Biology subjects

Merchant, K.

Publications and source records attributed to Merchant, K..

6 recordsLinked to original sources

bioRxiv2024

Investigating T cell Recruitment in Atherosclerosis using a novel Human 3D Tissue-Culture Model reveals the role of CXCL12 in intraplaque neovessels

BackgroundDevelopment of effective treatments for atherosclerosis requires new models that better predict the human immune response. Although T cells are abundant in human atherosclerotic lesions and play a key role in the pathogenesis, the mechanism involved in plaque infiltration remains ill defined. MethodsWe developed a three-dimensional tissue-culture model to study leukocyte recruitment to human atherosclerotic plaques. In this study, human atherosclerotic plaques obtained during carotid endarterectomy surgery were co-cultured with patient-matching T cells. Exogenous T cells were stained using a multi-factor staining strategy, which involved intracellular fluorescent cell tracker dyes combined with nuclear labels. Flow cytometry was used to assess the presence of the labeled cells within the plaques, and microscopic analysis was performed to examine their localization. ResultsFlow cytometry and microscopy cell-tracking analysis demonstrated that exogenous T cells successfully migrated into atherosclerotic plaques. Furthermore, infiltrated CD8+ T cells displayed a significant increase of CD69 expression, indicating their activation within the tissue. Blocking chemokine receptors, particularly CXCR4, significantly impaired T cell infiltration, demonstrating that exogenous CD8+ T cells invade plaques through chemotactic migration. Surprisingly, 3D microscopy combined with optical tissue clearing strategy revealed that CXCL12, the sole ligand of CXCR4, mainly accumulated in intraplaque neovessels. Single-cell RNA sequencing (scRNAseq) analysis further confirmed that endothelial cells from intraplaque neovessels were the primary source for CXCL12. Additionally, exogenous T cells were found within and in proximity to these neovessels, suggesting that the CXCL12/CXCR4 axis regulates T cell recruitment through intraplaque neovessels. ConclusionsOverall, these findings shed new light on the mechanism of action of CXCL12 in atherosclerosis and demonstrated the potential of the model to advance our understanding of leukocyte accumulation in human atherosclerosis and assist in testing novel pharmacological therapies.

molecular biology↗

bioRxiv2022

Elaboration of a MALDI-TOF Mass Spectrometry-based Assay of Parkin Activity and High-Throughput screening platform for Parkin Activators

Parkinsons disease (PD) is a progressive neurological disorder that manifests clinically as alterations in movement (bradykinesia, postural instability, loss of balance, and resting tremors) as well as multiple non-motor symptoms including but not limited to cognitive and autonomic abnormalities. Mitochondrial dysfunction has been linked to sporadic PD and loss-of-function mutations in genes encoding the ubiquitin E3 ligase Parkin and protein kinase, PTEN-induced kinase 1 (PINK1), that regulate mitophagy, are causal for familial and juvenile PD1-3. Among several therapeutic approaches being explored to treat or improve PD patients prognosis, the use of small molecules able to reinstate or boost Parkin activity represents a potential pharmacological treatment strategy4. A major barrier is the lack of high throughput platforms based on robust and accurate quantification of Parkin activity in vitro. Here we present two different and complementary Matrix Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-TOF MS) based approaches for the quantification of Parkin E3 ligase activity in vitro. These methods recapitulate distinct aspects of ubiquitin conjugation: Parkin auto-ubiquitylation and Parkin-catalysed discharge on lysine residues. Both approaches are scalable for high-throughput primary screening to facilitate the identification of Parkin modulators.

biochemistry↗

bioRxiv2021

Proteome Profiling of Cerebrospinal Fluid Reveals Novel Biomarker Candidates for Parkinson's Disease

Parkinsons disease (PD) is a growing burden worldwide, and despite ongoing efforts to find reliable biomarkers for early and differential diagnosis, prognosis and disease monitoring, there is no biofluid biomarker used in clinical routine to date. Cerebrospinal fluid (CSF) is collected often and should closely reflect structural and functional alterations in PD patients brains. Here we describe a scalable and sensitive mass spectrometry (MS)-based proteomics workflow for CSF proteome profiling to find specific biomarkers and identify disease-related changes in CSF protein levels in PD. From two independent cohorts consisting of more than 200 individuals, our workflow reproducibly quantified over 1,700 proteins from minimal sample amounts. Combined with machine learning, this identified a group of several proteins, including OMD, CD44, VGF, PRL, and MAN2B1 that were altered in PD patients or significantly correlate with clinical scores, indicative of disease progression. Interestingly, we uncovered signatures of enhanced neuroinflammation in patients with familial PD (LRRK2 G2019S carriers) as indicated by increased levels of CTSS, PLD4, HLA-DRA, HLA-DRB1, and HLA-DPA1. A comparison with urinary proteome changes in PD patients revealed a large overlap in protein composition PD-associated changes in these body fluids, including lysosomal factors like CTSS. Our results validate MS-based proteomics of CSF as a valuable strategy for biomarker discovery and patient stratification in a neurodegenerative disease like PD. Consistent proteomic signatures across two independent CSF cohorts and previously acquired urinary proteome profiles open up new avenues to improve our understanding of PD pathogenesis.

systems biology↗

bioRxiv2020

Urinary proteome profiling for stratifying patients with familial Parkinsons disease

The prevalence of Parkinsons disease (PD) is increasing but the development of novel treatment strategies and therapeutics altering the course of the disease would benefit from specific, sensitive and non-invasive biomarkers to detect PD early. Here, we describe a scalable and sensitive mass spectrometry (MS)-based proteomic workflow for urinary proteome profiling. Our workflow enabled the reproducible quantification of more than 2,000 proteins in more than 200 urine samples using minimal volumes from two independent patient cohorts. The urinary proteome was significantly different between PD patients and healthy controls, as well as between LRRK2 G2019S carriers and non-carriers in both cohorts. Interestingly, our data revealed lysosomal dysregulation in individuals with the LRRK2 G2019S mutation. When combined with machine learning, the urinary proteome data alone was sufficient to classify mutation status and disease manifestation in mutation carriers remarkably well, identifying VGF, ENPEP and other PD-associated proteins as the most discriminating features. Taken together, our results validate urinary proteomics as a valuable strategy for biomarker discovery and patient stratification in PD.

systems biology↗

bioRxiv2019

An assessment of LRRK2 serine 935 phosphorylation in human peripheral blood mononuclear cells in idiopathic Parkinson’s disease and G2019S LRRK2 cohorts

The phosphorylated form of LRRK2, pS935 LRRK2, has been proposed as a target modulation biomarker for LRRK2 inhibitors. To qualify the biomarker for therapeutic trials, we assessed pS935 LRRK2 levels in Peripheral Blood Mononuclear Cells (PBMCs). Analyses of PBMCs from healthy controls, idiopathic Parkinsons disease (iPD), and G2019S carriers with and without PD showed significant reductions in pS935 LRRK2 levels normalized to total LRRK2 levels in G2019S carriers with PD compared to those without PD or iPD. Neither analyte correlated with age, gender, or disease severity. Thus, pS935 LRRK2 in PBMCs may reflect a state marker for G2019S LRRK2-driven PD.

neuroscience↗

bioRxiv2018

LRRK2 kinase inhibitors induce a reversible effect in the lungs of non-human primates with no measurable pulmonary deficits

Putative gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2), resulting in increased kinase activity and cellular toxicity, are a leading genetic cause of Parkinsons disease (PD). Hence, there is strong interest in developing LRRK2 kinase inhibitors as a disease-modifying therapy. Published reports that repeat dosing with two LRRK2 kinase inhibitors (GNE-7915 and GNE-0877) induce histopathological changes in the lung of non-human primates Fuji et al. 2015 (1) raised concerns about potential safety liability of LRRK2 kinase inhibitors. In the present study, we sought to determine whether previously observed effects in the lung: (a) represent on-target pharmacology, but with the potential for margin of safety, (b) are reversible upon drug withdrawal, and (c) are associated with pulmonary function deficits. To this end, we evaluated the histopathological effects, toxicokinetics and target inhibition of three structurally diverse LRRK2 kinase inhibitors, GNE-7915 (30 mg/kg, BID, as a positive control), MLi-2 (15 and 50 mg/kg, QD) and PFE-360 (3 and 6 mg/kg, QD) following 2 weeks of dosing in non-human primates. Subsets of animals dosed with GNE-7915 or MLi-2 were evaluated after 2-week dose-free periods. All three LRRK2 kinase inhibitors induced mild cytoplasmic vacuolation of type II pneumocytes, as reported previously, confirming an on-target effect of these compounds. Interestingly, despite lower doses of both PFE-360 and MLi-2 producing nearly complete inhibition of LRRK2 kinase activity in the brain as assessed by levels of pS935-LRRK2, histopathological changes in lung were absent in animals treated with low-dose PFE-360 and observed only sporadically in the low-dose MLi-2 group. The lung effect was fully reversible at 2 weeks post-dosing of GNE-7915. In a second study of identical dosing with MLi-2 and GNE-7915, no deficits were observed in a battery of translational pulmonary functional tests. In aggregate, these results do not preclude the development of LRRK2 kinase inhibitors for clinical investigation in Parkinsons disease.

pharmacology and toxicology↗