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.