Periodic Reporting for period 1 - Asymmetric fates (The role of degradation pathways on cell stemness and fate determination)
Reporting period: 2020-07-06 to 2022-07-05
The haematopoietic system relies on the potential of haematopoietic stem cells (HSCs) to self-renew and differentiate into all lineages of mature blood cells, and is a reference model to study differentiation hierarchies. Cell fate determination results from different layers of regulation, including transcriptional, translational, epigenetic, metabolic, and cell biological changes. Autophagy, a cell degradation mechanism, plays mechanistically relevant roles that in principle may impact on all these layers. Here we address why autophagy depletion leads to a drastic loss of the stem cell compartment. Using inducible deletion of autophagy specifically in adult hematopoietic stem cells (HSCs) and in mice chimeric for autophagy-deficient and normal HSCs, we demonstrate that the stem cell loss is cell-intrinsic. Mechanistically, autophagy-deficient HSCs showed higher expression of several amino acid transporters (AAT) when compared to autophagy-competent cells, resulting in increased amino acid (AA) uptake. This was followed by sustained mTOR (mammalian target of rapamycin) activation, with enlarged cell size, glucose uptake and translation, which is detrimental to the quiescent HSCs. mTOR inhibition by rapamycin treatment in vivo was able to rescue autophagy-deficient HSC loss and bone marrow failure and resulted in better reconstitution after transplantation. Our results suggest that targeting mTOR may improve aged stem cell function, promote reprogramming and stem cell transplantation.
The original proposal had 3 different aims:
1. Investigate the mechanism that drives haematopoietic stem cell (HSC) recovery by rapamycin in scenarios of autophagy impairment
2. Establish an efficient strategy to image asymmetric cell division (ACD) in autophagy-deficient HSCs by long-term ex vivo HSCs expansion
3. Unravel the possible impact of degradation on ACD and HSCs maintenance and differentiation in vivo
During the timeframe of the fellowship, we were able to address Aim 1 and Aim 3 (with modifications). Aim 2 proved to be not feasible, as autophagy-deficient HSCs are vulnerable and could not be culture in vitro.
Concerning Aim 1, using different strategies to address the cell-intrinsic role of autophagy in HSC homeostasis (murine models where autophagy is deleted in HSCs specifically and/or bone marrow chimeras), we could show that upon autophagy loss, HSCs uptake an excessive amount of amino acids, which is followed by mTOR activation. This results in higher translation rates, which is detrimental to quiescent cells, such as HSCs. By inhibiting mTOR (rapamycin treatment) we could rescue the HSC compartment in autophagy-deficient scenarios.
Concerning Aim 3, as HSCs were not suitable to address whether asymmetric inheritance of cell cargoes is regulated by autophagy (due to technical reasons and low cell viability in vitro), we alternatively performed experiments in T cells. Adoptive transfer of isolated daughter cells containing or not aged mitochondria revealed that these organelles are indeed cell fate determinants and that their inheritance impacts on T cell fate.
1. Investigate the mechanism that drives haematopoietic stem cell (HSC) recovery by rapamycin in scenarios of autophagy impairment
2. Establish an efficient strategy to image asymmetric cell division (ACD) in autophagy-deficient HSCs by long-term ex vivo HSCs expansion
3. Unravel the possible impact of degradation on ACD and HSCs maintenance and differentiation in vivo
During the timeframe of the fellowship, we were able to address Aim 1 and Aim 3 (with modifications). Aim 2 proved to be not feasible, as autophagy-deficient HSCs are vulnerable and could not be culture in vitro.
Concerning Aim 1, using different strategies to address the cell-intrinsic role of autophagy in HSC homeostasis (murine models where autophagy is deleted in HSCs specifically and/or bone marrow chimeras), we could show that upon autophagy loss, HSCs uptake an excessive amount of amino acids, which is followed by mTOR activation. This results in higher translation rates, which is detrimental to quiescent cells, such as HSCs. By inhibiting mTOR (rapamycin treatment) we could rescue the HSC compartment in autophagy-deficient scenarios.
Concerning Aim 3, as HSCs were not suitable to address whether asymmetric inheritance of cell cargoes is regulated by autophagy (due to technical reasons and low cell viability in vitro), we alternatively performed experiments in T cells. Adoptive transfer of isolated daughter cells containing or not aged mitochondria revealed that these organelles are indeed cell fate determinants and that their inheritance impacts on T cell fate.
This project allowed us to finally determine that autophagy regulates HSC homeostasis in a cell-intrinsic manner. Understanding the molecular and cellular pathways that allow one single HSC to generate all hematopoietic cell types has an impact on both elucidating evolutionary ontogeny of hematopoietic cell types and developing new therapeutic approaches for haematopoietic malignancies. Furthermore, the results here obtained suggest that modulating the negative consequences of autophagy impairment, which happens during ageing, such as excessive mTOR activation and translation in quiescent cells, may improve stem cell function and improve stem cell transplantation strategies.