Mutations in TSC1 and TSC2 cause tuberous sclerosis complex (TSC), a multi-systemic genetic disease characterized by a variety of symptoms such as non-malignant brain or heart tumors, kidney and lung cysts, and neurocognitive defects. TSC1 and TSC2 have well-characterized roles in regulating cellular energy balance and protein translation, but also a number of non-canonical functions, for example in primary cilium formation. However, these functions remain poorly understood and it is currently unknown to what extent each of these TSC1/2 functions contributes to the development of TSC. Previous results from our and other research groups suggest that TSC1 and TSC2 may exert important aspects of their functions at the centrosome and/or in the context of regulating primary cilium structure and function. We hypothesize that TSC is, at least in part, a ciliopathy and that compromising this TSC- centrosome-primary cilium axis may contribute substantially to the development of TSC. Therefore, we aim to characterize the functions of TSC1/2 at the centrosome and to correlate impaired TSC1/2 functions with alterations in ciliary signaling. We will use genome-engineered mouse and human cell lines to test which of the canonical and non- canonical TSC1 or TSC2 functions can be rescued by versions of TSC1/2 that strictly localize to the centrosome in cells that otherwise lack TSC1 or TSC2 function, respectively. To control for TSC1/2 functions that are specifically linked to their centrosomal localization, we will test these functions in cells that lack centrosomes, but express wt TSC1/2. To test whether TSC1/2 loss-of-function phenotypes are a consequence of alterations in centrosome or primary cilium structure or function, we will induce centrosome overduplication and formation of supernumerary primary cilia in cells that have normal TSC1/2 function and test whether these cells mimic TSC1/2-associated phenotypes. In a complementary approach, we will use the BioID proximity labeling method in combination with centrosome purification to identify proteins that are spatially linked to centrosomal TSC1 and TSC2. We will use this approach to create the centrosomal interaction map of TSC1 and TSC2. Ultimately, the proposed work will help in developing diagnostic tools and therapies for TSC that specifically target the TSC1/2 functions that are relevant to the development of TSC phenotypes. In particular, unraveling the functional relationship between mutations in TSC1/2 and disrupted ciliary signaling will open up new routes for TSC treatments that aim to correct these signaling defects, for example by drugs that act as agonists for ciliary signaling receptors. Therefore, harnessing the TSC- centrosome-primary cilium axis for therapeutic purposes may have a significant impact on TSC treatment.
In order to treat patients that suffer from tuberous sclerosis complex (TSC) effectively, we need to know what the two genes mutated in TSC patients, do in normal, healthy cells. In this research project, we will address the question as of whether these two genes carry our some of their function at a small structure in the cell that is important for cell signaling: the centrosome. Ultimately, this research will help to better understand the TSC symptoms and to develop diagnostic tools and therapies for TSC that take advantage of these new connections to cell signaling.
