SP4: K. Lenhard Rudolph - Signalling pathways regulating asymmetric segregation of damaged proteins in stem cells

Background and previous work
Given the pivotal role of stem cells in ageing and diseases, it is of particular interest to
analyse mechanisms that influence the accumulation of damaged proteins in stem cells. An age-dependent increase in carbonlyated proteins was observed in hematopoietic stem and progenitor cells during human ageing. Asymmetric segregation of proteins during cell division is an emerging factor, which may contribute to self-renewal of adult mammalian stem cells. Asymmetric segregation of carbonylated proteins occurs when embryonic stem cells (ES) differentiate. In this case low levels of damaged proteins segregate into differentiated daughter cells but are retained in the ES cell. The molecular machinery that controls asymmetric segregation of carbonylated proteins and AGEs in adult mammalian stem cells remains largely unknown.
Specific aims
The main aim of this project is to use ES cell cultures to identify molecular pathways
regulating asymmetric segregation of long-lived protein damages at stem cell level and to test whether the same pathways contribute to asymmetric division of damaged proteins in adult stem cells.
Working programme
The work programme focuses on: (1) Identification and functional analysis of genes that influence the rate of asymmetric segregation of damaged proteins in ES cells and adult stem cells – We will use ES cell cultures to conduct lentiviral shRNA screens to identify candidate genes that regulate asymmetric segregation of damaged proteins indifferentiating ES cells. Sub-libraries of candidate genes will be tested to influence the asymmetric segregation of damaged proteins in hematopoietic and intestinal stem cells of adult mice. (2) Delineating ageing-associated changes in the expression and function of genes controlling asymmetric segregation of AGEs in murine stem cells – This analysis will be conducted on freshly isolated hematopoietic and intestinal stem cells from young versus old wild-type mice as well as from mouse models undergoing premature ageing due to specific molecular causes, e.g. telomere shortening in telomerase deficient mice or mitochondrial dysfunction due to impairments in mitochondrial biogenesis. (3) Identification and functional characterisation of checkpoints that limit stem cell functionality in response to AGE accumulation – We will conduct lentiviral shRNA screens on stem cells that lack key genes required for asymmetric segregation of damaged proteins to identify checkpoint genes that respond to such defects and limit stem cell self-renewal in this context. Collaborations within the consortium are planned with: SP1 – determine the profile of amide-AGEs changes in ageing stem cells, SP2 – determine the outcome of metabolic sialic acid engineering on intestinal and hematopoietic stem cells in mouse models of ageing, SP8 – determine ageing-associated PTMs in the nucleus of hematopoietic and intestinal stem cells during ageing, SP9 – analyse PTM (glycation/alkylation) targets of the FOXO signalling cascade in stem cell ageing. 

Prof. Dr. Karl Lenhard Rudolph
Leibniz Institute on Aging (FLI)
Beutenbergstr. 11
07745 Jena
e-mail: Lenhard Rudolph
Homepage: AG-Rudolph