The overall goal of research in the Wolfrum lab is to elucidate the function and dysfunction of vertebrate sensory cells, respectively, in health and disease. Our studies aim to understand the basic cellular mechanisms by which molecules of the cytoskeleton and protein complexes contribute to the function and viability of sensory cells. In our research, we focus on wing-like sensory cells: Olfactory cells in the nose, hair cells in the inner ear, but especially photoreceptor cells in the retina. To this end, we use multidisciplinary approaches that combine molecular biology and transcriptomics with protein biochemistry, proteomics, and cell biology using appropriate cell models and relevant animal models. A main expertise in the lab is immunocytochemistry by epifluorescence, confocal, STED microscopy and transmission electron microscropy, which we use as analytical tools for the spatial localization of molecules in cells and tissues.

The data from our basic research enable us to perform well-grounded translational work on retinal ciliopathies with a focus on the human Usher syndrome. In this regard, we aim to elucidate the underlying mechanisms of retinal degeneration and our therapy team led by Kerstin Nagel-Wolfrum assesses preclinical gene-based therapeutic strategies.

Main research themes:

I. Analysis of transport complexes in sensory cells.

Sensory cells are specialized neurons, which display an extraordinary high activity. In rod and cone photoreceptor cells, the photo-transductive disc membranes in the outer segments are continually renewed throughout lifetime. For the maintenance of photoreceptor cell function effective and well-regulated robust transport processes are necessary. In cellular systems, intracellular transport processes function in close association with the cytoskeleton. In our lab, we study the cellular function of these transport complexes, e.g., molecular motor proteins (myosin VIIa, cytoplasmic dynein) and intraflagellar transport (IFT) molecules as well as the associated structural cytoskeletal components in primary ciliated cells and photoreceptor cells. Since, molecules related to the human Usher syndrome and other retinal ciliopathies are associated with transport modules this research theme is strongly interconnected with the second theme.

 

II. Deciphering the cellular function of proteins and protein networks related to the human Usher syndrome (USH) and other retinal ciliopathies.

The human Usher syndrome (USH) is the most frequent cause of combined deaf-blindness. In human, the loss of vision and hearing is one of the most feared handicaps of all; affecting not only individual social interactions and independency, but also the possibility to uphold stimulating professional or domestic activities, and thus influences the very core of life. Currently, there are rehabilitative treatments for hearing loss with hearing aids or cochlear implants, but no treatment for vision loss. While the genetic defects leading to USH are widely understood, the molecular function of the USH proteins and mechanisms that cause the photoreceptor dysfunction and degeneration are almost unknown.

USH is genetically heterogeneous with at least 12 chromosomal loci assigned to three clinical types, USH1-3. Although these USH types exhibit similar phenotypes in human, the corresponding gene products belong to very different protein classes and families.

The main goal of our USH team is to elucidate the functions of USH proteins in the cellular context and to decipher protein networks related to USH - the USH interactome. Our analyses include the role of USH proteins at the connecting cilium, at synapses as well as protein networks associated with cell adhesions and intracellular transport of photoreceptor cells. Since recent work highlighted that USH molecules are additionally highly expressed in Müller glia cells we also include research on their functions in these redial glia cells.

From our results, we expect fundamental insights not only into the cellular function of USH molecules in health but also into the mechanisms, which lead to the cellular defects causing USH. These data are prerequisites for founded therapeutic strategies for the treatment and cure of USH, which are in the centre of the translational research of our therapy team.

 

Specific projects:

Deciphering the function of the orphan aGPCR VLGR1/ADGRV1 - USH2C

The very large G protein-coupled receptor 1 (VLGR1/ADGRV1) is a member of the adhesion G protein-coupled receptor (ADGR) family and one of the largest surface receptor in human, characterized by the common 7-transmembrane receptor domain and an extraordinary long extracellular adhesion domain. Mutations in VLGR1/ADGRV1 cause the human Usher syndrome (USH2C) and its haploinsufficience have been also linked to childhood absence epilepsy. In the absence of tangible knowledge of the molecular function and signaling of VLGR1, the pathomechanisms underlying these diseases are still unknown. Applying affinity proteomics, we have identified numerous novel potential binding partners and ligands of VLGR1 indicative for different pathways and functions of VLGR1. The obtained data paved the way for three research projects on VLGR1 (USH2C):

a. VLGR1 signaling at focal adhesions.

b. The role of VLGR1 in the formation of autophagosomes.

c. The function of VLGR1 at mitochondria-ER contact sites (MERCSs or MAMs).

 

Elucidating the divers functions of the multipotent scaffold-protein SANS - USH1G

The USH1G protein SANS (scaffold protein containing ankyrin repeats and SAM domain) is a small scaffold protein. Its numerous interaction partners, identified by “fishing expeditions”, such as yeast-2-hybrids or affinity proteomics tandem, define the different cellular functions of SANS. For further enlightening these functions, we carry on following studies:

a. SANS as part of intracellular transport modules

Our data indicate that SANS is a component of transport carriers, which transport cargoes along microtubule tracks from the trans-Golgi network (TGN) to the base of the cilium in photoreceptor cells. At the ciliary base SANS controls MAGi2-mediated endocytoses and the ciliary import of the microtubule depolymerizing kinesin KIF2A.

 

b. SANS as a regulator of pre-mRNA splicing

We have shown that SANS regulates splicing of pre-mRNA by interacting with key spliceosome components and controlling the transfer of the tri-snRNP spliceosome subcomplex from the Cajal bodies to the splicing speckles. Defects in USH1G/SANS or its deficiency can lead to defective splicing of target genes. Current research focuses on identifying genes whose mRNA splicing is affected by USH1G/SANS dysfunction. Initial data indicate that other USH and USH-related genes are affected.

c. The role of SANS in the dynamics of membrane-less organelles derived by phase separation

The phenomenon of liquid-liquid phase separation (LLPS) enables the compartmentalization of biopolymers into membrane-less organelles (MLOs). Cellular processes can proceed and be regulated within these compartments by increasing the local concentration of the molecular constituents and modulating their structure and dynamics. In our project we aim to decipher the roles of SANS in the formation of polymer condensates in MLOs at the base of primary cilia and in the nucleus, namely the Cajal bodies and the splicing speckles derived by LLPS.

 

Unraveling the retinal function of harmonin - USH1C

The USH1C/harmonin is a scaffold protein consisting of one harmonin homology, three PDZ, coiled-coil, and one proline-serine-threonine-rich (PST) domain through which numerous proteins including all other USH proteins bind. Extensive alternative splicing modulates harmonin´s modular composition and thus its molecular function in the cell. In the retina, specific harmonin isoforms are expressed in the photoreceptor cells and Müller glia cells where they are localized in the photosensitive outer segments, calyceal processes, synapses and in adhesions complexes, respectively. We study harmonin´s functions in vitro and in cells, e.g., cells derived from patients or animal models and in vivo in our USH1C pig model.

Generation, establishment and phenotypical characterization of porcine models for human Usher syndrome - USH1 pig models

Animal models are essential not only for unraveling the mechanistic understanding of protein functions and the mechanisms leading to diseases but also for preclinical evaluation of therapeutic options. The lack of an adequate animal model for the ocular phenotype in USH is the main reason why, to date, there is no therapy for vision loss and why the molecular function of USH proteins in the retina and the mechanisms causing retinal degeneration are still largely unknown.

All mouse models studied so far show if at all only a relatively mild visual deficit, which is probably due to structural differences in retinal cells between man and mice. Given the large molecular, morphologic and physiologic similarities between pig and human eyes, we have decided to establish large animal pig models for USH. In collaboration with Nik Klymiuk´s lab, Munich, we engineered a humanized knock-in pig model for USH1C by introducing the human USH1C exon 2 bearing the disease-causing nonsense mutation p.R31*. This USH1C^p.R31* pig model displays all three phenotypical hallmarks of USH1 in patients comprising pronounced hearing loss, vestibular dysfunction, and, more importantly, significantly impaired vision in the first year of life. Since the ocular phenotype found is very similar to that in USH1C patients, the USH1C pig model represents the first true animal model for USH1. Preliminary data prove the suitability of this model for preclinical evaluation of ocular gene therapy and other treatment options. Besides on-going translational work we utilize the USH1C^p.R31* pig model to analyze harmonin functions in retinal cells.

As a second pig model we currently analyze a naturally occurring USH1B/MYO7A pig model bearing a biallelic nonsense mutation (p.Q181*) in the MYO7A gene. This project is in close collaboration with Michael Wendt, Hanover, and Doris Höltig, Berlin. We will make use of this model to define the mechanisms underlying the visual dysfunction in USH1B and to provide a large animal for preclinical evaluation of therapies for USH1B patients.

For most of our projects, recent reviews are available which are indicated in the list of publications. Our projects are embedded in international collaborations as well as in scientific networks at JGU Mainz.