W.K. Engel

In sporadic inclusion body myositis muscle fibres TDP-43-positive inclusions are less frequent and robust than p62 inclusions, and are not associated with paired helical filaments.

Transactive response DNA-binding protein 43 (TDP-43) is a predominantly nuclear RNA/DNA-binding protein, also present in the cytoplasm (reviewed in [1,2]). Its functions are not fully understood, but it was reported to be involved in RNA processing, transcription and exon skipping [1,2]. Recent interest in TDP-43 was provoked by (i) discovering TDP-43-immunoreactive inclusions in the most common subtype of cerebral frontotemporal lobar degeneration (FTLD) associated with ubiquitin-positive inclusions, and in sporadic amyotrophic lateral sclerosis (ALS) [1,2]; and (ii) identification of mainly missense mutations of TDP-43 in familial and sporadic ALS [1,2]. In SOD1-mutant genetic ALS, TDP-43-immunoreactive inclusions are not present [2]. Although it was initially suggested that all ubiquitin-positive, tau-negative FTLD brains contain TDP-43-immunoreactive inclusions, subsequently some ubiquitin-positive, but TDP-43-negative, FTLD cases were identified [3,4]. Among other neurodegenerative diseases, TDP-43-immunoreactive inclusions were also demonstrated in brains of: 20–50% of sporadic Alzheimer disease (AD) brains [5,6], about 14% of familial AD and of Down syndrome, and in Lewy body disease [6,7]. In AD brain TDP-43-immunoreactive inclusions are usually considered secondary pathology [2]; in most cases they were immunonegative for phosphorylated tau and not associated with neurofibrillary tangles (NFTs) [5–8]. Also in contrast to FTLD-TDP brains, which contain a low molecular weight, 25 kDa TDP-43 cleaved fragment, which has been considered pathogenic and linked to TDP-43 cytotoxicity [1,9], AD brains do not contain this fragment [10]. p62, also known as sequestosome-1 (SQSTM1), is a ubiquitin-binding protein that is a common component of ubiquitinated multiprotein inclusions in protein aggregation disorders of the central nervous system, including AD and FTLD [11–13]. p62 is considered an integral component of NFTs in AD brain. In NFTs, p62 co-localizes with phosphorylated tau and is considered to participate in tau phosphorylation [12,14]. Because of the prominent presence of p62 in brains of patients with various neurological disorders, p62 immunostaining was recommended for neuropathological diagnosis [13]. Sporadic inclusion body myositis (s-IBM) is the most common muscle disease of persons age 50 years [15,16]. Characteristics of the s-IBM muscle biopsy are mononuclear cell inflammation and muscle fibres containing autophagic vacuoles (reviewed in 15–17). s-IBM abnormal muscle fibres, vacuolated and nonvacuolated, have accumulated ubiquitin-positive multiprotein aggregates containing misfolded proteins in the b-pleated sheet conformation of congophilic amyloid [15–17]. Intriguingly, s-IBM muscle fibres have several phenotypic similarities with brain tissue of AD and Parkinson disease Lewy bodies. These include accumulated aggregated amyloid-b (Ab), phosphorylated tau (p-tau) in the form of paired helical filaments (PHFs), which form intramuscle fibre bundles very similar to AD intraneuronal PHFs, accumulated a-synuclein and several other proteins (recently reviewed in 15,16). Inclusions immunoreactive for p62 were prominently accumulated in muscle fibres of all s-IBM muscle biopsies studied – consequently immunostaining for p62 was proposed as a pathological diagnostic marker for s-IBM [18]. In s-IBM muscle biopsies, most of the p62-immunopositive inclusions were linear or squiggly, and all co-localized with p-tau by light microscopy [18]. By immunoelectronmicroscopy, in s-IBM both p62 and p-tau were specifically associated with PHFs [18]. p62 was not immunoreactive in muscle fibre nuclei or in nuclei of inflammatory or connective tissue cells. In s-IBM muscle fibres p62 was significantly increased both on the protein and mRNA levels [18]. Transactive response DNA-binding protein 43immunoreactive cytoplasmic inclusions within s-IBM muscle fibres were first reported by Weihl et al. [19] and

Chaperone-mediated autophagy components are upregulated in sporadic inclusion-body myositis muscle fibres

Sporadic inclusion‐body myositis (s‐IBM) is an age‐associated degenerative muscle disease. Characteristic features are muscle‐fibre vacuolization and intramuscle‐fibre accumulations of multiprotein aggregates, which may result from the demonstrated impairments of the 26S proteasome and autophagy. Chaperone‐mediated autophagy (CMA) is a selective form of lysosomal degradation targeting proteins carrying the KFERQ motif. Lysosome‐associated membrane protein type 2A (LAMP2A) and the heat‐shock cognate protein 70 (Hsc70) constitute specific CMA components. Neither CMA components nor CMA activity has been studied in normal or disease human muscle, to our knowledge.

Myostatin precursor protein is increased and associates with amyloid-β precursor protein in inclusion-body myositis culture model

Myostatin, also called growth and differentiation factor-8, is a member of the transforming growth factor-b superfamily [1,2]. Myostatin is a secreted protein considered a negative regulator of muscle growth during development and of muscle mass during adulthood [1]. In mouse models, knocking out the myostatin gene, overexpressing proteins neutralizing myostatin, or natural mutations of the myostatin gene cause increased muscle mass [1]. In cattle, naturally occurring myostatin gene mutations leading to inactive protein cause ‘double-muscle cattle’ [2]. Recently reported was a child in whom a homozygous myostatin gene mutation that results in reduced production of myostatin protein, was associated with increased muscle bulk and strength [3]. Conversely, mature myostatin protein has been reported increased in muscle tissue of patients with HIV-associated muscle wasting [4], and increased myostatin-precursor protein (MstnPP) mRNA reported in muscle wasting associated with osteoarthritis [5]. Within muscle fibres, myostatin is synthesized as a MstnPP [6,7]. MstnPP and its mRNA, and mature myostatin, are predominantly expressed in skeletal muscle tissue (reviewed in [1]). MstnPP, a 375-amino-acid protein translated from a 3.1 kb mRNA [4], consists of three structural domains: a signal sequence; an N-terminal 28 kDa propeptide, also referred to as latency associated peptide [7]; and a C-terminal 12 kDa mature myostatin peptide [6,7]. Intracellular processing of myostatin from MstnPP has been proposed to occur through furin [6,8]. We recently showed in biopsied sporadic inclusion-body myositis (s-IBM) muscle fibres that both MstnPP and myostatin dimer were significantly increased, and MstnPP was physically associated with amyloid-b precursor protein (AbPP) [9]. Moreover, by lightand electron-microscopic immunocytochemistry, MstnPP/myostatin colocalized with amyloid-b (Ab)/AbPP [9]. s-IBM is severely progressive, the most common muscle disease of older persons, and there is no successful treatment [10]. Histological hallmarks of s-IBM include: (1) vacuolar degeneration and atrophy of muscle fibres, accompanied by intramuscle-fibre accumulations of ubiquitinated protein aggregates, including Ab/AbPP; (2) muscle-fibre atrophy; and (3) mononuclear lymphocytic inflammation [10–12]. Recently demonstrated in s-IBM muscle fibres were inhibition of 26S proteasome activity and presence of aggresomes [13]. Accumulation of AbPP/Ab appears to be an early upstream step in the s-IBM pathogenesis, because: (i) abnormal accumulation of AbPP epitopes appears to precede other abnormalities in IBM muscle fibres [11]; and (ii) several aspects of the s-IBM phenotype, including Ab accumulation, proteasome inhibition and aggresome formation, were produced in cultured normal human muscle fibres (CHMFs) after long-term overexpression of AbPP in them [13–15]. The latter provides a useful IBM human-muscle tissue-culture model. The aim of the present study was to utilize this model to investigate possible mechanisms responsible for increased MstnPP and myostatin within the biopsied s-IBM muscle fibres. We cultured human muscle fibres from satellite cells obtained from six normal diagnostic muscle biopsies, as described [13–15] and referenced therein. Into well-differentiated 3-week-old cultured muscle fibres we transferred a 3 kb human AbPP-cDNA encoding 751-AbPP using a replication-deficient adenovirus vector at 0.3 ¥ 10 pfu/ml culture medium, as detailed previously [14,15]. In addition, we treated some of the cultures with 1 mm epoxomicin (Biomol Research Laboratories, Plymouth Meeting, PA, USA) [13], an irreversible proteasome inhibitor [16]. Four days after AbPP gene transfer and 24 h after epoxomicin treatment, control and AbPP-overexpressing CHMFs (AbPP+ CHMFs) were processed for lightand electron-microscopic immunocytochemistry, immunoblotting, combined immunoprecipitation/immunoblotting, and reverse transcriptase polymerase chain reaction (RT-PCR), as described [13–15,17]. For all the myostatin studies, we used an anti-myostatin rabbit polyclonal antibody (Chemicon,