The primary finding of the present study is that conditionally induced proteasome inhibition in muscles rapidly causes muscle wasting, formation of rod-like nuclear and fibrillar cytoplasmic aggregates and autophagocytosis. The present study was conducted using mutant proteasome subunits that dominantly block proteasome function, and the effects assessed in the well-characterized Drosophila larval musculature system. The transgenic approach employed here has the benefits of in vivo analyses, specific targeting of proteasome inhibition only within the muscle cells and tight temporal control of the period of proteasome inhibition via induction with the transcriptional activator RU486. Within 6–12 h of RU486 induction of proteasome mutants, there were early indications of muscle atrophy and loss of muscle contractile machinery organization, and ultrastructural evidence of loss of muscle sarcomeric architecture, vacuolization and cytoplasmic aggregate formation. These changes became progressively more severe until, by 24 h, animals were unable to move, showed both gross and ultrastructural muscle disorganization, and had perinuclear accumulation of both polyubiquitinated proteins and a well-characterized indicator of the UPR machinery, GRP78.
In addition, transgenic proteasome inhibition led to a rapid loss of the NMJ SSR. Although the specific function of the SSR is not known, it has been hypothesized to serve to amplify voltage-triggered ionic flux and to be an important organizing centre for postsynaptic densities. The SSR is known to be maintained through the function of DLG (discs large), a muscle membrane-associated PDZ-domain-containing scaffolding protein which shows prominent enrichment in the postsynaptic SSR (Lahey et al., 1994
; Guan et al., 1996
). Although loss of synaptic DLG after proteasome inhibition could explain SSR loss, preliminary experiments suggest that DLG and synaptic function are relatively maintained in the face of dramatic muscle atrophy (K.F.Haas and K. Broadie, unpublished data). It has been shown that the SSR development correlates with the size of the muscle (Lahey et al., 1994
). Thus a similar mechanism could be acting to reduce the SSR during the rapid muscle atrophy caused by proteasome inhibition. In addition to the reorganization of muscle nuclei, this loss of SSR suggests a regression to architectures characteristic of earlier stages in muscle development.
In mammalian studies in vitro
(Lang-Rollin et al., 2004
; Goldbaum et al., 2006
), proteasome inhibition has been shown to trigger apoptotic degeneration in muscle cells. Similarly, in parkin mutant-expressing flies, defects in the function of the UPS lead to gross muscle degeneration (Greene et al., 2003
). In contrast, in the present study, there was no ultrastructural evidence of an apoptotic degenerative process observed in the study by (Greene et al. 2003
), as the nuclear envelope, chromatin pattern and mitochondrial architecture were maintained after proteasome inhibition. Proteasome inhibition for up to 24 h caused severe muscle atrophy, but no detectable loss of muscle cells. Individual muscle cells, likewise, showed no loss of nuclei and ultrastructural analyses indicated preservation of nuclear architecture. Similarly, there was no ultrastructural evidence of compromised mitochondrial architecture. Instead, proteasome inhibition resulted in progressive loss of sarcomere organization with the concurrent appearance of tangled cytoplasmic protein aggregates, as well as the progressive appearance of autophagocytic vacuoles engaged in engulfing these aggregates in addition to cytoplasmic organelles, especially mitochondria. Most tellingly, proteasome inhibition caused the progressive accumulation of polyubiquitinated proteins in punctate perinuclear aggregates, as well as the accumulation of the endoplasmic reticulum chaperone protein GRP78 in the same apparent structures. Taken together, these results strongly suggest that acute proteasome inhibition in the musculature triggers a rapid UPR that may provide a mechanistic cause for the rapid dissolution of muscle architecture and cellular atrophy.
One primary UPS function is to mediate ERAD, a critical pathway for protein quality control that clears unfolded and misfolded proteins (Kostova and Wolf, 2003
; Meusser et al., 2005
). Studies in muscle culture systems suggest that the UPS ERAD pathway may account for >60% of protein degradation, with the remainder of protein degradation processed through other UPS and lysosomal pathways (Purintrapiban et al., 2003
). When the ERAD pathway is overwhelmed, aggregates of unfolded proteins trigger an autophagocytic response, leading to aggresome formation, phagocytosis of protein aggregates and lysosomal degradation (Friedlander et al., 2000
; Iwata et al., 2005
; Yorimitsu et al., 2006
). Thus the results of the present study are consistent with defects caused by disruption of the UPS-dependent ERAD pathway.
The progressive autophagic muscle degradation after genetic proteasome inhibition in this Drosophila
model is similar to the sequence of changes identified in degenerative myopathies. A hereditary form of IBM is caused by mutations in p97/VCP (valosin-containing protein), a chaperone protein involved in the translocation and trafficking of unfolded and misfolded proteins to the proteasome (Weihl et al., 2006
). In muscle biopsies from patients with IBM and myofibrillar myopathies, there is an accumulation and co-localization of multiple proteins involved in the UPR (Ferrer et al., 2004
; Vattemi et al., 2004
). EM analysis of muscle biopsies from patients with myofibrillar myopathies show a very similar progression to the one described in the present study, with loss of normal myofibrillar architecture, accumulation of cytoplasmic filamentous material, aggregation of membranous organelles in vacuoles and autophagic degradation of organelles and fibrillar aggregates (Selcen et al., 2004
). Moreover, the rod-like nuclear aggregates described in the present study are similar to nuclear and cytoplasmic tubulofilamentous aggregates that have been described previously for IBM(Fidzianska and Glinka, 2006
models of sporadic IBM, combining over expression of amyloid-β or amyloid-β precursor protein with proteasome inhibition, display formation of fibrillar aggregates and autophagic muscle cell degeneration (Fratta et al., 2005
; Wojcik et al., 2006
). The inhibition of proteasome function at a time of a high level of protein synthesis during Drosophila
larval development is probably triggering a similar response. This Drosophila
genetic system offers the advantage of a readily malleable in vivo
preparation to further study the-mechanisms of muscle autophagy and the UPR, processes critical to understanding the pathophysiology of degenerative myopathies.