Dysregulation of the unfolded protein response of the ER in nemaline myopathy

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Project Summary Hallmarks of nemaline myopathy (NM) are electron dense rods in myofibers, muscle weakness, and lack of muscle regeneration (Sanoudou et al., 2006; Wallgren-Pettersson et al., 2011). Twelve genes have been closely linked to NM (Jungbluth et al., 2018). Despite our advanced understanding of NM that defects of the sarcomeric thin filament cause sarcomeric weakness, it still remains unclear how these structural flaws trigger muscle atrophy and defective muscle regeneration. There is, therefore, an urgent need to identify the mechanisms by which the NM-linked molecules influence muscle growth and survival. Our long-term goal is to understand roles of the endoplasmic reticulum (ER) in human development. We recently uncovered a novel activity of CUL3-KLHL41, a NM-linked ubiquitin ligase complex, that it regulates the sensors of the unfolded protein response (UPR) of the ER (Kim et al., 2018). In particular, the CUL3-KLHL41 complex strongly regulates the PERK signaling pathway of the UPR in C2C12 myotubes. The UPR plays a critical role in muscle growth/regeneration and has been implicated in congenital myopathies (Bohnert et al., 2018; Ebert et al., 2012; Miyake et al., 2017; Zhang et al., 2002). However, UPR dysregulation has not been examined in NM until now. Thus, we are in a unique position to reveal a new connection among CUL3, the UPR, and NM. The objective of this application is to define how CUL3-KLHL41 and other CUL3 adaptor molecules (i.e., KLHL40, KBTBD13, etc.) regulate the UPR in muscles. Our central hypothesis is that CUL3 adaptor molecules and possibly other NM-linked molecules regulate muscle growth via the UPR. The rationale that underlies the proposed research is that once we achieve the goal we will be able to provide a new concept for pathogenesis, diagnosis, and new treatment approaches for NM. To objectively test the hypothesis, we will pursue the following specific aims: 1) Establish the mechanism by which CUL3 regulates the UPR in myotubes; 2) Identify additional CUL3 adaptor genes whose mutations cause a myopathy in zebrafish. Under the first aim, we will identify muscle- specific substrates of CUL3-KLHL41 that regulates the UPR in C2C12 myotubes. We will employ proven RNAi methodology and evaluate changes in the levels of PERK. For the second aim, we will determine PERK dysregulation in vivo in stable klhl41 knockout zebrafish lines. Additionally, we will employ RNAi methodology to screen 51 muscle-specific putative CUL3 adaptor molecules (Deshmukh et al., 2015) for PERK regulation in C2C12 myotubes. Top three candidates will be evaluated for PERK dysregulation in zebrafish. Our proposed research is innovative, in our opinion, because the notion that aberrant UPR is an underlying mechanism of pathological atrophy in NM is new and unexplored. This knowledge is significant because while defects of the thin filaments of the sarcomere are difficult to restore, the UPR is amenable to pharmacological interventions. Thus, our research will lay a foundation for new pharmacological interventions of NM.