技术资料

Parkinson’s disease (PD) is a debilitating neurodegenerative disease characterized by the loss of midbrain dopaminergic neurons (DaNs) and the abnormal accumulation of ?-Synuclein (?-Syn) protein. Currently,no treatment can slow nor halt the progression of PD. Multiplications and mutations of the ?-Syn gene (SNCA) cause PD-associated syndromes and animal models that overexpress ?-Syn replicate several features of PD. Decreasing total ?-Syn levels,therefore,is an attractive approach to slow down neurodegeneration in patients with synucleinopathy. We previously performed a genetic screen for modifiers of ?-Syn levels and identified CDK14,a kinase of largely unknown function as a regulator of ?-Syn. To test the potential therapeutic effects of CDK14 reduction in PD,we ablated Cdk14 in the ?-Syn preformed fibrils (PFF)-induced PD mouse model. We found that loss of Cdk14 mitigates the grip strength deficit of PFF-treated mice and ameliorates PFF-induced cortical ?-Syn pathology,indicated by reduced numbers of pS129 ?-Syn-containing cells. In primary neurons,we found that Cdk14 depletion protects against the propagation of toxic ?-Syn species. We further validated these findings on pS129 ?-Syn levels in PD patient neurons. Finally,we leveraged the recent discovery of a covalent inhibitor of CDK14 to determine whether this target is pharmacologically tractable in vitro and in vivo. We found that CDK14 inhibition decreases total and pathologically aggregated ?-Syn in human neurons,in PFF-challenged rat neurons and in the brains of ?-Syn-humanized mice. In summary,we suggest that CDK14 represents a novel therapeutic target for PD-associated synucleinopathy. View Publication

BackgroundX-linked juvenile retinoschisis (XLRS) is an inherited disease caused by RS1 gene mutation,which leads to retinal splitting and visual impairment. The mechanism of RS1-associated retinal degeneration is not fully understood. Besides,animal models of XLRS have limitations in the study of XLRS. Here,we used human induced pluripotent stem cell (hiPSC)-derived retinal organoids (ROs) to investigate the disease mechanisms and potential treatments for XLRS.MethodshiPSCs reprogrammed from peripheral blood mononuclear cells of two RS1 mutant (E72K) XLRS patients were differentiated into ROs. Subsequently,we explored whether RS1 mutation could affect RO development and explore the effectiveness of RS1 gene augmentation therapy.ResultsROs derived from RS1 (E72K) mutation hiPSCs exhibited a developmental delay in the photoreceptor,retinoschisin (RS1) deficiency,and altered spontaneous activity compared with control ROs. Furthermore,the delays in development were associated with decreased expression of rod-specific precursor markers (NRL) and photoreceptor-specific markers (RCVRN). Adeno-associated virus (AAV)-mediated gene augmentation with RS1 at the photoreceptor immature stage rescued the rod photoreceptor developmental delay in ROs with the RS1 (E72K) mutation.ConclusionsThe RS1 (E72K) mutation results in the photoreceptor development delay in ROs and can be partially rescued by the RS1 gene augmentation therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03767-4. View Publication

The stimulator of interferon genes (STING) pathway has been implicated in neurodegenerative diseases,including Parkinson’s disease and amyotrophic lateral sclerosis (ALS). While prior studies have focused on STING within immune cells,little is known about STING within neurons. Here,we document neuronal activation of the STING pathway in human postmortem cortical and spinal motor neurons from individuals affected by familial or sporadic ALS. This process takes place selectively in the most vulnerable cortical and spinal motor neurons but not in neurons that are less affected by the disease. Concordant STING activation in layer V cortical motor neurons occurs in a mouse model of C9orf72 repeat-associated ALS and frontotemporal dementia (FTD). To establish that STING activation occurs in a neuron-autonomous manner,we demonstrate the integrity of the STING signaling pathway,including both upstream activators and downstream innate immune response effectors,in dissociated mouse cortical neurons and neurons derived from control human induced pluripotent stem cells (iPSCs). Human iPSC-derived neurons harboring different familial ALS-causing mutations exhibit increased STING signaling with DNA damage as a main driver. The elevated downstream inflammatory markers present in ALS iPSC-derived neurons can be suppressed with a STING inhibitor. Our results reveal an immunophenotype that consists of innate immune signaling driven by the STING pathway and occurs specifically within vulnerable neurons in ALS/FTD.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00401-024-02688-z. View Publication

Background & aimsHumans with WNT2B deficiency have severe intestinal disease,including significant inflammatory injury,highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.MethodsWe investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon,and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue.ResultsMice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased,epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6,and biopsy tissue from humans showed increased neutrophils.ConclusionsWNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation,particularly in the colon. Graphical abstract View Publication

To comprehend the volumetric neural connectivity of a brain organoid,it is crucial to monitor the spatiotemporal electrophysiological signals within the organoid,known as intra-organoid signals. However,previous methods risked damaging the three-dimensional (3D) cytoarchitecture of organoids,either through sectioning or inserting rigid needle-like electrodes. Also,the limited numbers of electrodes in fixed positions with non-adjustable electrode shapes were insufficient for examining the complex neural activity throughout the organoid. Herein,we present a magnetically reshapable 3D multi-electrode array (MEA) using direct printing of liquid metals for electrophysiological analysis of brain organoids. The adaptable distribution and the softness of these printed electrodes facilitate the spatiotemporal recording of intra-organoid signals. Furthermore,the unique capability to reshape these soft electrodes within the organoid using magnetic fields allows a single electrode in the MEA to record from multiple points,effectively increasing the recording site density without the need for additional electrodes. Conventional platforms for electrophysiological recording of organoids have limited recording site density. Here,the authors present the magnetically reshapable 3D liquid metal-based electrode array for high-resolution analysis on neural activities of brain organoids. View Publication

SummaryEpigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing,here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay,intellectual disability,hypotonia,and motor issues such as coordination problems,feeding difficulties,and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions,including autism spectrum disorder,and to a lesser extent,seizures,connective tissue disease signs,sleep disturbance,vision problems,and other organ anomalies,were observed in affected individuals. As a molecular biomarker,a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably,while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac,global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless,MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features,a specific blood DNA episignature,and a distinct,MSL2-specific molecular etiology compared to other MSL complex-related disorders. Graphical abstract MSL2 encodes a member of the MSL complex,an epigenetic regulator acetylating histone H4. We identify MSL2 variants leading to a neurodevelopmental disorder with intellectual disability,developmental delay,motor issues,seizures,dysmorphisms,and a specific blood methylation episignature. Patient-derived reprogrammed cells reveal developmental gene dysregulation without altered global H4 acetylation. View Publication

BackgroundAtrial fibrillation has an estimated prevalence of 1.5–2%,making it the most common cardiac arrhythmia. The processes that cause and sustain the disease are still not completely understood. An association between atrial fibrillation and systemic,as well as local,inflammatory processes has been reported. However,the exact mechanisms underlying this association have not been established. While it is understood that inflammatory macrophages can influence cardiac electrophysiology,a direct,causative relationship to atrial fibrillation has not been described. This study investigated the pro-arrhythmic effects of activated M1 macrophages on human induced pluripotent stem cell (hiPSC)-derived atrial cardiomyocytes,to propose a mechanistic link between inflammation and atrial fibrillation.MethodsTwo hiPSC lines from healthy individuals were differentiated to atrial cardiomyocytes and M1 macrophages and integrated in an isogenic,pacing-free,atrial fibrillation-like coculture model. Electrophysiology characteristics of cocultures were analysed for beat rate irregularity,electrogram amplitude and conduction velocity using multi electrode arrays. Cocultures were additionally treated using glucocorticoids to suppress M1 inflammation. Bulk RNA sequencing was performed on coculture-isolated atrial cardiomyocytes and compared to meta-analyses of atrial fibrillation patient transcriptomes.ResultsMulti electrode array recordings revealed M1 to cause irregular beating and reduced electrogram amplitude. Conduction analysis further showed significantly lowered conduction homogeneity in M1 cocultures. Transcriptome sequencing revealed reduced expression of key cardiac genes such as SCN5A,KCNA5,ATP1A1,and GJA5 in the atrial cardiomyocytes. Meta-analysis of atrial fibrillation patient transcriptomes showed high correlation to the in vitro model. Treatment of the coculture with glucocorticoids showed reversal of phenotypes,including reduced beat irregularity,improved conduction,and reversed RNA expression profiles.ConclusionsThis study establishes a causal relationship between M1 activation and the development of subsequent atrial arrhythmia,documented as irregularity in spontaneous electrical activation in atrial cardiomyocytes cocultured with activated macrophages. Further,beat rate irregularity could be alleviated using glucocorticoids. Overall,these results point at macrophage-mediated inflammation as a potential AF induction mechanism and offer new targets for therapeutic development. The findings strongly support the relevance of the proposed hiPSC-derived coculture model and present it as a first of its kind disease model.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-024-03814-0. View Publication

SummaryThe cornea and sclera are distinct adjacent tissues,yet their stromal cells originate from common neural crest cells (NCCs). Sclerocornea is a disease characterized by an indistinguishable boundary between the cornea and sclera. Previously,we identified a RAD21 mutation in a sclerocornea pedigree. Here,we investigated the impacts of RAD21 on NCC activities during eye development. RAD21 deficiency caused upregulation of PCDHGC3. Both RAD21 knockdown and PCDHGC3 upregulation disrupted the migration of NCCs. Transcriptome analysis indicated that WNT9B had 190.9-fold higher expression in scleral stroma than in corneal stroma. WNT9B was also significantly upregulated by both RAD21 knockdown and PCDHGC3 overexpression,and knock down of WNT9B rescued the differentiation and migration of NCCs with RAD21 deficiency. Consistently,overexpressing wnt9b in Xenopus tropicalis led to ocular developmental abnormalities. In summary,WNT9B is a determinant factor during NCC differentiation into corneal keratocytes or scleral stromal cells and is affected by RAD21 expression. Graphical abstract Highlights•Established a stable differentiation protocol from hESCs to corneal keratocytes•RAD21 deficiency affected the proliferation and migration ability of NCCs•Increased scleral markers after RAD21 knockdown during NCC differentiation to cornea•WNT9B is a crucial mediator during ocular NCC differentiation Cell biology; Developmental biology View Publication

Cytosine base editors (CBEs) revolutionize genome editing by enabling precise C-to-T conversions without double-strand breaks. Sdd7,a recently developed cytosine deaminase,exhibits high activity across a broad protospacer range but induces unintended off-target effects,including bystander mutations within and upstream of the protospacer and both gRNA-dependent and independent deamination. Here,we report that BE4max and Sdd7 induce bystander editing upstream of the protospacer. To overcome this,we engineer two Sdd7 variants,Sdd7e1 and Sdd7e2,enhancing specificity while preserving on-target efficiency. These variants display reduced bystander editing,narrowed editing windows,and significantly lower off-target activity. Delivery as ribonucleoproteins via engineered virus-like particles (eVLPs) further improves specificity,nearly eliminating bystander edits and increasing precise single-point mutations. Our findings establish Sdd7e1 and Sdd7e2,especially when delivered via eVLP,as high-fidelity CBEs poised for safe,precise therapeutic genome editing. CRISPR base editors enable precise DNA changes but often cause off-target edits. Here,authors engineer two Sdd7 variants that minimize bystander and off-target mutations and show enhanced precision when delivered as ribonucleoproteins via engineered virus-like particles. View Publication

Single-cell technologies can measure the expression of thousands of molecular features in individual cells undergoing dynamic biological processes. While examining cells along a computationally-ordered pseudotime trajectory can reveal how changes in gene or protein expression impact cell fate,identifying such dynamic features is challenging due to the inherent noise in single-cell data. Here,we present DELVE,an unsupervised feature selection method for identifying a representative subset of molecular features which robustly recapitulate cellular trajectories. In contrast to previous work,DELVE uses a bottom-up approach to mitigate the effects of confounding sources of variation,and instead models cell states from dynamic gene or protein modules based on core regulatory complexes. Using simulations,single-cell RNA sequencing,and iterative immunofluorescence imaging data in the context of cell cycle and cellular differentiation,we demonstrate how DELVE selects features that better define cell-types and cell-type transitions. DELVE is available as an open-source python package: https://github.com/jranek/delve. Characteristic genes or proteins driving continuous biological processes are difficult to uncover from noisy single-cell data. Here,authors present DELVE,an unsupervised feature selection method to identify core molecular features driving cell fate decisions. View Publication

Current approaches to the treatment of schizophrenia have mainly focused on the protein-coding part of the genome; in this context,the roles of microRNAs have received less attention. In the present study,we analyze the microRNAome in the blood and postmortem brains of schizophrenia patients,showing that the expression of miR-99b-5p is downregulated in both the prefrontal cortex and blood of patients. Lowering the amount of miR-99b-5p in mice leads to both schizophrenia-like phenotypes and inflammatory processes that are linked to synaptic pruning in microglia. The microglial miR-99b-5p-supressed inflammatory response requires Z-DNA binding protein 1 (Zbp1),which we identify as a novel miR-99b-5p target. Antisense oligonucleotides against Zbp1 ameliorate the pathological effects of miR-99b-5p inhibition. Our findings indicate that a novel miR-99b-5p-Zbp1 pathway in microglia might contribute to the pathogenesis of schizophrenia. Synopsis The involvement of microRNAs in the pathogenesis of schizophrenia is not well-understood. This study shows that miR-99b-5p regulates Z-DNA binding protein 1 (Zbp1) to control inflammatory responses in microglia and the development of schizophrenia-like symptoms in mice. miR-99b-5p is downregulated in the blood and brains of schizophrenia patients.miR-99b-5p inhibition induces schizophrenia-like phenotypes in mice and microglial inflammation.Zbp1 is a novel miR-99b-5p target in microglia.Zbp1 antisense oligos ameliorate the pathological outcomes of decreased miR-99b-5p levels. Dysregulation of a novel miR-99b-5p-Zbp1 (Z-DNA binding protein 1) pathway in microglia induces inflammatory responses and schizophrenia-like phenotypes in mice. View Publication

Advancements in human-engineered heart tissue have enhanced the understanding of cardiac cellular alteration. Nevertheless,a human model simulating pathological remodeling following myocardial infarction for therapeutic development remains essential. Here we develop an engineered model of myocardial repair that replicates the phased remodeling process,including hypoxic stress,fibrosis,and electrophysiological dysfunction. Transcriptomic analysis identifies nine critical signaling pathways related to cellular fate transitions,leading to the evaluation of seventeen modulators for their therapeutic potential in a mini-repair model. A scoring system quantitatively evaluates the restoration of abnormal electrophysiology,demonstrating that the phased combination of TGF? inhibitor SB431542,Rho kinase inhibitor Y27632,and WNT activator CHIR99021 yields enhanced functional restoration compared to single factor treatments in both engineered and mouse myocardial infarction model. This engineered heart tissue repair model effectively captures the phased remodeling following myocardial infarction,providing a crucial platform for discovering therapeutic targets for ischemic heart disease. Engineered human models of hearts are needed to study pathology and repair. Here,the authors develop a model which replicates the phased remodelling process. The model is then used to study signalling pathway modulators for their therapeutic potential in a mini-repair model. View Publication