Muscle disorders: from bench to bedside
For two decades my research has focused on the genetics and pathophysiological processes of hereditary neuromuscular diseases, particularly hereditary inclusion body myopathy (HIBM). The disorder was first characerized in Hadassah more than 30 years ago (Prof Argov, with whom I collaborate throughout the period). My laboratory has identified the gene responsible for this unique disorder characterized by adult-onset, slowly progressive distal and proximal muscle weakness, with a typical muscle pathology. This disease is the most common form of ethnic-related familial degenerative myopathy in Israel, with a prevalence of 1:1500 in the Jewish Iranian (Persian) community, although we now know that it can occur in other communities in the Middle East.The identification in our laboratory of GNE as the gene causing HIBM allowed the recognition of that same disorder worldwide, creating a new classification of this group of diseases, and recently its new denomination worldwide as GNE Myopathy. Furthermore it serves as a model for late onset muscle diseases and was recently used by the Israeli National Bioethic committee for genetic screening guidelines in such conditions.
Our identification of the "Middle Eastern" founder mutation in the GNE, (M743T), has enabled testing for genetic counselling in Israel and abroad.
GNE encodes a bifunctional enzyme which plays a key role in the biosynthetic pathway of sialic acid,an essential molecule involved in many biological and pathological processes. One of the hypotheses for the development of the muscle disease is deficiency of sialylation. As a result, a clinical therapy trial (phase III) was run in 4 centers in the world, the largest at Hadassah (HCRC, Co-Pi Prof Argov and Prof Caraco), by a US based company (Ultragenyx). However, the final results did not show improvement.
Indeed it is not clear if sialic acid deficiency is the only pathophysiological process in GNE Myopathy. Our research is aimed towards the understanding of potential new functions of GNE in muscle, to try and understand and intervene in the pathophysiology of the disease. We investigate different aspects of GNE activity using biochemical and molecular technologies in muscle tissue, and as essential tools for our reseach, we have established muscle cell cultures and animal models (mice and zebrafish), carrying the most frequent mutation in GNE.
These in vitro and in vivo systems specifically designed for a genomic and proteomic approach will provide us with the necessary tools to unravel the mechanisms of GNE protein in normal muscle tissue and possible steps for the eventual correction of the mutation effect in GNE Myopathy.
In addition for the last few years we have developed an Adeno associated virus (AAV) mediated gene therapy platform for GNE Myopathy which we have shown to be efficient and safe in mice models. We have established the translational strategy to go from preclinical to clinical studies. This specific project was supported by the Ministry of Industry and Commerce, through its Kamin program. We are now in the process of submitting a full IND request for gene therapy with this platform in collaboration with Dr Jerry Mendell from the Nationwide Children Hospital in Columbus, Ohio.
GNE Myopathy Muscle histology
Tibialis anterior muscle sections stained with hematoxylin and eosin (H&E) demonstrating (A) variability of muscle fiber size, central nuclei, fiber splitting (arrow) and (B) multiple rimmed vacuoles in periphery and in the center of the atrophic fibers which are also illustrated with the modified Gomori trichrome staininig (C) (original magnification x160). (D) Fibers with vacuoles at the periphery (upper arrow) or at the center with extensions through the length of the fiber (lower arrow). (semithin sections, X200). The pictures were kindly provided by Prof. Zohar Argov (Dept. of Neurology, Hadassah Hospital) and Prof. Dov Soffer (Dept. of Pathology, Hadassah Hospital).
Muscle extracted proteins of normal and GNE Myopathy patients were analyzed by 2D gels using ph4 to pH7 strips. Second dimension was run on 12% acrylamide. Comparative analysis was performed with the Delta 2D Bio Imaging program. Differentially expressed spots were extracted and identified by mass spectrometry.
GNE role in Zebrafish muscle histology
Whole-mount immunostaining of 7 dpf larvae with antibody against slow muscle myosin (F59). Embryos were injected with either 1mM control-MO, 1mM gne E3I3-MO (morphants showing normal/mild or intermediate phenotypes are presented) or co-injected with 40ng/µl gne mRNA + 1mM E3I3-MO. Lateral view, scale bar=50µm. Note the disruption of the myofibers structure after anti GNE morpholinos injection and its rescue by injection of GNE mRNA.
BiFC visualization of the GNE - α-actinin2 interactions
HeLa cells transfected with constructs encoding the WT GNE and α-actinin2 were grown for 24hrs. Cells were fixed and stained with DAPI to visualize the nucleus and analyzed on a fluorescence microscope. BiFC of GNE with α-actinin2; note the unique interaction pattern.
Left to Right:
Hagay Livne, Avi Harazi, Stella Mitrani-Rosenbaum, Miriam Rapport, Esther Kadosh, Lena Yakovlev