across different cell types.

Keywords:  4R tau; CP: Neuroscience; CP: Stem cell research; MAPT; astrocytes; iPSC models; inflammation; mitochondria; splicing; synapses; tauopathy

DOI:  https://doi.org/10.1016/j.celrep.2024.115013
Int J Mol Sci. 2024 Nov 12. pii: 12114. [Epub ahead of print]25(22):

Protective Effects of Ambroxol on Aβ and α-Synuclein-Induced Neurotoxicity Through Glucocerebrosidase Activation in HT-22 Hippocampal Neuronal Cells.

Sheng-Chieh Lin, Ching-Chi Chang, Sing-Hua Tsou, Pai-Yi Chiu, Ju-Fang Cheng, Hui-Chih Hung, Wei-Jen Chen, Ying-Jui Ho, Chih-Li Lin.

  Dementia with Lewy bodies (DLB) is a progressive neurodegenerative disorder marked by the accumulation of α-synuclein (αSyn), often co-existing with amyloid β (Aβ) pathology. Current treatments are largely symptomatic, highlighting a critical need for disease-modifying therapies. Evidence suggests that αSyn aggregates contribute to neuronal death in DLB, particularly when exacerbated by Aβ. Given the role of autophagy in clearing misfolded proteins, exploring agents that promote this pathway is essential for developing effective treatments. Ambroxol (AMBX), a mucolytic drug, has demonstrated potential in activating glucocerebrosidase (GCase), an enzyme that enhances lysosomal function and facilitates the autophagic clearance of toxic protein aggregates, including αSyn. This study aims to evaluate AMBX's neuroprotective effects in a cellular model of DLB, with the goal of identifying new therapeutic agents that target the underlying pathology of DLB. In this study, HT-22 hippocampal neuronal cells were exposed to αSyn and Aβ, followed by AMBX treatment. Our results showed that AMBX significantly improved cell viability and reduced apoptosis in cells co-treated with αSyn and Aβ. Additionally, AMBX restored GCase activity, promoted autophagy, and reduced oxidative stress, which in turn mitigated αSyn aggregation and phosphorylation. These findings suggest that by activating GCase and enhancing autophagy, AMBX may help alleviate DLB-associated neurodegeneration. This study underscores the potential of AMBX as a therapeutic agent for DLB and supports further investigation in animal models and clinical trials to validate its efficacy in neurodegenerative disease contexts.

Keywords:  Ambroxol (AMBX); amyloid β (Aβ); autophagy; glucocerebrosidase; αSyn (αSyn)

DOI:  https://doi.org/10.3390/ijms252212114
Cell Rep. 2024 Nov 28. pii: S2211-1247(24)01333-0. [Epub ahead of print]43(12): 114982

Microglial APOE3 Christchurch protects neurons from Tau pathology in a human iPSC-based model of Alzheimer's disease.

Guoqiang George Sun, Cheng Wang, Randall C Mazzarino, Paula Andrea Perez-Corredor, Hayk Davtyan, Mathew Blurton-Jones, Francisco Lopera, Joseph F Arboleda-Velasquez, Yanhong Shi.

  Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder characterized by extracellular amyloid plaques and neuronal Tau tangles. A recent study found that the APOE3 Christchurch (APOECh) variant could delay AD progression. However, the underlying mechanisms remain unclear. In this study, we established neuron-microglia co-cultures and neuroimmune organoids using isogenic APOE3 and APOECh microglia derived from human induced pluripotent stem cells (hiPSCs) with PSEN1 mutant neurons or brain organoids. We show that APOECh microglia are resistant to Aβ-induced lipid peroxidation and ferroptosis and therefore preserve the phagocytic activity and promote pTau clearance, providing mechanistic insights into the neuroprotective role of APOE3Ch microglia. Moreover, we show that an APOE mimetic peptide can mimic the protective effects of APOECh microglia. These findings demonstrate that the APOECh microglia plays a causal role in microglial neuroprotection, which can be exploited for therapeutic development for AD.

Keywords:  APOE Christchurch; CP: Neuroscience; Tau; brain organoids; ferroptosis; iPSCs; induced pluripotent stem cells; lipid droplet; lipid peroxidation; microglia; phagocytosis; presenilin; resilience

DOI:  https://doi.org/10.1016/j.celrep.2024.114982
Front Neurosci. 2024 ;18 1498801

Epileptiform activity in brain organoids derived from patient with Glucose Transporter 1 Deficiency Syndrome.

Y Müller, L Lengacher, F Friscourt, C Quairiaux, L Stoppini, P J Magistretti, S Lengacher, C Finsterwald.

   Introduction: Glucose Transporter 1-Deficiency Syndrome (GLUT1-DS) is a rare genetic disorder caused by mutations in the gene encoding for GLUT1 and characterized by impaired glucose uptake in the brain. This leads to brain hypometabolism and the development of symptoms that include epilepsy, motor dysfunctions and cognitive impairment. The development of patient-specific in vitro models is a valuable tool for understanding the pathophysiology of rare genetic disorders and testing new therapeutic interventions.
Methods: In this study, we generated brain organoids from induced pluripotent stem cells (iPSCs) derived either from a GLUT1-DS patient or a healthy individual. The functional organoids were analyzed for cellular composition, maturity, and electrophysiological activity using a custom-made microelectrode array (MEA) platform, which allowed for the detection of spikes, burst patterns, and epileptiform discharges.
Results: Immunostaining revealed a similar distribution of neurons and astrocytes in both healthy and GLUT1-DS brain organoids, though GLUT1-DS brain organoids exhibited reduced cellular density and smaller overall size. Electrophysiological recordings demonstrated functional spike profiles in both organoid types. Notably, our study demonstrates that brain organoids derived from a GLUT1-DS patient exhibit distinct epileptiform activity and heightened sensitivity to glucose deprivation, reflecting key features of the disorder.
Discussion: These findings validate the use of brain organoids as a model for studying GLUT1-DS and highlight their potential for testing novel therapeutic strategies aimed at improving glucose metabolism and managing epilepsy in patients.

Keywords:  GLUT1-DS; astrocyte; brain energy metabolism; brain organoid; drug development; epilepsy; glucose; multielectrode array

DOI:  https://doi.org/10.3389/fnins.2024.1498801
Exp Dermatol. 2024 Nov;33(11): e70017

Generating Skin-Derived Precursor-Like Cells From Human-Induced Pluripotent Stem Cell-Derived Skin Organoids.

Imaan A Ahmed, Jane Sun, Min Jie Kong, Kiarash Khosrotehrani, Abbas Shafiee.

  Skin-derived precursor (SKPs) cells are multipotent stem cells found in the dermis that contribute to wound healing and induce hair follicle neogenesis when transplanted. The clinical application of adult human SKPs, however, is hindered by their loss of potency after in vitro expansion. To overcome this challenge, we aimed to isolate SKPs from human-induced pluripotent stem cell-derived skin organoids (SKOs), to enable mass production of these cells for therapeutics. We developed a protocol to isolate skin-derived precursor-like cells (SKP-like cells) from human SKOs. SKP-like cells derived from SKOs exhibited characteristic spheroid morphology and were capable of self-renewal in defined SKP growth medium. Immunofluorescence analysis confirmed the expression of key markers, including SOX2, fibronectin and S100β, within the SKP-like cells. The findings of this pilot study shed light on the potential of SKO-derived SKP-like cells for future hair regenerative applications. Furthermore, this research highlights the application of human SKOs as a valuable source for isolating progenitor cells, aiming to advance hair regeneration and restore skin function.

Keywords:  SOX2; hair follicle; regeneration; skin‐derived precursor; wound healing

DOI:  https://doi.org/10.1111/exd.70017
Commun Biol. 2024 Nov 23. 7(1): 1561

Modeling early phenotypes of Parkinson's disease by age-induced midbrain-striatum assembloids.

Kyriaki Barmpa, Claudia Saraiva, Diego Lopez-Pigozzi, Gemma Gomez-Giro, Elisa Gabassi, Sarah Spitz, Konstanze Brandauer, Juan E Rodriguez Gatica, Paul Antony, Graham Robertson, Rahman Sabahi-Kaviani, Alessandro Bellapianta, Florentia Papastefanaki, Regina Luttge, Ulrich Kubitscheck, Ahmad Salti, Peter Ertl, Mario Bortolozzi, Rebecca Matsas, Frank Edenhofer, Jens C Schwamborn.

Parkinson's disease, an aging-associated neurodegenerative disorder, is characterised by nigrostriatal pathway dysfunction caused by the gradual loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain. Human in vitro models are enabling the study of the dopaminergic neurons' loss, but not the dysregulation within the dopaminergic network in the nigrostriatal pathway. Additionally, these models do not incorporate aging characteristics which potentially contribute to the development of Parkinson's disease. Here we present a nigrostriatal pathway model based on midbrain-striatum assembloids with inducible aging. We show that these assembloids can develop characteristics of the nigrostriatal connectivity, with catecholamine release from the midbrain to the striatum and synapse formation between midbrain and striatal neurons. Moreover, Progerin-overexpressing assembloids acquire aging traits that lead to early neurodegenerative phenotypes. This model shall help to reveal the contribution of aging as well as nigrostriatal connectivity to the onset and progression of Parkinson's disease.

DOI: https://doi.org/10.1038/s42003-024-07273-4
bioRxiv. 2024 Nov 14. pii: 2024.11.12.623183. [Epub ahead of print]

Single-cell transcriptomic landscape of the neuroimmune compartment in amyotrophic lateral sclerosis brain and spinal cord.

John F Tuddenham, Masashi Fujita, Anthony Khairallah, Claire Harbison, Xena E Flowers, Guillermo Coronas-Samano, Silas Maniatis, Aidan Daly, Julie A Schneider, Andrew F Teich, Jean Paul G Vonsattel, Peter A Sims, Wassim Elyaman, Elizabeth M Bradshaw, Hemali Phatnani, Neil Shneider, David A Bennett, Philip L De Jager, Serge Przedborski, Vilas Menon, Marta Olah.

Development of therapeutic approaches that target specific microglia responses in amyotrophic lateral sclerosis (ALS) is crucial due to the involvement of microglia in ALS progression. Our study identifies the predominant microglia subset in human ALS primary motor cortex and spinal cord as an undifferentiated phenotype with dysregulated respiratory electron transport. Moreover, we find that the interferon response microglia subset is enriched in donors with aggressive disease progression, while a previously described potentially protective microglia phenotype is depleted in ALS. Additionally, we observe an enrichment of non-microglial immune cell, mainly NK/T cells, in ALS central nervous system, primarily in the spinal cord. These findings pave the way for the development of microglia subset-specific therapeutic interventions to slow or even stop ALS progression.

DOI: https://doi.org/10.1101/2024.11.12.623183
Cells. 2024 Nov 14. pii: 1882. [Epub ahead of print]13(22):

Loss of Dnah5 Downregulates Dync1h1 Expression, Causing Cortical Development Disorders and Congenital Hydrocephalus.

Koichiro Sakamoto, Masakazu Miyajima, Madoka Nakajima, Ikuko Ogino, Kou Horikoshi, Ryo Miyahara, Kaito Kawamura, Kostadin Karagiozov, Chihiro Kamohara, Eri Nakamura, Nobuhiro Tada, Akihide Kondo.

  Dnah5 is associated with primary ciliary dyskinesia in humans. Dnah5-knockout (Dnah5-/- mice develop acute hydrocephalus shortly after birth owing to impaired ciliary motility and cerebrospinal fluid (CSF) stagnation. In contrast to chronic adult-onset hydrocephalus observed in other models, this rapid ventricular enlargement indicates additional factors beyond CSF stagnation. Herein, we investigated the contributors to rapid ventricular enlargement in congenital hydrocephalus. Dnah5-/- mice were generated using CRISPR/Cas9. The expression of dynein, N-cadherin, and nestin in the cerebral cortex was assessed using microarrays and immunostaining. Real-time PCR and Western blotting were performed for gene and protein quantification, respectively. All Dnah5-/- mice developed hydrocephalus, confirmed by electron microscopy, indicating the absence of axonemal outer dynein arms. Ventricular enlargement occurred rapidly, with a 25% reduction in the number of mature neurons in the motor cortex. Dync1h1 expression was decreased, while cytoplasmic dynein levels were 56.3% lower. Levels of nestin and N-cadherin in the lateral ventricular walls decreased by 31.7% and 33.3%, respectively. Reduced cytoplasmic dynein disrupts neurogenesis and axonal growth and reduces neuron cortical density. Hydrocephalus in Dnah5-/- mice may result from cortical maldevelopment due to cytoplasmic dynein deficiency, further exacerbating ventricular enlargement due to CSF stagnation caused by impaired motile ciliary function.

Keywords:  Dnah5; cytoplasmic dynein; motile cilia; neurogenesis; primary cilia

DOI:  https://doi.org/10.3390/cells13221882
Proc Natl Acad Sci U S A. 2024 Dec 03. 121(49): e2407375121

Molecular architecture of synaptic vesicles.

Uljana Kravčenko, Max Ruwolt, Jana Kroll, Artsemi Yushkevich, Martina Zenkner, Julia Ruta, Rowaa Lotfy, Erich E Wanker, Christian Rosenmund, Fan Liu, Mikhail Kudryashev.

  Synaptic vesicles (SVs) store and transport neurotransmitters to the presynaptic active zone for release by exocytosis. After release, SV proteins and excess membrane are recycled via endocytosis, and new SVs can be formed in a clathrin-dependent manner. This process maintains complex molecular composition of SVs through multiple recycling rounds. Previous studies explored the molecular composition of SVs through proteomic analysis and fluorescent microscopy, proposing a model for an average SV (1). However, the structural heterogeneity and molecular architecture of individual SVs are not well described. Here, we used cryoelectron tomography to visualize molecular details of SVs isolated from mouse brains and inside cultured neurons. We describe several classes of small proteins on the SV surface and long proteinaceous densities inside SVs. We identified V-ATPases, determined a structure using subtomogram averaging, and showed them forming a complex with the membrane-embedded protein synaptophysin (Syp). Our bioluminescence assay revealed pairwise interactions between vesicle-associated membrane protein 2 and Syp and V-ATPase Voe1 domains. Interestingly, V-ATPases were randomly distributed on the surface of SVs irrespective of vesicle size. A subpopulation of isolated vesicles and vesicles inside neurons contained a partially assembled clathrin coat with an icosahedral symmetry. We observed V-ATPases under clathrin cages in several isolated clathrin-coated vesicles (CCVs). Additionally, from isolated SV preparations and within hippocampal neurons we identified clathrin baskets without vesicles. We determined their and CCVs preferential location in proximity to the cell membrane. Our analysis advances the understanding of individual SVs' diversity and their molecular architecture.

Keywords:  V-ATPase; clathrin; cryo-ET; synaptic vesicles

DOI:  https://doi.org/10.1073/pnas.2407375121
RNA Biol. 2024 Jan;21(1): 37-51

RNA-containing extracellular vesicles in infection.

Kayo Schemiko Almeida, Suélen Andreia Rossi, Lysangela Ronalte Alves.

  Extracellular vesicles (EVs) are membrane-bound particles released by cells that play vital roles in intercellular communication by transporting diverse biologically active molecules, including RNA molecules, including mRNA, miRNA, lncRNA, and other regulatory RNAs. These RNA types are protected within the lipid bilayer of EVs, ensuring their stability and enabling long-distance cellular interactions. Notably, EVs play roles in infection, where pathogens and host cells use EV-mediated RNA transfer to influence immune responses and disease outcomes. For example, bacterial EVs play a crucial role in infection by modulating host immune responses and facilitating pathogen invasion. This review explores the complex interactions between EV-associated RNA and host-pathogen dynamics in bacteria, parasites, and fungi, aiming to uncover molecular mechanisms in infectious diseases and potential therapeutic targets.

Keywords:  Extracellular vesicles; RNA; bacteria; fungi; host-response; infection; parasites

DOI:  https://doi.org/10.1080/15476286.2024.2431781
bioRxiv. 2024 Nov 18. pii: 2024.11.18.623405. [Epub ahead of print]

Profiling Human iPSC-Derived Sensory Neurons for Analgesic Drug Screening Using a Multi-Electrode Array.

Christian Fofie Kuete, Rafael Granja-Vazquez, Vincent Truong, Patrick Walsh, Theodore Price, Swati Biswas, Gregory Dussor, Joseph Pancrazio, Benedict Kolber.

Chronic pain is a major global health issue, yet effective treatments are limited by poor translation from preclinical studies to humans. To address this, we developed a high-content screening (HCS) platform for analgesic discovery using hiPSC-derived nociceptors. These cells were cultured on multi-well micro-electrode arrays to monitor activity, achieving nearly 100% active electrodes by week two, maintaining stable activity for at least two weeks. After maturation (28 days), we exposed the nociceptors to various drugs, assessing their effects on neuronal activity, with excellent assay performance (Z' values >0.5). Pharmacological tests showed responses to analgesic targets, including ion channels (Nav, Cav, Kv, TRPV1), neurotransmitter receptors (AMPAR, GABA-R), and kinase inhibitors (tyrosine, JAK1/2). Transcriptomic analysis confirmed the presence of these drug targets, although expression levels varied compared to primary human dorsal root ganglion cells. This HCS platform facilitates the rapid discovery of novel analgesics, reducing the risk of preclinical-to-human translation failure.
Motivation: Chronic pain affects approximately 1.5 billion people worldwide, yet effective treatments remain elusive. A significant barrier to progress in analgesic drug discovery is the limited translation of preclinical findings to human clinical outcomes. Traditional rodent models, although widely used, often fail to accurately predict human responses, while human primary tissues are limited by scarcity, technical difficulties, and ethical concerns. Recent advancements have identified human induced pluripotent stem cell (hiPSC)-derived nociceptors as promising alternatives; however, current differentiation protocols produce cells with inconsistent and physiologically questionable phenotypes.To address these challenges, our study introduces a novel high-content screening (HCS) platform using hiPSC-derived nociceptors cultured on multi-well micro-electrode arrays (MEAs). The "Anatomic" protocol, used to generate these nociceptors, ensures cells with transcriptomic profiles closely matching human primary sensory neurons. Our platform achieves nearly 100% active electrode yield within two weeks and demonstrates sustained, stable activity over time. Additionally, robust Z' factor analysis (exceeding 0.5) confirms the platform's reliability, while pharmacological validation establishes the functional expression of critical analgesic targets. This innovative approach improves both the efficiency and clinical relevance of analgesic drug screening, potentially bridging the translational gap between preclinical studies and human clinical trials, and offering new hope for effective pain management.

DOI: https://doi.org/10.1101/2024.11.18.623405