Biology subjects

Mahjoub, M. R.

Publications and source records attributed to Mahjoub, M. R..

13 recordsLinked to original sources

bioRxiv2026

Progressive Actomyosin Remodeling Defining a Structured Podocyte Injury Response in Nephrotic Syndrome

Nephrotic syndrome, the leading cause of chronic kidney disease worldwide, is associated with injury and eventual loss of podocytes, the specialized epithelial cells that form the final layer of the kidney's filtration apparatus. The progression from injury to loss is largely irreversible. However, some forms of nephrotic syndrome undergo remission, suggesting that injured podocytes may retain a capacity for repair, the structural basis of which remains unknown. Podocytes respond to injury by the stereotypical morphological change known as foot process effacement (FPE), which can be visualized using transmission electron microscopy. Using modified ultrastructure expansion microscopy (U-ExM) protocol combined with super-resolution microscopy, we discovered that injured podocytes exhibit a three-stage progression of FPE maturation, with kinetics determined by the degree of actomyosin contractility. Through longitudinal study of adriamycin-induced nephropathy in mice, we identified three sequential stages of actomyosin reorganization within the sarcomere-like structures (SLSs) that define the injury response: focal infiltration of myosin IIA into synaptopodin-rich foot processes (Stage 1), disordered intermingling of synaptopodin/myosin IIA (Stage 2), and the formation of highly organized periodic contractile arrays oriented perpendicular to the direction of capillary blood flow (Stage 3). Longitudinal quantification revealed predictable temporal dynamics, with mature Stage 3 predominating by day 7-9 post-injury. To determine whether this sequence is conserved in human disease, we analyzed kidney biopsies from patients with minimal change disease, focal segmental glomerulosclerosis, membranous nephropathy, and diabetic nephropathy. The same three stages were present across all four diseases, and their distribution tracked disease course: acute podocytopathies exhibited heterogeneous early-stage patterns, whereas chronic injuries converged on uniform, mature SLS organization. These findings demonstrate that podocyte injury is a graded, potentially reversible process rather than a binary switch. They identify structured actomyosin remodeling as a conserved wound response across mechanistically distinct kidney diseases and suggest that therapeutic strategies targeting specific stages of this progression could alter the trajectory of diverse glomerular diseases.

cell biology↗

bioRxiv2026

Automated Ventricle Assessment via Three-dimensional Anatomical Reconstruction (AVA-TAR): a computational toolkit for autonomous lateral ventricle assessment in preclinical hydrocephalus models

IntroductionCurrent workflows for studying hydrocephalus in rodent models rely on manual segmentation or qualitative assessment of ventricular size on small animal magnetic resonance imaging, which are both inefficient and prone to variability. Atlas-based methods enable more streamlined segmentation, but their analysis is limited to morphologically normal samples. ObjectiveThis study aimed to develop and internally validate a deep learning model that performs automated segmentation of lateral ventricles in rodent brain MRIs, allowing for 3D ventricle reconstruction, morphological analysis, and ventriculomegaly detection. MethodsFour U-Net++ neural networks, each with different encoder backbones, were trained using 307 rodent brain MRIs (262 rats, 45 mice), each with manually segmented lateral ventricles serving as the ground truth. Model performance was evaluated using the Dice coefficient, intersection over union (IoU), and Hausdorff index. The most optimal model was evaluated further for its ability to quantify ventricle volume, convexity, surface area, and symmetry. ResultsThe U-Net++ model with an EfficientNet-B1 encoder achieved high accuracy (Dice: 0.823 {+/-} 0.136; IoU: 0.721 {+/-} 0.85). Further assessment of its morphological predictions found strong correlations with manual measurements of ventricular morphology, with Pearson and interclass correlation coefficients exceeding 0.96 across all metrics. The full validated pipeline was packaged into a publicly available application, hosted at https://ava-tar.org. ConclusionThis study introduces a deep learning tool for automated segmentation and morphological analysis of lateral ventricles in rodent MRIs. The tools efficiency and accuracy in quantifying ventricle morphology offers significant utility in preclinical hydrocephalus research with potential future application in the clinical setting.

neuroscience↗

bioRxiv2024

Cilia dysfunction in the lateral ventricles after neonatal intraventricular hemorrhage does not lead to functional changes in cilia-based CSF flow networks

Intraventricular hemorrhage (IVH) has long been thought to lead to motile cilia dysfunction whereby intraventricular blood breakdown products damage and slough cilia from the ependymal wall. However, specifically how IVH may affect cilia development, structure, and transcriptional activation is not well-understood. Moreover, the impact of blood breakdown product-mediated cilia damage on the functional organization of cilia-based CSF flow networks is unknown. Here, we show hemoglobin exposure affects the number of ciliated ependymal cells in the lateral ventricle (LV) but does not impact in vitro beat frequency of the remaining cilia. Ultrastructurally, IVH decreases the total number of ciliary tufts without impacting axoneme structure. IVH does not result in changes in the expression of cilia-related genes and instead leads to downregulation of neurogenesis markers in parallel with innate immune upregulation. Functionally, we identify three previously uncharacterized cilia-mediated CSF flow domains in the LV lateral wall and show that IVH does not result in widespread disruption of their functional organization. These data de-emphasize cilia as a major contributor to global CSF dysfunction after IVH, and instead call attention to preserving the neurodevelopmental environment and preventing runaway innate immune system activation, as considerations to developing treatment strategies to prevent posthemorrhagic hydrocephalus and other neurodevelopmental sequelae.

neuroscience↗

bioRxiv2024

Multi-omics profiling of mouse polycystic kidney disease progression at a single cell resolution

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and causes significant morbidity, ultimately leading to end-stage kidney disease. PKD pathogenesis is characterized by complex and dynamic alterations in multiple cell types during disease progression, hampering a deeper understanding of disease mechanism and the development of therapeutic approaches. Here, we generate a single nucleus multimodal atlas of an orthologous mouse PKD model at early, mid and late timepoints, consisting of 125,434 single-nucleus transcriptomic and epigenetic multiomes. We catalogue differentially expressed genes and activated epigenetic regions in each cell type during PKD progression, characterizing cell-type-specific responses to Pkd1 deletion. We describe heterogeneous, atypical collecting duct cells as well as proximal tubular cells that constitute cyst epithelia in PKD. The transcriptional regulation of the cyst lining cell marker GPRC5A is conserved between mouse and human PKD cystic epithelia, suggesting shared gene regulatory pathways. Our single nucleus multiomic analysis of mouse PKD provides a foundation to understand the earliest changes molecular deregulation in a mouse model of PKD at a single-cell resolution.

genomics↗

bioRxiv2024

Loss of an extensive ciliary connectome induces proteostasis and cell fate switching in a severe motile ciliopathy

Motile cilia have essential cellular functions in development, reproduction, and homeostasis. Genetic causes for motile ciliopathies have been identified, but the consequences on cellular functions beyond impaired motility remain unknown. Variants in CCDC39 and CCDC40 cause severe disease not explained by loss of motility. Using human cells with pathological variants in these genes, Chlamydomonas genetics, cryo-electron microscopy, single cell RNA transcriptomics, and proteomics, we identified perturbations in multiple cilia-independent pathways. Absence of the axonemal CCDC39/CCDC40 heterodimer results in loss of a connectome of over 90 proteins. The undocked connectome activates cell quality control pathways, switches multiciliated cell fate, impairs microtubule architecture, and creates a defective periciliary barrier. Both cilia-dependent and independent defects are likely responsible for the disease severity. Our findings provide a foundation for reconsidering the broad cellular impact of pathologic variants in ciliopathies and suggest new directions for therapies.

cell biology↗

bioRxiv2024

Ultrastructure expansion microscopy (U-ExM) of mouse and human kidneys for analysis of subcellular structures

Ultrastructure expansion microscopy (U-ExM) involves the physical magnification of specimens embedded in hydrogels, which allows for super-resolution imaging of subcellular structures using a conventional diffraction-limited microscope. Methods for expansion microscopy exist for several organisms, organs, and cell types, and used to analyze cellular organelles and substructures in nanoscale resolution. Here, we describe a simple step-by-step U-ExM protocol for the expansion, immunostaining, imaging, and analysis of cytoskeletal and organellar structures in kidney tissue. We detail the critical modified steps to optimize isotropic kidney tissue expansion, and preservation of the renal cell structures of interest. We demonstrate the utility of the approach using several markers of renal cell types, centrioles, cilia, the extracellular matrix, and other cytoskeletal elements. Finally, we show that the approach works well on mouse and human kidney samples that were preserved using different fixation and storage conditions. Overall, this protocol provides a simple and cost-effective approach to analyze both pre-clinical and clinical renal samples in high detail, using conventional lab supplies and standard widefield or confocal microscopy.

cell biology↗

bioRxiv2023

DLG1 functions upstream of SDCCAG3 and IFT20 to control targeting of polycystin-2 to the primary cilium

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

cell biology↗

bioRxiv2023

Impaired centrosome biogenesis in kidney stromal progenitors reduces abundance of interstitial lineages and accelerates injury-induced fibrosis

Defective centrosome function can disrupt embryonic kidney development, by causing changes to the renal interstitium that leads to fibrocystic disease pathologies. Yet, it remains unknown how mutations in centrosome genes impact kidney interstitial cells. Here, we examined the consequences of defective centrosome biogenesis on stromal progenitor cell growth, differentiation and fate. Conditional deletion of Cep120, a ciliopathy gene essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of pericytes, interstitial fibroblasts and mesangial cells. This was due to delayed mitosis, increased apoptosis, and changes in Wnt and Hedgehog signaling essential for differentiation of stromal lineages. Cep120 ablation resulted in hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis via enhanced TGF-{beta}/Smad3-Gli2 signaling after renal injury. Our study defines the cellular and developmental defects caused by centrosome dysfunction in embryonic kidney stroma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/535583v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@a3b059org.highwire.dtl.DTLVardef@8ed18corg.highwire.dtl.DTLVardef@5f573borg.highwire.dtl.DTLVardef@1581c20_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDefective centrosome biogenesis in kidney stroma causes: C_LIO_LIReduced abundance of stromal progenitors, interstitial and mesangial cell populations C_LIO_LIDefects in cell-autonomous and paracrine signaling C_LIO_LIAbnormal/delayed nephrogenesis and tubular dilations C_LIO_LIAccelerates injury-induced fibrosis via defective TGF-{beta}/Smad3-Gli2 signaling axis C_LI

developmental biology↗

bioRxiv2023

Aberrant centrosome biogenesis disrupts nephron progenitor cell renewal and fate resulting in fibrocystic kidney disease

Mutations that disrupt centrosome structure or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet, it remains unclear how mutations in proteins essential for centrosome biogenesis impact embryonic kidney development. Here, we examined the consequences of conditional deletion of a ciliopathy gene, Cep120, in the two nephron progenitor niches of the embryonic kidney. Cep120 loss led to reduced abundance of both metanephric mesenchyme and ureteric bud progenitor populations. This was due to a combination of delayed mitosis, increased apoptosis, and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis, and decline in filtration function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in pathways essential for branching morphogenesis, cystogenesis and fibrosis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney development, and identifies new therapeutic targets for renal centrosomopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/535568v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@198c93forg.highwire.dtl.DTLVardef@1c4750dorg.highwire.dtl.DTLVardef@e0c829org.highwire.dtl.DTLVardef@17512c3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsDefective centrosome biogenesis in nephron progenitors causes: O_LIReduced abundance of metanephric mesenchyme and premature differentiation into tubular structures C_LIO_LIAbnormal branching morphogenesis leading to reduced nephron endowment and smaller kidneys C_LIO_LIChanges in cell-autonomous and paracrine signaling that drive cystogenesis and fibrosis C_LIO_LIUnique cellular and developmental defects when compared to Pkd1 knockout models C_LI

developmental biology↗

bioRxiv2023

The effect of Dnaaf5 gene dosage on primary ciliary dyskinesia phenotypes

DNAAF5 is a dynein motor assembly factor associated with the autosomal heterogenic recessive condition of motile cilia, primary ciliary dyskinesia (PCD). The effects of allele heterozygosity on motile cilia function are unknown. We used CRISPR-Cas9 genome editing in mice to recreate a human missense variant identified in patients with mild PCD and a second, frameshift null deletion in Dnaaf5. Litters with Dnaaf5 heteroallelic variants showed distinct missense and null gene dosage effects. Homozygosity for the null Dnaaf5 alleles was embryonic lethal. Compound heterozygous animals with the missense and null alleles showed severe disease manifesting as hydrocephalus and early lethality. However, animals homozygous for the missense mutation had improved survival, with partial preserved cilia function and motor assembly observed by ultrastructure analysis. Notably, the same variant alleles exhibited divergent cilia function across different multiciliated tissues. Proteomic analysis of isolated airway cilia from mutant mice revealed reduction in some axonemal regulatory and structural proteins not previously reported in DNAAF5 variants. While transcriptional analysis of mouse and human mutant cells showed increased expression of genes coding for axonemal proteins. Together, these findings suggest allele-specific and tissue-specific molecular requirements for cilia motor assembly that may affect disease phenotypes and clinical trajectory in motile ciliopathies. Brief SummaryA mouse model of human DNAAF5 primary ciliary dyskinesia variants reveals gene dosage effects of mutant alleles and tissue-specific molecular requirements for cilia motor assembly.

cell biology↗

bioRxiv2022

Inhibition of Centrosome Clustering Reduces Cystogenesis and Improves Kidney Function in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited monogenic disorder accounting for [~]5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display defects in the biogenesis of the centrosome which causes genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors that drive cyst growth and fibrosis. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which often causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering", a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that blocking centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibition of centrosome clustering activates a p53-mediated mitotic surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results provide the first evidence that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

developmental biology↗

bioRxiv2018

Super-resolution Molecular Map of Basal Foot Reveals Novel Cilium in Airway Multiciliated Cells

Motile cilia are beating machines that play a critical role in airway defense. During airway cell differentiation, hundreds of motile cilia are templated from basal bodies that extend a basal foot, an appendage that links motile cilia together to ensure beating coordination. This assembly has thus far escaped structural analysis because its size is below the resolution limit. Here, we determine the molecular architecture and identify basal foot proteins using a super-resolution-driven approach. Quantitative super-resolution image analysis shows that the basal foot is organized in three main regions linked by elongated coiled-coil proteins. FIB-SEM tomography and comparative super-resolution mapping of basal feet reveal that, among hundreds of motile cilia of an airway cell, a hybrid cilium with features of primary and motile cilia is harbored. The hybrid cilium is conserved in mammalian multiciliated cells and originates from parental centrioles. We further demonstrate that this novel cilium is a signalling centre whose cellular position is dependent on flow.

cell biology↗

bioRxiv2018

The Heterotaxy Gene, CCDC11, is Essential for Cytokinesis and Cell-Cell Adhesion Via RhoA Regulation

Mutations in CCDC11 have been identified in multiple patients with heterotaxy (Htx), a disorder of left-right (LR) patterning of the internal organs. In Xenopus, depletion of Ccdc11 causes defects in LR patterning, recapitulating the patient phenotype. Upon Ccdc11 depletion, normally monociliated cells of the Left-Right Organizer (LRO) exhibit multiple cilia per cell. Unexpectedly, we found that Ccdc11 is necessary for successful cytokinesis, and the multiciliation observed in Ccdc11-depleted cells was due to failed cytokinesis. Furthermore, CCDC11 depletion alters cell-cell adhesion with reduction in junctional localization of adhesion molecules. The small GTPase RhoA is critical for cytokinesis and cell-cell adhesion. Because the CCDC11 depletion phenotypes are reminiscent of RhoA loss of function, we investigated a possible connection to regulation of RhoA signaling. We demonstrate that CCDC11 is localized to the cytokinetic contractile ring overlapping with RhoA during cytokinesis and regulates total RhoA protein levels. Our results suggest that CCDC11 connects cytokinesis and LR patterning via RhoA regulation, providing a potential mechanism for heterotaxy disease pathogenesis.

cell biology↗