Biology subjects

El Khatib, M.

Publications and source records attributed to El Khatib, M..

7 recordsLinked to original sources

bioRxiv2025

Integrative analysis of pooled CRISPR screens provide functional insights into AD GWAS risk genes

Emerging research has implicated Alzheimers disease (AD) pathology with dysregulation of many key pathways in microglia, including lipid transport and metabolism, phagocytosis of plaques, and lysosomal function. However, the exact mechanisms underlying these pathways remain poorly understood. Leveraging high-throughput CRISPR screens to understand the interplay between these pathways may enable novel therapeutic strategies for AD and other neurological diseases. Here, we constructed activation and interference CRISPRa/i libraries targeting 203 genes, 71 of which were identified through neurodegenerative GWAS, and 132 additional genes linked to microglial functions. We used this library to conduct pooled CRISPRa/i screens across a range of functional assays relating to lipid metabolism and lysosomal function using a monocytic cell line, THP-1. We identified a core set of lipid and lysosome mediators and validated a subset in primary macrophages. To gain insights into transcriptional states modulated by these genes we also applied the CRISPRa/i libraries to Perturb-seq, enabling us to capture transcriptomic changes. Through non-negative matrix factorization, we identified five gene programs altered by our perturbation library. We then used an integrative analysis of functional screen data with Perturb-seq data that enabled us to uncover novel functions and genetic relationships between perturbations. This multidimensional resource links genetic perturbations to phenotypes and transcriptional programs, establishing a scalable framework for systematic gene discovery in neurodegeneration and beyond. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=162 SRC="FIGDIR/small/660041v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@5b2acborg.highwire.dtl.DTLVardef@156a0b1org.highwire.dtl.DTLVardef@4fdbb8org.highwire.dtl.DTLVardef@e2166c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG

genetics↗

bioRxiv2025

Multimodal retinal imaging by visible light optical coherence tomography and phosphorescence lifetime ophthalmoscopy in the mouse eye

SignificanceOxygen metabolism is important to retinal disease development, but current imaging methods face challenges in resolution, throughput, and depth sectioning to spatially map microvascular oxygen. AimTo develop a multimodal system capable of simultaneous phosphorescence lifetime imaging scanning laser ophthalmoscopy (PLIM-SLO) and visible light optical coherence tomography (VIS-OCT) to capture capillary-level oxygen partial pressure (pO2) and structural volumes in rodents. ApproachC57BL/6 mice were imaged by VIS-OCT with high-definition (10 kHz raster) and Doppler (100 kHz circular) protocols. Phosphorescent probe Oxyphor 2P was retro-orbitally injected to enable intravascular PLIM-SLO imaging (200 {micro}s pixel dwell time), while a tunable lens was used to adjust the focal depth. The extracted phosphorescence lifetimes were used for pO2 calculation. Simultaneous imaging utilized a shared imaging path and synchronized data collection. ResultsVIS-OCT images revealed detailed anatomy and Doppler shifts, while PLIM-SLO provided capillary pO2 at multiple depths. A hemoglobin oxygen dissociation curve related retinal arterial pO2 to systemic oxygen saturation as inhaled oxygen was varied. Registered simultaneous images were captured and pO2 was empirically adjusted for the combined excitation. ConclusionsDetailed anatomical structures and capillary pO2 levels can be simultaneously imaged, providing a useful tool to study oxygen metabolism in rodent disease models.

bioengineering↗

bioRxiv2025

Shallow-angle intracranial cannula for repeated infusion and in vivo imaging with multiphoton microscopy

Multiphoton microscopy serves as an essential tool for high-resolution imaging of the living mouse brain. To facilitate optical access to the brain during imaging, the cranial window surgery is commonly used. However, this procedure restricts physical access above the imaging area and hinders the direct delivery of imaging agents and drugs. To overcome this limitation, we have developed a cannula delivery system that enables the implantation of a low-profile cannula nearly parallel to the brain surface at angles as shallow as 8 degrees, while maintaining compatibility with multiphoton microscopy. To validate this approach, we perform direct infusion and imaging of various fluorescent cell markers in the brain. Additionally, we successfully demonstrate tracking of degenerating neurons over time in Alzheimers disease mice using Fluoro-Jade C. Furthermore, we show longitudinal imaging of brain tissue partial pressure of oxygen using a phosphorescent oxygen sensor. Our developed technique should enable a wide range of new longitudinal imaging studies in the mouse brain.

neuroscience↗

bioRxiv2024

The Alzheimers Disease Risk Genes MS4A4A And MS4A6A Cooperate to Negatively Regulate Trem2 and Microglia states

Genetic variations in MS4A4A and MS4A6A are linked to the regulation of cerebrospinal fluid soluble TREM2 (sTREM2) levels and are associated with Alzheimers disease (AD) risk and progression. Using CRISPR knockout and MS4A4A-degrading antibodies in primary human microglia, non-human primates (NHP), and a xenotransplantation model of amyloid pathology, we provide evidence that MS4A4A and MS4A6A are negative regulators of both the transmembrane and soluble TREM2 proteins. They also negatively regulate microglia proliferation, survival, metabolism, lysosomal function, energetics, phagocytosis, and disease-fighting states. Mechanistically, we find that MS4A4A exerts negative regulation by interacting with MS4A6A and protecting it from degradation. MS4A6A in turn forms a complex with and blocks the co-receptor DAP12, which is required for the stability, cell surface localization, and signaling of TREM2 and other receptors. Taken together, the data indicate that MS4A4A and MS4A6A are cooperating, post-transcriptional negative regulators of TREM2 and microglial function, and potential drug targets for AD.

neuroscience↗

bioRxiv2024

Carbonic anhydrase inhibitors prevent presymptomatic capillary flow disturbances in a model of cerebral amyloidosis

Structured abstractO_ST_ABSINTRODUCTIONC_ST_ABSDisturbances in microvascular flow dynamics are hypothesized to precede the symptomatic phase of Alzheimers disease (AD). However, evidence in presymptomatic AD remains elusive, underscoring the need for therapies targeting these early vascular changes. METHODSWe employed a multimodal approach, combining in vivo optical imaging, molecular techniques, and ex vivo MRI, to investigate early capillary dysfunction in Tg-SwDI mice without memory impairment. We also assessed the efficacy of carbonic anhydrase inhibitors (CAIs) in preventing capillary flow disturbances. RESULTSOur study revealed capillary flow disturbances associated with alterations in capillary morphology, adhesion molecule expression, and Amyloid-{beta} (A{beta}) load in 9-10-month-old Tg-SwDI mice without memory impairment. CAI treatment ameliorated these capillary flow disturbances, enhanced oxygen availability, and reduced A{beta} load. DISCUSSIONThese findings underscore the importance of capillary flow disturbances as early biomarkers in presymptomatic AD and highlight the potential of CAIs for preserving vascular integrity in the early stages of AD.

neuroscience↗

bioRxiv2021

Socialization of Providencia stuartii enables resistance toenvironmental insults.

Providencia stuartii is a highly-social pathogen responsible for nosocomial chronic urinary tract infections. The bacterium indeed forms floating communities of cells (FCC) besides and prior-to canonical surface-attached biofilms (SAB). Within P. stuartii FCC, cells are riveted one to another owing to by self-interactions between its porins, viz. Omp-Pst1 and Omp-Pst2. In pathophysiological conditions, P. stuartii is principally exposed to high concentrations of urea, ammonia, bicarbonate, creatinine and to large variations of pH, questioning how these environmental cues affect socialization, and whether formation of SAB and FCC protects cells against those. Results from our investigations indicate that FCC and SAB can both form in the urinary tract, endowing cells with increased resistance and fitness. They additionally show that while Omp-Pst1 is the main gateway allowing penetration of urea, bicarbonate and ammonia into the periplasm, expression of Omp-Pst2 enables resistance to them.

microbiology↗

bioRxiv2021

Rational control of structural off-state heterogeneity in a photoswitchable fluorescent protein provides switching contrast enhancement

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, a key parameter that largely dictates the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

biophysics↗