Biology subjects

Chua, J.

Publications and source records attributed to Chua, J..

7 recordsLinked to original sources

bioRxiv2026

Defining and rescuing pathomechanisms of myotubularin and autophagy disruption in a novel human cell model of Charcot-Marie-Tooth Type 4B3

Charcot-Marie-Tooth Type 4B3 (CMT4B3) is a genetic disorder leading to peripheral axon degeneration and clinical manifestations of distal weakness and gait impairment. CMT4B3 is caused by mutations in SBF1/MTMR5, a negative regulator of phosphoinositide signaling and autophagy. Although SBF1 mutations are ubiquitously expressed, how and why loss of SBF1/MTMR5 exerts deleterious effects predominantly in neurons of the peripheral nervous system (PNS) is unknown. To investigate the effects of mutant SBF1/MTMR5 on PNS neurons compared to non-neurons, we engineered a novel and unique model system of CMT4B3 using human induced pluripotent stem cells (iPSCs) differentiated into key components of the PNS: motor neurons (iMNs), sensory neurons (iSNs), or skeletal muscle (iMuscle). To model CMT4B3, we used iPSCs derived from a CMT43B patient, or genetically knocked down SBF1 in WT cells. Strikingly, CMT4B3 iMNs showed the highest degree of cell degeneration among all cell types, concordant with the clinical phenotype of patients. We also found that CMT4B3 iMNs and iSNs showed attenuated expression of MTMR5 and related paralogs MTMR2 and MTMR13. Knockdown of SBF1 most significantly augmented autophagy in iMNs than other cell types. Finally, we tested treatment with VPS34-IN1, a pharmacologic inhibitor of the Class III PI3-Kinase functioning in opposition to MTMR5 in regulating phosphoinositides, and found that VPS34-IN1 rescued cell death in CMT4B3 iMNs. Together, our results for the first time confirm PNS cell type-specific differences in myotubularin expression, autophagy, and vulnerabilities to SBF1 mutations, and identify a novel therapeutic strategy of high disease-modifying potential for CMT4B3.

neuroscience↗

bioRxiv2025

A two-component system signaling hub controls enterococcal membrane remodeling in response to daptomycin

Daptomycin is a last resort antibiotic used to treat vancomycin-resistant enterococcal infections, but daptomycin resistance (DAPR) arises quickly during treatment. Resistance is due to sequential acquisition of point mutations in the two-component system LiaFSR and in cardiolipin synthases and is associated with alteration of phospholipid and glycolipid membrane composition. The molecular mechanisms underlying these lipid changes are currently unknown. Similarly, it is unclear why mutations in liaFSR occur prior to mutations in cls. We discovered that Enterococcus faecalis remodels membrane composition as a phenotypic response to daptomycin that parallels the membrane composition of DAPR strains. The enrichment in glycolipids that follows antibiotic exposure is due to LtaS1, the main LTA synthase of E. faecalis. Moreover, LtaS1 activity is governed by a network of two-component systems formed by LiaFSR, SapRS, and BsrRS that couple antibiotic sensing with membrane lipid remodeling. Together, our results provide a unifying mechanism that drives phenotypic membrane fortification in a Gram-positive pathogen which simultaneously predisposes the cell to acquire genetic high-level daptomycin resistance. Significance StatementDaptomycin is the preferred alternative to treat vancomycin-resistant Enterococcus infections. However, the efficacy of daptomycin is limited by the acquisition of daptomycin resistance. Alterations in lipid membrane composition represent a conserved strategy to fortify the Gram-positive membrane. We discovered that E. faecalis phenotypically remodels membrane composition in response to daptomycin and that the enrichment in glycolipids is dependent on LTA synthesis. We expand our limited knowledge of enterococcal LTA synthases, confirming LtaS1 as the main LTA synthase and placing LTA biogenesis under control of a network of antibiotic-responsive two-component systems. Our work provides a molecular explanation for the sequential evolution of daptomycin resistance and may support the use of existing LtaS inhibitors to prevent acquisition of daptomycin resistance.

microbiology↗

bioRxiv2025

Lgr5+ Stem Cells Maintain Apex Position in Cell Hierarchy of the Intestinal Epithelium During Homeostasis and Injury

The cellular origin of intestinal epithelial homeostasis and regeneration has been a subject of continued debate, with recent models challenging the primacy of WNT-dependent Lgr5 crypt base columnar (CBC) cells as the central intestinal stem cell population. Here, we revisit this question through quantitative integration of single-cell transcriptomic, chromatin accessibility, spatial, and lineage-tracing analyses across the proximal-to-distal axis of the small intestinal epithelium. Our data show that under homeostatic conditions, Lgr5 cells exclusively sustain epithelial self-renewal in nearly all crypt-villus units along the entire length of the small intestine, a process for which R-spondin is indispensable. Following irradiation or chemotoxic injury, surviving Lgr5 cells and their progeny reprogram into transient fetal-like cell states that initiate epithelial repair. Crucially, successful regeneration depends on the reactivation of canonical WNT/{beta}-catenin signaling, as evidenced by increased TCF motif accessibility and upregulation of WNT target genes in newly forming Lgr5+ stem cells. Accordingly, pharmacological inhibition of WNT signaling blocks the reconstitution of Lgr5 cells and crypt regeneration, leading to epithelial collapse. These findings reconcile prior controversies by demonstrating the central role of Lgr5 CBC cells in epithelial self-renewal and regeneration following injury.

cell biology↗

bioRxiv2025

Aberrant NOTUM+ Program Induced in LGR5+ Crypt Base Columnar Cells Maintains an Immunosuppressive Niche in Colorectal Cancer

Colorectal cancer (CRC) remains a leading cause of cancer-related mortality, with treatment failure largely driven by cancer stem-like cells that resist conventional chemoradiation and subsequently initiate tumor recurrence. While immune checkpoint blockade is effective in microsatellite instability-high (MSI-H) CRCs, the majority of CRCs are microsatellite stable (MSS) and exhibit immune exclusion, rendering them refractory to immunotherapy. Here, we identify a previously uncharacterized cancer cell subtype, which we term cancerous Crypt Base Columnar (canCBC) cells. These cells transcriptionally resemble normal LGR5+ CBC cells but activate an aberrant WNT/{beta}-catenin signalling inhibitory program, marked by NOTUM expression. We show that canCBC cells are specifically enriched in MSS tumors, where their presence correlates with reduced CD8 T cell infiltration, broader immune exclusion, and a propensity for regional lymphatic dissemination. Consistently, targeted ablation of canCBCs enhances the tumor-clearing potential of CD8 T cells. This study identifies a novel therapeutic target for overcoming immune exclusion and improving immunotherapy responses in MSS CRCs.

cancer biology↗

bioRxiv2024

The DJ-1-derived peptide, ND-13, confers cardioprotection by inhibiting mitochondrial fission and preserving mitochondrial bioenergetics via the RhoA-ROCK1-Drp1 pathway

BackgroundAcute myocardial infarction (AMI) and post-infarct heart failure (HF) are among the leading causes of death and disability worldwide. As such, new treatments are urgently needed to protect the heart against the detrimental effects of acute ischemia/reperfusion injury (IRI), in order to prevent the onset of HF and improve clinical outcomes following AMI. Given that mitochondrial dysfunction is a key determinant of IRI-induced cardiomyocyte death in AMI, we investigated ND-13, a 13-amino acid peptide derived from the pro-survival protein DJ-1, as a novel mitoprotective strategy for limiting myocardial infarct size (IS) following AMI. Methods and ResultsIn isolated adult Dendra-2 mice cardiomyocytes subjected to simulated IRI, treatment with ND-13 peptide reduced cell death by 42%, decreased phosphorylation of Drp1 at Ser616 and inhibited mitochondrial fission, improved mitochondrial respiratory function, decreased oxidative stress, and preserved ATP levels. Treatment of cardiomyocytes with ND-13 decreased levels of RhoA and reduced activity of downstream ROCK1, the latter of which is known to phosphorylate Drp1 at Ser616. In ex vivo Langendorff-perfused hearts subjected to IRI, treatment with ND-13 at reperfusion reduced IS by 43%, attenuated oxidative stress and preserved post-infarct cardiac contractile function. Finally, in vivo administration of ND-13 peptide at reperfusion in mice subjected to acute myocardial IRI reduced IS by 35% at 72-hours of reperfusion, and restored mitochondrial bioenergetics in cardiomyocytes isolated from the area-at-risk following 2-hours reperfusion as evidenced by preservation of key metabolites involved in glucose oxidation and fatty acid oxidation. The cardioprotective phenotype translated to improved cardiac function and less adverse left ventricular remodelling at 28 days post-infarction. ConclusionsWe show for the first time the mitoprotective effects of the DJ-1-derived peptide, ND-13, administered at the onset of reperfusion following AMI. We found that ND-13 protects mitochondria by inhibiting IRI-induced mitochondrial fission and preserved mitochondrial function following IRI via the RhoA-ROCK-Drp1 pathway. These findings highlight ND-13 as a novel mitoprotective agent which has the therapeutic potential for limiting IS and preventing HF in patients with AMI. Clinical PerspectiveO_ST_ABSWhat Is New?C_ST_ABSO_LIAdministration of the DJ-1-derived peptide, ND-13, at the onset of reperfusion reduced myocardial infarct size and preserved cardiac contractile function following acute myocardial ischemia/reperfusion injury (IRI). C_LIO_LIThe cardioprotective effect of ND-13 treatment was mediated by inhibition of IRI-induced mitochondrial fission and improvement in mitochondrial bioenergetics via the RhoA-ROCK-Drp1 pathway. C_LI What are the Clinical Implications?O_LITreatment with ND-13 peptide at the onset of reperfusion has the therapeutic potential to reduce myocardial infarct size and prevent the onset of heart failure in ST-segment elevation myocardial infarction patients undergoing primary percutaneous coronary intervention. C_LI

physiology↗

bioRxiv2022

Neuroimaging within the Dominantly Inherited Alzheimer's Network (DIAN): PET and MRI

The Dominantly Inherited Alzheimer Network (DIAN) Observational Study is an international collaboration studying autosomal dominant Alzheimer disease (ADAD). This rare form of Alzheimer disease (AD) is caused by mutations in the presenilin 1 (PSEN1), presenilin 2 (PSEN2), or amyloid precursor protein (APP) genes. As individuals from these families have a 50% chance of inheriting the familial mutation, this provides researchers with a well-matched cohort of carriers vs non-carriers for case-control studies. An important trait of ADAD is that the age at symptom onset is highly predictable and consistent for each specific mutation, allowing researchers to estimate an individuals point in their disease time course prior to symptom onset. Although ADAD represents only a small proportion (approximately 0.1%) of all AD cases, studying this form of AD allows researchers to investigate preclinical AD and the progression of changes that occur within the brain prior to AD symptom onset. Furthermore, the young age at symptom onset (typically 30-60 years) means age-related comorbidities are much less prevalent than in sporadic AD, thereby allowing AD pathophysiology to be studied independent of these confounds. A major goal of the DIAN Observational Study is to create a global resource for AD researchers. To that end, the current manuscript provides an overview of the DIAN magnetic resonance imaging (MRI) and positron emission tomography (PET) protocols and highlights the key imaging results of this study to date.

neuroscience↗

bioRxiv2020

Multiplex CRISPRi-Cas9 silencing of planktonic and stage-specific biofilm genes in Enterococcus faecalis

Enterococcus faecalis is an opportunistic pathogen, which can cause multidrug-resistant life-threatening infections. Gaining a complete understanding of enterococcal pathogenesis is a crucial step in identifying a strategy to effectively treat enterococcal infections. However, bacterial pathogenesis is a complex process often involving a combination of genes and multi-level regulation. Compared to established knockout methodologies, CRISPRi approaches enable rapid and efficient silencing of genes to interrogate gene products and pathways involved in pathogenesis. As opposed to traditional gene inactivation approaches, CRISPRi can also be quickly repurposed for multiplexing or used to study essential genes. Here we have developed a novel dual-vector nisin-inducible CRISPRi system in E. faecalis that can efficiently silence via both non-template and template strand targeting. Since nisin-controlled gene expression system is functional in various Gram-positive bacteria, the developed CRISPRi tool can be extended to other genera. This system can be applied to study essential genes, genes involved in antimicrobial resistance, and genes involved in biofilm formation and persistence. The system is robust, and can be scaled up for high-throughput screens or combinatorial targeting. This tool substantially enhances our ability to study enterococcal biology and pathogenesis, host-bacteria interactions, and inter-species communication. IMPORTANCEEnterococcus faecalis causes multidrug resistant life-threatening infections, and is often co-isolated with other pathogenic bacteria from polymicrobial biofilm-associated infections. Genetic tools to dissect complex interactions in mixed microbial communities are largely limited to transposon mutagenesis and traditional time- and labour-intensive allelic exchange methods. Built upon streptococcal dCas9, we developed an easily-modifiable, inducible CRISPRi system for E. faecalis that can efficiently silence single and multiple genes. This system can silence genes involved in biofilm formation, antibiotic resistance, and can be used to interrogate gene essentiality. Uniquely, this tool is optimized to study genes important for biofilm initiation, maturation, and maintenance, and can be used to perturb pre-formed biofilms. This system will be valuable to rapidly and efficiently investigate a wide range of aspects of complex enterococcal biology.

microbiology↗