Elucidating the Effect of Myopathy-Causing Mutations and Second-Site Suppressors on Client Processing by J-Domain Proteins

Defects in protein quality control may lead to protein misfolding and aggregation often associated with protein conformational disorders such as Alzheimerճ Disease and Limb Girdle Muscular Dystrophy, among others. Molecular chaperones protect against protein misfolding and aggregation. A chaperone of interest is the ubiquitously expressed type II Hsp40 co-chaperone DNAJB6, which assists in protein folding and disaggregation. Mutations within the DNAJB6 G/F domain have been associated with the dominantly inherited disease Limb-Girdle Muscular Dystrophy type 1D (LGMD1D), now referred to as LGMDD1. Our collaborators recently discovered novel LGMDD1-associated mutations in the J-domain of DNAJB6. In the enclosed body of work, we used yeast as a model to perform phenotypic, biochemical and functional assays to elucidate the effect of the J-domain mutations on canonical chaperone function with the goal of beginning to understand how mutations in this domain may affect LGMDD1 pathogenesis. Moreover, we have identified second-site suppressors that rescue a viability defect in yeast that is associated with a myopathy-causing mutation. With this work we have begun to assess the ways in which second-site suppressors may be therapeutic for inherited myopathies such as LGMDD1. The heat shock response is a highly conserved program from yeast to mammals, thus, we have used a yeast model system to study disease-causing mutations. The yeast type II Hsp40 co-chaperone, Sis1, is homologous to DNAJB6 and has an important role in yeast for the propagation of two yeast prions, [RNQ+] and [PSI+]. The True lab has previously published work showing that when LGMDD1-associated mutations in the G/F domain are present in Sis1, its client processing function is altered. Since novel J-domain mutations have yet to be characterized, we assessed the effect of these mutations using our yeast model. Here, we provide evidence that novel variants in the Hsp40 J-domain lead to aberrant chaperone function and altered protein homeostasis in a client and conformer specific manner. Moreover, we identified a novel client-dependent viability defect when one of the J-domain mutants is expressed. This is the first time, to our knowledge, that steady-state levels of a mutated chaperone have been shown to be dependent on stabilization by a client. Lastly, we have identified and began to characterize second-site suppressors which may lead future studies into using second-site suppressors for therapeutic purposes. This body of work enables direct comparisons between disease-associated mutants in different domains so that we may begin to not only understand how LGMDD1 mutants could impact disease severity and pathogenesis, but also whether similar therapeutic avenues could be explored to treat patients with different mutations in the future

Client processing is altered by novel myopathy-causing mutations in the HSP40 J domain

The misfolding and aggregation of proteins is often implicated in the development and progression of degenerative diseases. Heat shock proteins (HSPs), such as the ubiquitously expressed Type II Hsp40 molecular chaperone, DNAJB6, assist in protein folding and disaggregation. Historically, mutations within the DNAJB6 G/F domain have been associated with Limb-Girdle Muscular Dystrophy type 1D, now referred to as LGMDD1, a dominantly inherited degenerative disease. Recently, novel mutations within the J domain of DNAJB6 have been reported in patients with LGMDD1. Since novel myopathy-causing mutations in the Hsp40 J domain have yet to be characterized and both the function of DNAJB6 in skeletal muscle and the clients of this chaperone are unknown, we set out to assess the effect of these mutations on chaperone function using the genetically tractable yeast system. The essential yeast Type II Hsp40, Sis1, is homologous to DNAJB6 and is involved in the propagation of yeast prions. Using phenotypic, biochemical, and functional assays we found that homologous mutations in the Sis1 J domain differentially alter the processing of specific yeast prion strains, as well as a non-prion substrate. These data suggest that the newly-identified mutations in the J domain of DNAJB6 cause aberrant chaperone function that leads to the pathogenesis in LGMDD1

Metabotropic glutamate receptor 2/3 (mGluR2/3) activation suppresses TRPV1 sensitization in mouse, but not human sensory neurons

AbstractThe use of human tissue to validate putative analgesic targets identified in rodents is a promising strategy for improving the historically poor translational record of preclinical pain research. We recently demonstrated that in mouse and human sensory neurons, agonists for metabotropic glutamate receptors 2 and 3 (mGluR2/3) reduce membrane hyperexcitability produced by the inflammatory mediator prostaglandin E2(PGE2). Previous rodent studies indicate that mGluR2/3 can also reduce peripheral sensitization by suppressing inflammation-induced sensitization of TRPV1. Whether this observation similarly translates to human sensory neurons has not yet been tested. We found that activation of mGluR2/3 with the agonist APDC suppressed PGE2-induced sensitization of TRPV1 in mouse, but not human, sensory neurons. We also evaluated sensory neuron expression of the gene transcripts for mGluR2 (Grm2), mGluR3 (Grm3), and TRPV1 (Trpv1). The majority ofTrpv1+mouse and human sensory neurons expressedGrm2and/orGrm3, and in both mice and humans,Grm2was expressed in a greater percentage of sensory neurons thanGrm3. Although we demonstrated a functional difference in the modulation of TRPV1 sensitization by mGluR2/3 activation between mouse and human, there were no species differences in the gene transcript colocalization of mGluR2 or mGluR3 with TRPV1 that might explain this functional difference. Taken together with our previous work, these results suggest that mGluR2/3 activation suppresses only some aspects of human sensory neuron sensitization caused by PGE2. These differences have implications for potential healthy human voluntary studies or clinical trials evaluating the analgesic efficacy of mGluR2/3 agonists or positive allosteric modulators.</jats:p

Optogenetic silencing of nociceptive primary afferents reduces evoked and ongoing bladder pain

Abstract Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) suffer from chronic pain that severely affects quality of life. Although the underlying pathophysiology is not well understood, inhibition of bladder sensory afferents temporarily relieves pain. Here, we explored the possibility that optogenetic inhibition of nociceptive sensory afferents could be used to modulate bladder pain. The light-activated inhibitory proton pump Archaerhodopsin (Arch) was expressed under control of the sensory neuron-specific sodium channel (sns) gene to selectively silence these neurons. Optically silencing nociceptive sensory afferents significantly blunted the evoked visceromotor response to bladder distension and led to small but significant changes in bladder function. To study of the role of nociceptive sensory afferents in freely behaving mice, we developed a fully implantable, flexible, wirelessly powered optoelectronic system for the long-term manipulation of bladder afferent expressed opsins. We found that optogenetic inhibition of nociceptive sensory afferents reduced both ongoing pain and evoked cutaneous hypersensitivity in the context of cystitis, but had no effect in uninjured, naïve mice. These results suggest that selective optogenetic silencing of nociceptive bladder afferents may represent a potential future therapeutic strategy for the treatment of bladder pain