PINK1 in the regulation of terminal erythroid maturation and enucleation

The National Institute of Diabetes and Digestive and Kidney Diseases awarded Icahn School of Medicine at Mount Sinai $100,228 on February 13, 2027, under the Diabetes, Digestive, and Kidney Diseases Extramural Research program (CFDA 93.847) to conduct research on PINK1 regulation of terminal erythroid maturation and red blood cell enucleation.

The research tests the physiological role of PINK1 (PTEN-induced kinase 1) in terminal erythroid maturation and enucleation—the process by which developing red blood cells remove their nucleus. The investigator will examine how PINK1 regulates this process via the PINK1/parkin mitophagy pathway and independent mechanisms, building on prior findings that PINK1-knockout mice produce more red blood cells and exhibit higher enucleation rates in orthochromatic erythroblasts with accumulated mitochondria. Understanding these mechanisms addresses the understudied gap in erythropoiesis that hinders comprehension of diseases arising from ineffective erythropoiesis and advancement of in vitro red blood cell production.

Performance occurs in New York, New York. The period of performance runs from February 13, 2027, through August 31, 2029. The assistance type is a Project Grant.

Abstract

Rbcs are the most abundant cell type of blood, and are produced via erythropoiesis.2 terminal erythroid maturation involves a series of step-wise, mitotic divisions of erythroid progenitors to form rbcs (pro- erythroblast >basophilic erythroblast > polychromatic erythroblast > orthochromatic erythroblast> reticulocyte > RBC).7 IT is characterized by a dramatic increase in nuclear condensation and hemoglobinization, followed by enucleation (removal of the nucleus), organelle clearance, and rbcs with a hemoglobin-rich cytoplasm - critical to its function as an oxygen carrier.7 enucleation occurs at the orthochromatic erythroblast stage, and is considered a rate-limiting step of terminal erythroid maturation. Terminal erythroid maturation is an understudied part of erythropoiesis, which hinders our understanding of diseases that arise due ineffective erythropoiesis and to the advancement of in vitro RBC production.2,6,7 the ghaffari laboratory previously found that mitochondrial aggregation adjacent to the nucleus is critical to murine erythroblast enucleation.2 this indicates that mitochondria play a dynamic function in aiding terminal erythroid maturation, prior to their own removal from the cell following enucleation via mitophagy (the autophagic removal of mitochondria). PINK1 (pten-induced kinase 1) regulation of parkin via PINK1/parkin mitophagy pathway is a main and well-known regulator of mitophagy.14,15 the physiological role of PINK1 in terminal erythroid maturation, via PINK1/parkin pathway and/or independent of IT, is not well understood. I found that PINK1-/- mice produce more rbcs compared to WT. I also found that mitochondria are accumulated, and enucleation rates are higher in PINK1-/- orthochromatic erythroblasts. In this project, i will test the role of PINK1 as a novel regulator of terminal erythroid maturation and erythroid cell enucleation (aim 1). I will specifically employ fluorescence activated cell sorting (facs) techniques to sort PINK1-/- versus WT terminal erythroblasts at each stage of maturation and then probe (1.1) mitochondrial quality, (1.2) rates of in vivo mitophagy (using a mitophagy reporter mouse model: mitochondrial keima),27 and (1.3) the overall transcriptomic profiles. I will further study a direct downstream target of PINK1, MIRO1 (mitochondrial RHO-gtpase 1) in mechanistically regulating mitochondrial movement during erythroid cell enucleation via its link to microtubules (aim 2).18 in the absence of PINK1, MIRO1 levels accumulate and may result in abnormal mitochondrial movement. I found that MIRO1 is accumulated at early stages of terminally maturing PINK1-/- erythroblasts relative to WT. To expand this, i will employ crispr/CAS9 gene editing to (2.1) knockdown MIRO1 in PINK1-/- murine erythroblasts to levels comparable to WT, and determine if this rescues the observed increased enucleation. (2.2) knockdown PINK1 in human peripheral blood mononuclear cells (pbmcs)-derived erythroblasts to determine if the terminal erythroid maturation phenotype is recapitulated, and to further explore MIRO1's role in this. Overall, this project will characterize PINK1 and MIRO1 as novel regulators of terminal erythroid maturation and erythroid enucleation.