In vivo and in vitro genome editing to explore GNE functions

Abstract

GNE myopathy is an adult onset neuromuscular disorder characterized by slowly progressive distal and proximal muscle weakness, caused by missense recessive mutations in the GNE gene. Although the encoded bifunctional enzyme is well known as the limiting factor in the biosynthesis of sialic acid, no clear mechanisms have been recognized to account for the muscle atrophic pathology, and novel functions for GNE have been hypothesized. Two major issues impair studies on this protein. First, the expression of the GNE protein is minimal in human and mice muscles and there is no reliable antibody to follow up endogenous expression. Second, no reliable animal model is available for the disease and cellular models from GNE myopathy patients’ muscle cells (expressing the mutated protein) are less informative than expected. In order to broaden our knowledge on GNE functions in muscle, we have taken advantage of the CRISPR/Cas9 method for genome editing to first, add a tag to the endogenous Gne gene in mouse, allowing the determination of the spatiotemporal expression of the protein in the organism, using well established and reliable antibodies against the specific tag. In addition we have generated a Gne knock out murine muscle cell lineage to identify the events resulting from the total lack of the protein. A thorough multi-omics analysis of both cellular systems including transcriptomics, proteomics, phosphoproteomics and ubiquitination, unraveled novel pathways for Gne, in particular its involvement in cell cycle control and in the DNA damage/repair pathways. The elucidation of fundamental mechanisms of Gne in normal muscle may contribute to the identification of the disrupted functions in GNE myopathy, thus, to the definition of novel biomarkers and possible therapeutic targets for this disease.

GNE deficiency impairs Myogenesis in C2C12 cells and cannot be rescued by ManNAc supplementation

“…In the present study, we established a Gne -knockout cell model, using the murine myoblast cell line C2C12 to study the efficiency of exogenous ManNAc and sialic acid (Neu5Ac) administration in a skeletal muscular background. First, we found that the absence of GNE prevents the differentiation of myoblasts into mature myotubes, as described previously in other studies ( Ilouz et al. 2022 ; Schmitt et al.…”

“…HEK-293 (ACC 305) and C2C12 cells (ACC 565) were purchased from DSMZ (Braunschweig, Germany). Sol8 wild type and Sol8 Gne -knockout cells were kindly provided by Stella Mitrani-Rosenbaum (Hadassah–The Hebrew University Medical Center, Israel) ( Ilouz et al. 2022 ).…”

“…2022 ). Myogenic impairment was also observed in a cell model, using the murine Sol8 skeletal muscle cell line ( Ilouz et al. 2022 ).…”

Generation and characterization of a novel gne Knockout Model in Zebrafish

“…BRAT1 is known to be involved in the cellular response to DNA damage (Ertl et al, 2021). This result is particularly interesting since in a muscle cellular model knocked out for GNE that has been recently established in our laboratory, we also see this DNA damage/repair pathway affected (Ilouz et al, 2022). The human orthologue of si: ch211-198k9.6, is RBBP6 (retinoblastoma tumor suppressor 6binding protein), known to be involved in mRNA processing, cell cycle regulation, and ubiquitination activity (Xiao et al, 2019).…”

Increasing brain N‐acetylneuraminic acid alleviates hydrocephalus‐induced neurological deficits

“…However, there is currently no commercialized pharmacological intervention available to reduce brain Neu5Ac in animal models. There are gene‐editing animal models targeting endogenous Neu5Ac production being developed recently 77–79 . In some of these models, brain Neu5Ac decreases to varying extents and impaired motor function could be observed 77,80 .…”