Study of endoplasmic reticulum stress in Shwachman-Diamond syndrome and SRP-related congenital neutropenia – NeutroSTRESS

The Shwachman-Diamond syndrome (SDS) and SDS-like congenital neutropenia (CN) related to anomalies of the signal recognition particle (SRP-CN) complex are characterized by impaired granulocytic differentiation and apoptosis of neutrophil granulocytes and their precursors. SDS and SRP-CN are respectively caused by genetic defects of SBDS and SRP54/SRP68 affecting the ribosome biogenesis and the targeting of nascent proteins to the endoplasmic reticulum (ER). A key function of neutrophils is to synthesize a high amount of proteins with anti-infectious roles, all packaged into granules. From the promyelocyte stage of granulopoiesis in which granule biogenesis starts, it is obvious that the ribosomes and ER are essential organelles. Defects of the ribosome/ER axis could result in alterations of protein synthesis, protein transport into ER, protein maturation, and folding leading to cell apoptosis. Our recently published and preliminary results on SBDS, SRP54, and SRP68 showed evidence of enhanced ER stress, activation of unfolded protein response (UPR), and p53-dependent apoptosis of the granulocytic cells.
The objectives of this proposal are: (i) to characterize robustly the ER stress induced by defective SBDS or SRP complex in each compartment of the granulocytic differentiation and to test ER stress inhibitors to restore the granulocytic homeostasis, (ii) to decipher the molecular mechanisms causing the ER stress and UPR, and to identify which proteins are incorrectly translocated in the ER and iii) to identify novel genetic causes of CN with SDS-like features and understand their interaction with proteins of ER.
To reach these goals, the project is divided into 3 scientific work packages (WP).
In the first WP, using shRNA downmodulated and primary patient’s cells strategies already mastered for SRP54 and SBDS, we will characterize the impact of SBDS and SRPs defects on granulocytic differentiation. We will analyze different cell death processes and if they are dependent on p53 or not. We will study the functional consequences on ER stress at the different stages of granulopoiesis and at both transcript and protein levels. We will also perform in vitro assays to test chemical chaperones that restore ER homeostasis. Finally, as SDS and SRP-CN patients may present transient anemia and/or thrombocytopenia, we will also analyze the impact of the different mutations on erythroid and megakaryocytic lineages.
In the second WP, we will use three complementary approaches to dissect the molecular mechanisms of ER stress-induced by SBDS and SRP mutations. We will perform single-cell transcriptomes in granulocytic progenitor using an innovative approach combining the single-cell RNA-seq and the CITE-seq technologies. We will perform ribosome profiling to determine deregulated ribosomal pauses known to affect co-translational degradation of mRNA and nascent chain. Finally, we will do proteomics to find out which proteins are deregulated or incorrectly translocated in the ER.
In the third WP, as 30% of CN remain without etiology, we will search for novel genetic causes of CN using whole-genome and RNA sequencing focusing on genes from the proteostasis pathway.
The project relies on the tight collaboration of two partners who have previously worked together. They have robust and complementary expertise in hematopoiesis, single-cell RNA studies, ER stress study, and genomics. The project will also take advantage of the French Registry of CN integrating a centralized biobank and a database with exhaustive clinical information and, of two collaborations with research groups that are experts for the study of ER stress and ribosome profiling.
At the end of the project, we expect to have greatly improved our knowledge of molecular mechanisms leading to ER stress/UPR activation in granulopoiesis and of proteins impaired in SDS and SRP-related CN. Furthermore, the identification of new genes may reveal novel pathological pathways involved in CN.