Janis McFerrin

Proteasome Inhibition and Aggresome Formation in Sporadic Inclusion-Body Myositis and in Amyloid-β Precursor Protein-Overexpressing Cultured Human Muscle Fibers

The 26S proteasome system is involved in eliminating various proteins, including ubiquitinated misfolded/unfolded proteins, and its inhibition results in cellular accumulation of protein aggregates. Intramuscle-fiber ubiquitinated multiprotein-aggregates are characteristic of sporadic inclusion-body myositis (s-IBM) muscle fibers. Two major types of aggregates exist, containing either amyloid-beta (Abeta) or phosphorylated tau (p-tau). We have now asked whether abnormalities of the 26S proteasome contribute to s-IBM pathogenesis and whether the multiprotein aggregates have features of aggresomes. Using cultured human muscle fibers we also studied the effect of amyloid-beta precursor protein (AbetaPP) overexpression on proteasome function and the influence of proteasome inhibition on aggresome formation. We report that in s-IBM muscle biopsies 26S proteasome subunits were immunodetected in the gamma-tubulin-associated aggresomes, which also contained Abeta, p-tau, ubiquitin, and HSP70. In addition, a) expression of proteasome subunits was greatly increased, b) the 20Salpha proteasome subunit co-immunoprecipitated with AbetaPP/Abeta, and c) the three major proteasomal proteolytic activities were reduced. In cultured muscle fibers, AbetaPP-overexpressing fibers displayed diminished proteasomal proteolytic activities, and addition of proteasome inhibitor strikingly increased aggresome formation. Accordingly, proteasome dysfunction in s-IBM muscle fibers may play a role in accumulation of misfolded, potentially cytotoxic proteins and may be induced by increased intracellular AbetaPP/Abeta.

Homocysteine-induced endoplasmic reticulum protein (Herp) is up-regulated in sporadic inclusion-body myositis and in endoplasmic reticulum stress-induced cultured human muscle fibers.

Herp is a stress‐response protein localized in the endoplasmic reticulum (ER) membrane. Herp was proposed to improve ER‐folding, decrease ER protein load, and participate in ER‐associated degradation (ERAD). Intra‐muscle‐fiber ubiquitinated multiprotein‐aggregates containing, among other proteins, either amyloid‐β (Aβ) or phosphorylated tau are characteristic of sporadic inclusion‐body myositis (s‐IBM). ER stress and proteasome inhibition appear to play a role in s‐IBM pathogenesis. We have now studied Herp in s‐IBM muscle fibers and in ER‐stress‐induced or proteasome‐inhibited cultured human muscle fibers. In s‐IBM muscle fibers: (i) Herp was strongly immunoreactive in the form of aggregates, which co‐localized with Aβ, GRP78, and β2 proteasome subunit; (ii) Herp mRNA and protein were increased. In ER‐stress‐induced cultured human muscle fibers: (i) Herp immunoreactivity was diffusely increased; (ii) Herp mRNA and protein were increased. In proteasome‐inhibited cultured human muscle fibers: (i) Herp immunoreactivity was in the form of aggregates; (ii) Herp protein was increased, but its mRNA was not. Accordingly, in s‐IBM muscle fibers: (i) increase of Herp might be due to both ER‐stress and proteasome inhibition; (ii) co‐localization of Herp with Aβ, proteasome, and ER‐chaperone GRP78 could reflect its possible role in processing and degradation of cytotoxic proteins in ER.

Endoplasmic reticulum stress induces myostatin precursor protein and NF-κB in cultured human muscle fibers: Relevance to inclusion body myositis

Abstract Sporadic-inclusion body myositis (s-IBM) is the most common progressive muscle disease of older persons. It leads to pronounced muscle fiber atrophy and weakness, and there is no successful treatment. We have previously shown that myostatin precursor protein (MstnPP) and myostatin (Mstn) dimer are increased in biopsied s-IBM muscle fibers, and proposed that MstnPP/Mstn increase may contribute to muscle fiber atrophy and weakness in s-IBM patients. Mstn is known to be a negative regulator of muscle fiber mass. It is synthesized as MstnPP, which undergoes posttranslational processing in the muscle fiber to produce mature, active Mstn. To explore possible mechanisms involved in Mstn abnormalities in s-IBM, in the present study we utilized primary cultures of normal human muscle fibers and experimentally modified the intracellular micro-environment to induce endoplasmic-reticulum (ER)-stress, thereby mimicking an important aspect of the s-IBM muscle fiber milieu. ER stress was induced by treating well-differentiated cultured muscle fibers with either tunicamycin or thapsigargin, both well-established ER stress inducers. Our results indicate for the first time that the ER stress significantly increased MstnPP mRNA and protein. The results also suggest that in our system ER stress activates NF-κB, and we suggest that MstnPP increase occurred through the ER-stress-activated NF-κB. We therefore propose a novel mechanism leading to the Mstn increase in s-IBM. Accordingly, interfering with pathways inducing ER stress, NF-κB activation or its action on the MstnPP gene promoter might prevent Mstn increase and provide a new therapeutic approach for s-IBM and, possibly, for muscle atrophy in other neuromuscular diseases.

Presence of BACE1 and BACE2 in muscle fibres of patients with sporadic inclusion-body myositis

Sporadic inclusion-body myositis (IBM) is the most common, progressive muscle disease of older individuals. We investigated the presence of BACE1 and BACE2-two beta secretases that cleave amyloid-beta-precursor protein-in muscle-biopsy samples from patients with IBM and from controls. On immunofluorescence, BACE1 and BACE2 co-localised with amyloid beta in IBM vacuolated muscle fibres, but were not found in controls. Immunoblotting showed increased BACE2 but not BACE1 in patients with IBM compared with controls. Our study suggests that both of these proteases might participate in processing of amyloid-beta-precursor protein in IBM muscle fibres.

NOGO is increased and binds to BACE1 in sporadic inclusion-body myositis and in AβPP-overexpressing cultured human muscle fibers

Increased amyloid-β precursor protein (AβPP) and amyloid-β (Aβ) accumulation appear to be upstream steps in the pathogenesis of sporadic inclusion-body myositis (s-IBM). BACE1, participating in Aβ production is also increased in s-IBM muscle fibers. Nogo-B and Nogo-A belong to a family of integral membrane reticulons, and Nogo-B binding to BACE1 blocks BACE1 access to AβPP, decreasing Aβ production. We studied Nogo-B and Nogo-A in s-IBM muscle and in our IBM muscle culture models, based on AβPP-overexpression or ER-stress-induction in cultured human muscle fibers (CHMFs). We report that: (1) in biopsied s-IBM fibers, Nogo-B is increased, accumulates in aggregates, is immuno-co-localized with BACE1, and binds to BACE1; Nogo-A is undetectable. (2) In CHMFs, (a) AβPP overexpression increases Nogo-B, Nogo-A, and BACE1, (b) ER stress increases BACE1 but decreases Nogo-B and Nogo-A, (c) Nogo-B and Nogo-A associate with BACE1. Accordingly, two novel mechanisms, AβPP overexpression and ER stress, are involved in Nogo-B and Nogo-A expression in human muscle. We propose that in s-IBM muscle the Nogo-B increase may represent an attempt by muscle fiber to decrease Aβ production. However, the increase of Nogo-B seems insufficient because Aβ continues to accumulate and the disease progresses. We propose that manipulations, which increase Nogo-B in s-IBM muscle might offer a new therapeutic opportunity.

Cystatin C colocalizes with amyloid-β and coimmunoprecipitates with amyloid-β precursor protein in sporadic inclusion-body myositis muscles

Cystatin C (CC), an endogenous cysteine protease inhibitor, is accumulated within amyloid‐β (Aβ) amyloid deposits in Alzheimers disease (AD) brain and was proposed to play a role in the AD pathogenesis. Because the chemo‐morphologic muscle phenotype of sporadic inclusion‐body myositis (s‐IBM) has several similarities with the phenotype of AD brain, including abnormal accumulation of Aβ deposits, we studied expression and localization of CC in muscle biopsies of 10 s‐IBM, and 16 disease‐ and five normal‐control muscle biopsies. Physical interaction of CC with amyloid‐β precursor protein (AβPP) was studied by a combined immunoprecipitation/immunoblotting technique in the s‐IBM muscle biopsies and in AβPP‐overexpressing cultured human muscle fibers. In all s‐IBM muscle biopsies, CC‐immunoreactivity either colocalized with, or was adjacent to, the Aβ‐immunoreactive inclusions in 80–90% of the vacuolated muscle fibers, mostly in non‐vacuolated regions of their cytoplasm. Ultrastructurally, CC immunoreactivity‐colocalized with Aβ on 6–10 nm amyloid‐like fibrils and floccular material. By immunoblotting, CC expression was strongly increased in IBM muscle as compared to the controls. By immunoprecipitation/immunoblotting experiments, CC coimmunoprecipitated with AβPP, both in s‐IBM muscle and in AβPP‐overexpressing cultured normal human muscle fibers. Our studies (i) demonstrate for the first time that CC physically associates with AβPP, and (ii) suggest that CC may play a novel role in the s‐IBM pathogenesis, possibly by influencing AβPP processing and Aβ deposition.

BACE1 and BACE2 in pathologic and normal human muscle

BACE1 and BACE2 are recently discovered enzymes participating in processing of amyloid beta precursor protein (AbetaPP). Their discovery is contributing importantly to understanding the mechanism of amyloid-beta generation, and hence the pathogenesis of Alzheimers disease (AD). Sporadic inclusion-body myositis (s-IBM) and hereditary inclusion-body myopathy (h-IBM) are progressive muscle diseases in which overproduction of AbetaPP and accumulation of its presumably toxic proteolytic product amyloid-beta (Abeta) in abnormal muscle fibers appear to play an important upstream role in the pathogenic cascade. In normal human muscle AbetaPP was also shown to be present and presumably playing a role (a) at neuromuscular junctions and (b) during muscle development. To investigate whether BACE1 and BACE2 play a role in normal and diseased human muscle, we have now studied them by immunocytochemistry and immunoblotting in 35 human muscle biopsies, including: 5 s-IBM; 5 chromosome-9p1-linked quadriceps-sparing h-IBM; and 25 control muscle biopsies. In addition, expression of BACE1 and BACE2 was studied in normal cultured human muscle. Our studies demonstrate that BACE1 and BACE2 (a) are expressed in normal adult muscle at the postsynaptic domain of neuromuscular junctions, and in cultured human muscle; (b) are accumulated in the form of plaque-like inclusions in both s-IBM and h-IBM vacuolated muscle fibers; and (c) are immunoreactive in necrotizing muscle fibers. Accordingly, BACE1 and BACE2 participate in normal and abnormal processes of human muscle, suggesting that their functions are broader than previously thought.

Myostatin is increased and complexes with amyloid-β within sporadic inclusion-body myositis muscle fibers

Myostatin is a negative regulator of muscle mass and strength. Sporadic inclusion-body myositis (s-IBM) is the most common degenerative muscle disease of older persons and is characterized by pronounced muscle wasting. s-IBM is of unknown etiology and pathogenesis, and it lacks definitive treatment. We have now demonstrated in samples from 12 s-IBM biopsies that: (1) by light and electron microscopic immunocytochemistry, myostatin/myostatin precursor is accumulated within muscle fibers and co-localized with amyloid-β (Aβ); (2) by immunoblots, both myostatin and myostatin precursor are increased; and (3) by immunoprecipitation, myostatin precursor complexes with Aβ. Our study suggests that myostatin/myostatin precursor, either alone, or bound to Aβ, may play a novel role in the pathogenesis of s-IBM.

AβPP-overexpression and proteasome inhibition increase αB-crystallin in cultured human muscle: Relevance to inclusion-body myositis

Amyloid-beta precursor protein (AbetaPP) and its fragment amyloid-beta (Abeta) are increased in s-IBM muscle fibers and appear to play an important role in the pathogenic cascade. alphaB-Crystallin (alphaBC) was shown immunohistochemically to be accumulated in s-IBM muscle fibers, but the stressor(s) influencing alphaBC accumulation was not identified. We now demonstrate, using our experimental IBM model based on genetic overexpression of AbetaPP into cultured normal human muscle fibers, that: (1) AbetaPP overexpression increased alphaBC 3.7-fold (p=0.025); (2) additional inhibition of proteasome with epoxomicin increased alphaBC 7-fold (p=0.002); and (3) alphaBC physically associated with AbetaPP and Abeta oligomers. We also show that in biopsied s-IBM muscle fibers, alphaBC was similarly increased 3-fold (p=0.025) and physically associated with AbetaPP and Abeta oligomers. We propose that increased AbetaPP is a stressor increasing alphaBC expression in s-IBM muscle fibers. Determining the consequences of alphaBC association with Abeta oligomers could have clinical therapeutic relevance.

Glucocorticoid increases acetylcholinesterase and organization of the postsynaptic membrane in innervated cultured human muscle

We have studied the influence of hydrocortisone (HC) on the neuromuscular junctions (NMJs) established on cultured human muscle fibers that had been innervated by fetal rat spinal cord neurons. Treatment with HC was begun 4 weeks after innervation and continued for 1-28 days. Four weeks of treatment significantly increased (a) size of acetylcholinesterase (AChE)-positive sites, indicative of NMJs; (b) intensity of AChE staining; (c) A12-AChE (junctional) molecular fraction; and (d) organization of junctional postsynaptic folds. The effect of HC depended on the dose and duration of treatment. These effects on the molecular properties of the postsynaptic component of the human neuromuscular junction could be through an action of HC directly on the muscle fiber or indirectly by affecting the motor neuron. Because the increased organization of the postsynaptic folds and the increased AChE seem to be salutory effects on the NMJ of prolonged HC treatment, these changes of the NMJ itself might contribute to the long-term beneficial effect of prednisone, another glucocorticoid, in myasthenia gravis patients.