Schmugge, M. | Search bioRxiv

bioRxiv2023

Transcriptomic gene profiles in an ex vivo model of erythropoiesis to unravel molecular pathomechanisms in sickle cell disease

We characterized the transcriptional profiles of erythroid cells differentiated from peripheral blood mononuclear cells (PBMCs) from peripheral blood collected from patients diagnosed with Sickle Cell Disease (SCD), which have been treated with Hydroxyurea (HU) in comparison to untreated SCD patients and healthy controls (HC) using bulk RNAseq. We identified 1398 differentially expressed genes (DEGs) in SCD non-treated-derived erythroid cells and 495 DEGs in SCD HU-treated patient-derived erythroid cells compared to HC. We found biological processes such as oxidative phosphorylation pathway, proteasome, autophagy, natural killer cell (NK) cytotoxicity, adaptive immune response or inflammatory response to be significantly enriched in our patient study groups by using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Our findings collectively suggest different as well as common molecular signatures between our groups. We could validate 12 of our top DEGs in treated patients by qRT-PCR. We found similar regulation patterns when comparing the mRNA levels of mutS homolog 5-Suppressor APC Domain Containing 1 (MSH5-SAPCD1), G protein subunit gamma 4 (GNG4), stabilin 1/ clever-1 (STAB1) and Fas Binding Factor 1 (FBF1) from the bone marrow cells and spleen tissue from the Berkely SCD mouse model to the expressions observed in the transcriptome of our ex-vivo patient-derived erythropoiesis model.

molecular biology↗

bioRxiv2023

Clusterin can mediate apoptosis-induced molecular mechanisms in immune thrombocytopenia

Abnormalities in the apoptosis pathway are possible risk factors for various autoimmune diseases including immune thrombocytopenia (ITP). ITP is an autoimmune bleeding disorder characterized by a low platelet count and mostly mild but in rare occasions life threatening bleeding symptoms. Platelets and megakaryocytes (MKs) may be seen as the major targets of the pathogenic immune responses in ITP. A mechanistic understanding of the ITP pathogenesis is still lacking. Our data indicate that mechanisms associated with impaired clusterin-mediated apoptosis might play a role in ITP platelet pathophysiology and platelet production by MKs. We could demonstrate by apoptosis proteomic profiling significantly increased expression levels of some apoptotic genes such as clusterin (CLU), pro-caspase 3, catalase, TRAILR1/DR4, Bax, Bad and Bcl-2 compared to healthy controls in platelet-rich plasma (PRP) from 10 ITP patients. We could validate by both RT-qPCR and Western blotting that CLU, a stress-activated chaperone, is significantly increased in both newly diagnosed and chronic ITP. We used the human megakaryoblastic cell line MEG-01, treated for 4h with plasma from acute and chronic ITP patients and healthy controls. We performed chemical treatments in plasma treated MEG-01 by using pan-caspase inhibitors (Z-VAD-FMK), apoptosis inducer ABT-737, Rotenone and Rapamycin. We determined the expression at mRNA levels of apoptosis pathway regulatory genes Bax, caspase-3, -8, -9 as well as CLU, GRP78 and GRP94 by qRT-PCR. We could demonstrate significantly downregulation of mRNA expression levels of these apoptotic markers in ITP plasma treated and CLU siRNA transfected MEG-01 cells. Our results indicate a possible impairment of apoptosis pathway via upregulation of CLU and Bax in platelets and in their producers MKs that can lead to platelet destruction in ITP disease.

molecular biology↗