Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

Abstract

Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

Activation of the Unfolded Protein Response in Sporadic Inclusion-Body Myositis but Not in HereditaryGNEInclusion-Body Myopathy

“…Our results differ from those of Li et al who reported upregulation of the ER chaperones in patients with GNE-h-IBM (46). The discrepancies may be caused by a very limited number of control patients used for some analyses by Li et al, and the fact that their patients had a different genetic background and different GNE mutations from the patients in our studies.…”

“…Our group of patients is very homogenous, i.e. they all carry a homozygous mutation in the kinase domain of GNE , whereas Li et al had a more diverse group of patients (46). It is known that different GNE mutations cause different clinical phenotypes (21, 47).…”

Hereditary inclusion-body myopathies

“…Once an ER stress condition is established, the misfolded and unfolded proteins trapped in the ER are retrotranslocated to the cytoplasm and degraded by either the ubiquitin-proteasome system or through the autophagic process [22]. Indeed, there is evidence that ER stress and the unfolded protein response, two cellular mechanisms intended to cope with abnormal folding and accumulation of proteins, are activated in GNE myopathy muscle (Broccolini and Mirabella, personal observation 2010, and [23]). …”

Proteasome Dysfunction Induces Muscle Growth Defects And Protein Aggregation

“…Thus, we generated conditional Rpt3-knockout mice to specifically block proteasomal activity in skeletal muscle to clarify the role of the proteasomal system in skeletal muscle tissue. Additionally, because the dysregulation of autophagy is involved in the pathogenic mechanisms of several myopathies, such as Pompe disease (Raben et al, 2008), Danon disease (Nishino et al, 2000), VMA21 deficiency (Ramachandran et al, 2013), autosomal dominant inclusion body myopathy associated with Paget's disease of the bone and frontotemporal dementia with valosincontaining protein (VCP) mutation (Watts et al, 2004), GNE myopathy (Li et al, 2013) and collagen VI muscular dystrophy (Grumati et al, 2010), we also investigated morphologically similar anomalies using specific immunohistochemical markers of known myopathies in the conditional Rpt3-knockout mice.…”