Lamins in tissue progenitor cell regulation

Lamins are intermediate filament proteins that form a structural network at the nuclear envelope in multicellular eukaryotes, called the nuclear lamina. Mutations in lamins and some associated proteins are linked to human diseases, ranging from muscular dystrophies to accelerated aging syndromes. The molecular basis of these diseases is not understood. In these study we will test and molecularly characterize a novel disease-relevant function of lamins in adult stem cell regulation in regenerative tissues. While most previous studies have focused on the lamina network at the nuclear periphery, our recent work revealed a lamina-independent pool of lamins in the nuclear interior, which requires a specific lamin-binding partner, lamin-associated polypeptide LAP2a . Using LAP2a knockout mice and primary cells derived therefrom we found that the nucleoplasmic lamin-LAP2a complex affects cell cycle progression of early progenitor cells in regenerative tissues in a retinoblastoma protein (Rb)-dependent manner. The molecular mechanisms of these pathways, however, are still unknown and will be addressed in this study: Aim 1 will identify the molecular pathways regulating nucleoplasmic lamins. We will test the involvement of lamin modifications and analyze the effect of LAP2a on lamin stability and assembly in vitro. Aim 2 will test how LAP2a affects the Rb pathway at molecular level. We will generate Rb1+/- / LAP2a -/- double mutant mice to identify a genetic link of Rb and LAP2a in vivo. Furthermore, using primary myoblasts derived from LAP2a - deficient and overexpressing mice we will address the mechanistic details of LAP2a `s function in cell cycle regulation and during in vitro differentiation to myofibers. In particular, we will test the role of LAP2a in Rb modifications and in the regulation of transcriptional repressors and epigenetic pathways, and we will identify deregulated genes and promoters in LAP2a -mutant cells. Aim 3 will test the LAP2a gain-of-function phenotype at organismal level by overexpression of LAP2a in muscle satellite cells or muscle fibers in transgenic mice. We will analyze muscle physiology, function and regeneration in vivo and test myoblast differentiation ex vivo in primary cultures. Finally, Aim 4 will test how mutations in LAP2a linked to striated muscle disorders in patients affect lamin interactions, Rb regulation, myoblast self renewal and differentiation, using primary patient cells and mouse myoblasts expressing mutated LAP2a variants. Overall, our studies will change the current view on lamin functions providing a novel concept on the involvement of nucleoplasmic lamins in adult stem cell regulation, which is highly relevant for lamin-linked diseases.

In mammalian cells, lamins form a protein network at the nuclear envelope called the lamina, which provides mechanical stability and stiffness for the nucleus. Mutations in lamins have been linked to several rare diseases, including muscular dystrophies and the premature ageing Hutchinson Gilford Progeria syndrome (HGPS). The molecular disease mechanisms are poorly understood hindering the development of efficient therapeutic approaches.Our project aimed at the identification of novel molecular functions of lamins and how these functions may be affected in lamin-linked diseases. In contrast to most studies on lamins done world-wide in the past years, which focused predominantly on the lamina scaffold structure, we investigated a different cellular pool of lamins. Our experiments indicated that this lamin pool may be important for tissue regeneration in humans and may also be highly relevant for lamin-linked human diseases. We found that this barely studied lamin pool is not part of the lamina scaffold but diffuses freely throughout the nucleus and fulfills important roles in the regulation of adult stem cells. Adult stem cells are found in many mammalian tissues that have to be continuously renewed during lifetime, such as skin, muscle or blood cells. Proliferation of adult stem cells is important for the formation of new stem cells (self renewal) and for the formation of tissue progenitor cells, which will then form the specific cell types to replenish the tissue. A tightly regulated balance of stem cell renewal and tissue formation is essential for normal tissue homeostasis and for efficient regeneration after injury. In this project we addressed the molecular mechanisms, how the lamins affect the activity of adult stem cells and tissue progenitor cells. Unexpectedly we found that lamins have a global role in the organization and regulation of chromosomes and genes, which in turn affect signaling pathways that regulate self renewal and proliferation of adult stem cells.In addition we tested human cells derived from premature ageing disease patients that express mutant forms of lamins. We found that in these cells particularly the dynamic pool of lamins was lost, which led to defects in cell proliferation and tissue formation. These findings suggest that the expression of disease-linked lamin mutants affects adult stem cell renewal, which in turn can lead to impaired tissue homeostasis and disease specific defects, such as progressive muscle wasting or skin ageing seen in these patients. Overall, our findings identify a novel lamin-mediated mechanism that regulates adult stem cell function and tissue regeneration and open up new avenues for the development of novel drugs and efficient therapies of patients.