inclusion body myositis
Amyloid formation is a predominant feature of sporadic inclusion body myositis (sIBM) (Askanas and Engel 2008) and involves the Alzheimer’s disease protein Aβ (Wojcik, Engel et al. 2005; Vattemi, Nogalska et al. 2009). A role for MstnPP in sIBM was proposed after co-localisation and direct association with Aβ was observed in diseased cells (Wojcik, Engel et al. 2005), and because ER stress causes an upregulation of myostatin expression (Nogalska, Wojcik et al. 2007). The results presented here show that misfolded human MstnPP aggregates share the morphological characteristics of amyloid oligomers and the ability to bind the amyloid-specific dye thioflavin T. The α-helical predominant secondary structure of these aggregates suggests they are likely to represent an intermediate species that occurs in the transition between native protein and β-sheet-rich protofibrils. When subjected to the mildly denaturing conditions of pH 5.3 and 60 °C, the aggregates form amyloid protofibrils and after one week, are able to form long, linear and unbranching amyloid fibrils. At 37 °C, β-sheet rich protofibrils form over an increased time period. These in vitro results show that MstnPP is capable of amyloid protofibril and fibril formation, supporting a role for misfolding of the myostatin precursor in the pathogenesis of sIBM. Furthermore, MstnPP aggregates and protofibrils have a cytotoxic effect on mouse myoblasts when added to the culture medium.
9.1.1 Implications for the folding of MstnPP in vivo
After in vitro refolding of the myostatin precursor protein, misfolded yet soluble aggregates that form amyloid protofibrils and fibrils at a lowered pH, can be observed. Although elevated temperature has been used to produce mature MstnPP fibrils quickly in this study, a physiological temperature allows protofibril formation over an extended time period; by comparison, fibril formation is expected to occur over many years in vivo and sIBM is considered to be a disease of the elderly (Garlepp and Mastaglia 2007; Karpati and O'Ferrall 2009).
Growth factors such as myostatin and most other secreted proteins are folded in the ER. A major obstacle to folding of native TGF-β family proteins is the formation of the correct disulphide-bonded cysteine pairs (Heiring and Muller 2001; Muller and Heiring 2002); therefore, the correct redox environment and assistance from disulphide bond chaperones is critical. The myostatin propeptide has been proposed to have a chaperone-like function in the correct folding of the growth factor domain (Jin, Dunn et al. 2004; Funkenstein and Rebhan 2007). During ER-stressed situations, such as an altered redox environment, protein misfolding can be extensive (Pratico 2008; Schroder 2008). Although the unfolded protein response (UPR) results in the elimination of misfolded proteins and/or apoptosis if necessary (Paschen 2003; Lai, Teodoro et al. 2007; Schroder 2008), if the production of misfolded proteins overwhelms the UPR, protein aggregation may lead to amyloid formation (Bucciantini, Giannoni et al. 2002; Bucciantini, Calloni et al. 2004).
It is hypothesised that in an ER-stressed situation, newly-translated MstnPP may have the potential to misfold and form similar aggregates to those now observed in vitro. Although in most situations the UPR will function to return the ER to homeostasis thereby eliminating misfolded myostatin, overwhelming stress may promote the aggregation and structural rearrangement of misfolded forms of myostatin, allowing amyloid association and protofibril formation. This hypothesis is made more complex by the observation that the correctly folded MstnPP dimer is also able to form amyloid aggregates and fibrils (Fig. 8.11). These results imply that in an ER-stressed situation, MstnPP that has already undergone chaperone-assisted folding may also be capable of partial denaturation and structural rearrangements that allow amyloid formation.
9.1.2 Implications for sporadic inclusion body myositis
A role for myostatin and/or MstnPP in the pathogenesis of sIBM was suggested in 2005 when it was shown in 12 sIBM biopsies that myostatin/MstnPP was accumulated within muscle fibers and colocalised with Aβ (Wojcik, Engel et al.
2005). A subsequent study showed similar results in an inclusion body myositis culture model (Wojcik, Nogalska et al. 2007), and that overexpression of AβPP/Aβ
promoted the accumulation and aggresomal targeting of MstnPP. Further research suggested that MstnPP expression, at both the mRNA and protein level, was increased following ER stress in cultured human muscle fibres (Nogalska, Wojcik et al. 2007). This upregulation is most likely mediated by NF-κB; MstnPP has an NF-κB consensus site in its promoter and upregulation was not observed in the presence of NF-κB inhibitors (Nogalska, Wojcik et al. 2007). Furthermore, treatment with the anti-oxidant resveratrol reduced both NF-κB activation and myostatin expression (Nogalska, D'Agostino et al. 2008).
How might myostatin/MstnPP be involved in the pathogenesis of sIBM?
Increased activity of the myostatin growth factor
One possibility is that an increase in the level of circulating myostatin growth factor, due to increased expression and processing of MstnPP, contributes to the prominent muscle-fiber atrophy (Zimmers, Davies et al. 2002) as well as the diminished potential of satellite cells to proliferate (Karpati and O'Ferrall 2009). However, the observation that MstnPP is present as amorphous aggregates (Wojcik, Engel et al.
2005) and the possible aggresomal localisation (Wojcik, Nogalska et al. 2007) may inhibit processing by furin convertase. Aggresomes have been proposed to have a cytoprotective function, serving as recruitment centres to facilitate the degradation of toxic proteins (Taylor, Tanaka et al. 2003). Aggregated MstnPP may be targeted for degradation by the ubiquitin pathway, which is upregulated by the UPR (Schroder 2008), supporting a reduction in active growth factor levels.
Furin cleavage of soluble aggregates was not attempted in this thesis. However, after furin digestion, a proportion of undigested MstnPP was often observed despite the addition of extra furin (Chapter 3), which may represent misfolded precursor protein. It has been suggested that putative disorder in the region of the polypeptide chain immediately N-terminal to the furin cleavage site assists in access of furin to the polypeptide chain (Chapter 5); if this is the case, and aggregation of MstnPP results in an altered structure in this area, furin cleavage may be inhibited.
MstnPP aggregation
Post-translational modification of MstnPP, perhaps through association with Aβ/AβPP, may lessen its degradation and traffic, resulting in MstnPP accumulation
and an increase in ER stress (Wojcik, Nogalska et al. 2007). One scenario is that AβPP/Aβ accumulation precedes that of MstnPP at the protein level, as AβPP overexpression does not increase MstnPP mRNA. Another suggestion is that MstnPP negatively influences Aβ and other proteins to cause their oligomerization (Wojcik, Engel et al. 2005).
Is it possible that MstnPP is acting independently of Aβ in sIBM? The results presented in Chapter 8 suggest that MstnPP misfolding and amyloid aggregation may have a negative effect on cell viability without an effect on, or stimulation from, Aβ. The milieu of different events that occur in amyloid disease suggest that no aspect is completely in isolation and each has an influence on the others. Protein misfolding is both promoted by and contributes to ER stress and the production of reactive oxygen species (ROS) that act negatively on both ER and mitochondrial function (Schroder 2008). The exact cause of sIBM is unknown although ER stress is a common theme underlying degenerative disease (Paschen 2003). A likely scenario, and the suggestion favoured here, is that ER stress causes the misfolding and amyloid-like aggregation of newly-translated and/or chaperone-folded myostatin. Cytotoxicity assays in cultured mouse myoblasts show that aggregated myostatin has a negative effect on cell viability (Fig. 8.12); therefore, misfolded myostatin may contribute to sIBM via a cytotoxic mechanism.
9.1.3 MstnPP cytotoxicity
The cytotoxicity exhibited by MstnPP aggregates and protofibrils in C2C12 cells supports a mechanism in which aggregation by MstnPP in sIBM contributes to muscle degeneration; this may occur independently of atrophy that may result from increased myostatin growth factor signalling. The in vitro assays investigate the effect of extracellular protein on cell viability. In sIBM tissue and cell culture models, MstnPP aggregates were localised to the cytoplasm, aggresomes and nuclear regions (Wojcik, Engel et al. 2005; Wojcik, Nogalska et al. 2007). However, an absence of extracellular MstnPP was not shown. Since the myostatin precursor protein is secreted (Anderson, Goldberg et al. 2008), post-secretion aggregation or secretion of aggregated/misfolded MstnPP may contribute to muscle fiber atrophy in sIBM. Extracellular cytotoxicity studied for other aggregated proteins involves mechanisms
such as alterations to endo- and exocytosis (Walsh, Hartley et al. 1999), alterations to Ca2+ homeostasis and the generation of reactive oxygen species (Bucciantini, Calloni
et al. 2004). The presence of pore-like structures for MstnPP under TEM suggests that the cytotoxic mechanism may include membrane permeabilization (Lashuel 2005), the favoured hypothesis for amyloid oligomer cytotoxicity. A number of models place membrane permeabilization upstream of processes such as Ca2+ dyshomeostasis and ROS production (Glabe 2006).
Alternatively, MstnPP aggregates may exert toxic effects from an intracellular locality, with the observed cytotoxicity in cell culture due to internalisation of protein (Bucciantini, Calloni et al. 2004). Localisation in aggresomes suggests that the myostatin aggregates are targeted for degradation; if cell homeostasis is perturbed and degradation is not carried out, enhanced aggregation by MstnPP may add to cell stress and contribute to disease in this way. Although localisation to the ER was not shown, it was also not discounted; MstnPP aggregation within the ER may have a similar effect. The observation of MstnPP aggregates in the nucleus (Wojcik, Engel et al.
2005) is intriguing as a nuclear role for MstnPP or any TGF-β family member has not been shown to date. These results, coupled with the cytoplasmic staining shown for MstnPP (Wojcik, Nogalska et al. 2007), suggest that trafficking of the MstnPP protein may be altered in sIBM, a scenario that may be due to, or promote, MstnPP aggregation.
In any case, both extra- and intracellular cytotoxicity may occur via membrane permeabilization. The disruption of nuclear, mitochondrial, ER and Golgi membranes may occur in addition to that of the cell.
ER stress-induced upregulation of MstnPP
ER stress induces an upregulation of MstnPP at both the mRNA and protein levels, most likely via activation of NF-κB (Nogalska, Wojcik et al. 2007). However, it is not clear why, in a situation where ER homeostasis is perturbed, a protein requiring oxidative folding and Ca2+-regulated processing is upregulated.
As the goal of the UPR is to halt cell processes until homeostasis has been restored, an upregulation of MstnPP may occur to increase myostatin growth factor levels. Signalling by the myostatin growth factor may assist via upregulation of p21 and halting of the cell cycle (Thomas, Langley et al. 2000), locally inhibiting cell growth and/or development until homeostasis is restored. ER stress responses include both
protective and apoptotic mechanisms (Schroder 2008); an upregulation of myostatin is most likely protective. In a cultured sIBM cell model, NF-κB activation of MstnPP and the ER stress markers Grp78 and Herp was not accompanied by pro-apoptotic cleavage of procaspase-3, suggesting that the protective pathway was active (Nogalska, Wojcik et al. 2007). As there is currently no evidence to support these suggestions, further investigation is required to understand the mechanisms involved here.