Biology subjects

Sarrafha, L.

Publications and source records attributed to Sarrafha, L..

8 recordsLinked to original sources

bioRxiv2026

Chronic NLRP3 inflammasome activation drives neutrophil brain entry and interactions with microglia

NOD-like receptor family pyrin domain-containing 3 (NLRP3) is a cytosolic regulator of an inflammasome-mediated innate immune response. In the central nervous system (CNS), NLRP3 inflammasome activation has been implicated in multiple neurodegenerative diseases, yet the mechanisms by which it contributes to disease remain unclear. Here, we investigated the CNS effects of chronic NLRP3 activation using a humanized NLRP3 gain-of-function mouse model (hNLRP3D305N). Bulk brain analyses confirmed constitutive inflammasome activation, widespread cytokine induction, and the increased presence of blood-associated proteins suggestive of dysfunction at CNS border sites and the blood-brain barrier (BBB). Furthermore, cerebrospinal fluid (CSF) neurofilament light chain levels were elevated, indicating neuronal damage. Single-cell RNA-sequencing of CD45+ immune cells in the brain demonstrated that microglia adopt distinct reactive states and that peripheral immune cells infiltrate the CNS, with neutrophils emerging as the predominant infiltrating immune cell type. This finding was confirmed by untargeted bulk brain and CSF proteomics that also suggest neutrophil reactivity. Immunohistochemistry further revealed regional neutrophil entry into the brain parenchyma, concurrent with reactive microglia and engulfment of neutrophils, suggesting functional microglia-neutrophil interactions. Collectively, these findings establish a direct pathogenic role for the NLRP3 inflammasome in the CNS independent of other neurodegeneration-related disease pathologies.

neuroscience↗

bioRxiv2024

Transferrin receptor-mediated transport at the blood-brain barrier is elevated in early development but maintained across adult aging

Transferrin receptor (TfR)-mediated transcytosis across the blood-brain barrier (BBB) is a promising strategy to improve delivery of biologics to the central nervous system (CNS). However, it remains unclear whether age and aging-related diseases impact TfR expression and/or BBB transport capacity. Here, we used the TfR-targeted antibody transport vehicle (ATVTfR) to enhance CNS delivery in healthy mice and in the 5xFAD mouse model of Alzheimers disease (AD). Healthy neonates exhibited the highest vascular TfR expression and ATVTfR brain exposure, whereas BBB transport capacity remained stable across adulthood. Additionally, neither TfR expression nor ATVTfR brain uptake changed significantly in 5xFAD mice. Further, vascular TfR expression in AD patient brains was similar to age-matched controls, suggesting that TfR transport may be conserved for AD in humans. The elevated TfR-mediated brain delivery observed in early mouse development suggests the potential of added efficacy in utilizing TfR platforms for the treatment of early childhood diseases. Preservation of ATVTfR transport in adult mice across healthy aging and in an AD model supports continued application of TfR platforms in age-related diseases.

neuroscience↗

bioRxiv2024

Lysosomes cell autonomously regulate myeloid cell states and immune responses

Myeloid cells maintain tissue homeostasis via the recognition, engulfment, and lysosomal clearance of dying cells and cellular debris, which is often accompanied by changes from homeostatic to reactive states. While a role for phagocytic receptors in gating these transitions has been described1,2, less is known about if and how lysosomes can contribute to transcriptional and functional plasticity. To determine how lysosomal health impacts myeloid cell states, we evaluated microglia and macrophages deficient for progranulin (encoded by Grn), a lysosomal protein with pleiotropic functions whose loss is associated with several neurodegenerative diseases3-8. Single-cell RNA-sequencing of the aged mouse brain identified a Grn knockout (KO)-specific microglial subpopulation marked by high GPNMB expression that displays hallmarks of lysosomal dysfunction, including lipofuscin accumulation. Epigenetic analysis of aged microglia revealed MITF/TFE transcription factors as key mediators of the transcriptional states associated with Grn deficiency. In addition to identifying a core myeloid cell transcriptional response to diverse lysosomal stressors, targeted perturbations of various lysosomal properties in vitro uncovered a cell autonomous, TREM2- independent, response to lysosomal deacidification (via v-ATPase or VPS34 loss of function) that overlaps with Grn KO microglia phenotypes, including the induction of a lysosomal gene program, increased proliferation, and secretion of pro-inflammatory cytokines. Compound loss-of-function approaches established GPNMB upregulation upon lysosomal stress is required for the compensatory response to enhance lysosomal function via promoting acidification. Finally, pharmacological endolysosomal reacidification through sodium/proton exchanger inhibition partially rescued Grn KO microglia phenotypes. Overall, these data establish a fundamental link between lysosomal health and myeloid cell epigenetic, transcriptional, and functional states observed in neurodegeneration models.

neuroscience↗

bioRxiv2024

Fc-engineered large molecules targeting blood-brain barrier transferrin receptor and CD98hc have distinct central nervous system and peripheral biodistribution compared to standard antibodies

The blood-brain barrier (BBB) poses a significant challenge drug delivery to the brain. BBB-crossing molecules are emerging as a new class of therapeutics with significant potential for central nervous system (CNS) indications. In particular, transferrin receptor (TfR)- and CD98 heavy chain (CD98hc)-targeting molecules have been demonstrated to cross the BBB for enhanced brain delivery. Previously, we reported TfR and CD98hc antibody transport vehicles (ATVTfR and ATVCD98hc) that utilize these BBB receptors to improve CNS drug delivery1,2. Here, we provide a comprehensive and unbiased biodistribution characterization of ATVTfR and ATVCD98hc compared to a standard IgG at a multiscale level, ranging from whole-body to brain region- and cell type-targeting specificity. Mouse whole-body tissue clearing revealed distinct organ localization for each molecule. In the CNS, ATVTfR and ATVCD98hc not only achieves enhanced brain delivery but importantly, much broader parenchymal distribution in contrast to the severely limited distribution observed with a standard antibody that was not able to be improved even at very high dose levels. Using cell sorting and single-cell RNA sequencing of mouse brain, we revealed that standard IgG predominantly localizes to perivascular and leptomeningeal cells and reaches the CNS by entering the CSF, rather than crossing the BBB. In contrast, ATVTfR and ATVCD98hc enables broad parenchymal cell-specific distribution via transcytosis through brain endothelial cells (BECs) along the neurovasculature. Finally, we extended the translational relevance of our findings by revealing enhanced and broad brain and spinal cord biodistribution of ATVTfR compared to standard IgG in cynomolgus monkey. Taken together, this multiscale analysis reveals in-depth biodistribution differences between ATVTfR, ATVCD98hc, and standard IgG. These results may better inform platform selection for specific therapeutic targets of interest, optimally matching platforms to desired CNS target engagement, peripheral organ exposures, and predict or potentially reduce off-target effects.

neuroscience↗

bioRxiv2024

Genetically Encoded and Modular SubCellular Organelle Probes (GEM-SCOPe) reveal widespread lysosomal and mitochondrial dysfunction in human PRKN knockout astrocytes and neurons

Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development of many diseases. Quantitative visualization of mitochondria and lysosomes is crucial to understand how these organelles are dysregulated during disease. To address a gap in live-imaging tools, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), a modular toolbox of fluorescent markers designed to inform on localization, distribution, turnover, and oxidative stress of specific organelles. We expressed GEM-SCOPe in differentiated astrocytes and neurons from a human pluripotent stem cell PRKN-knockout model of Parkinsons disease and identified disease-associated changes in proliferation, lysosomal distribution, mitochondrial transport and turnover, and reactive oxygen species. We demonstrate GEM-SCOPe is a powerful panel that provide critical insight into the subcellular mechanisms underlying Parkinsons disease in human cells. GEM-SCOPe can be expanded upon and applied to a diversity of cellular models to glean an understanding of the mechanisms that promote disease onset and progression.

cell biology↗

bioRxiv2022

Disruption of lysosomal proteolysis in astrocytes facilitates midbrain proteostasis failure in an early-onset PD model.

Accumulation of advanced glycation end products (AGEs) on biopolymers accompany cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early on-set Parkinsons Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, -synuclein (-syn) phosphorylation to increase, and proteins to aggregate. These processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in PD.

neuroscience↗

bioRxiv2021

A reference induced pluripotent stem cell line for large-scale collaborative studies

Human induced pluripotent stem cell (iPSC) lines are a powerful tool for studying development and disease, but the considerable phenotypic variation between lines makes it challenging to replicate key findings and integrate data across research groups. To address this issue, we sub-cloned candidate iPSC lines and deeply characterised their genetic properties using whole genome sequencing, their genomic stability upon CRISPR/Cas9-based gene editing, and their phenotypic properties including differentiation to commonly-used cell types. These studies identified KOLF2.1J as an all-around well-performing iPSC line. We then shared KOLF2.1J with groups around the world who tested its performance in head-to-head comparisons with their own preferred iPSC lines across a diverse range of differentiation protocols and functional assays. On the strength of these findings, we have made KOLF2.1J and hundreds of its gene-edited derivative clones readily accessible to promote the standardization required for large-scale collaborative science in the stem cell field. SummaryThe authors of this collaborative study deeply characterized human induced pluripotent stem cell (iPSC) lines to rationally select a clonally-derived cell line that performs well across multiple modalities. KOLF2.1J was identified as a candidate reference cell line based on single-cell analysis of its gene expression in the pluripotent state, whole genome sequencing, genomic stability after highly efficient CRISPR-mediated gene editing, integrity of the p53 pathway, and the efficiency with which it differentiated into multiple target cell populations. Since it is deeply characterized and can be readily acquired, KOLF2.1J is an attractive reference cell line for groups working with iPSCs. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/472643v6_ufig1.gif" ALT="Figure 1"> View larger version (84K): org.highwire.dtl.DTLVardef@cef106org.highwire.dtl.DTLVardef@31d5d8org.highwire.dtl.DTLVardef@1ce6d8corg.highwire.dtl.DTLVardef@17a19d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2021

Identification and characterization of hPSC-derived FOXA2+ progenitor cells with ventricular cardiac differentiation potential

While much progress has been made in understanding early cardiac development, the precise mechanisms that specify the different cardiomyocyte subtypes remain poorly understood. Recent data from our lab have shown that transient Foxa2 expression identifies a progenitor population with exclusive ventricular differentiation potential in the mouse heart. Here we have translated this concept to the human pluripotent stem cell (hPSC) system. Using a FOXA2-GFP reporter cell line we characterized expression of FOXA2 during hPSC cardiac differentiation and found that a subset of cardiac mesoderm precursors transiently expresses FOXA2. Gene expression analysis of FOXA2+ and FOXA2- cardiac mesoderm revealed that both populations similarly express early cardiac specification markers such as PDGFRA, TBX5, and ISL1, while other key candidates including TBX20 and GATA4 are significantly upregulated in the FOXA2+ population. Isolation and subsequent differentiation of FOXA2+ and FOXA2- populations demonstrates their comparable differentiation potential to both cardiomyocytes and epicardial cells. However, cardiomyocytes derived from FOXA2+ precursors showed enhanced differentiation efficiency toward ventricular cardiomyocytes compared to cardiomyocytes derived from FOXA2- precursors. To identify new mechanisms that regulate ventricular specification, we performed small molecule screening and found that inhibition of the EGFR pathway strongly increased the cardiac mesoderm population in general, and the FOXA2+ precursors in particular. Finally, we have identified a combination of cell surface markers to specifically isolate FOXA2+ cardiac precursors. In summary, our results suggest that FOXA2+ cardiac mesoderm harbors ventricular-specific differentiation potential and isolation of these cells permits the generation of cultures enriched for ventricular cardiomyocytes. Generating such enriched cardiac populations will be relevant for regenerative medicine approaches, as well as for disease modeling from induced pluripotent stem cells.

developmental biology↗