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. 2010 Jan;112(2):389-96.
doi: 10.1111/j.1471-4159.2009.06461.x. Epub 2009 Oct 29.

In AbetaPP-overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AbetaPP751 and GSK3beta activation: effects mitigated by lithium and apparently relevant to sporadic inclusion-body myositis

Chiara Terracciano  1 , Anna Nogalska, W King Engel, Valerie Askanas
Affiliations

In AbetaPP-overexpressing cultured human muscle fibers proteasome inhibition enhances phosphorylation of AbetaPP751 and GSK3beta activation: effects mitigated by lithium and apparently relevant to sporadic inclusion-body myositis

Chiara Terracciano et al. J Neurochem. 2010 Jan.

Abstract

Muscle fiber degeneration in sporadic inclusion-body myositis (s-IBM) is characterized by accumulation of multiprotein aggregates, including aggregated amyloid-beta (Abeta)-precursor protein 751 (AbetaPP751), Abeta, phosphorylated tau, and other 'Alzheimer-characteristic' proteins. Proteasome inhibition is an important component of the s-IBM pathogenesis. In brains of Alzheimer's disease (AD) patients and AD transgenic-mouse models, phosphorylation of neuronal AbetaPP695 (p-AbetaPP) on Thr668 (equivalent to T724 of AbetaPP751) is considered detrimental because it increases generation of cytotoxic Abeta and induces tau phosphorylation. Activated glycogen synthase kinase3beta (GSK3beta) is involved in phosphorylation of both AbetaPP and tau. Lithium, an inhibitor of GSK3beta, was reported to reduce levels of both the total AbetaPP and p-AbetaPP in AD animal models. In relation to s-IBM, we now show for the first time that (1) In AbetaPP-overexpressing cultured human muscle fibers (human muscle culture IBM model: (a) proteasome inhibition significantly increases GSK3beta activity and AbetaPP phosphorylation, (b) treatment with lithium decreases (i) phosphorylated-AbetaPP, (ii) total amount of AbetaPP, (iii) Abeta oligomers, and (iv) GSK3beta activity; and (c) lithium improves proteasome function. (2) In biopsied s-IBM muscle fibers, GSK3beta is significantly activated and AbetaPP is phosphorylated on Thr724. Accordingly, treatment with lithium, or other GSK3beta inhibitors, might benefit s-IBM patients.

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Figures

Fig. 1
Fig. 1
Phosphorylated AβPP T724 (p-AβPPT724) in sporadic-inclusion body myositis (s-IBM) muscle fibers. (A) Immunofluorescence using the specific phospho-AβPP antibody illustrates pAβPPT724-immunoreactive aggregates in two muscle fibers (arrows). (B). Immunoprecipitation (IP) of p-AβPPT724 in s-IBM muscle biopsy. IP was performed using 6E10 antibody, which recognizes both AβPP and Aβ; immunoblots (IB) were subsequently probed with the specific anti p-AβPPT724 rabbit polyclonal antibody. There is a distinct band corresponding to AβPPT724. In # the primary antibody was omitted from the immunoprecipitation reaction to determine the specificity of the reaction.
Fig. 2
Fig. 2
GSK3β is activated in s-IBM biopsied muscle. (A) Representative immunoblots of normal control and s-IBM muscle biopsies. B) The ratio of GSK3βY216 to total GSK3β, obtained by densitometric analysis of protein bands, shows that in s-IBM, as compared to controls, GSK3βY216 is significantly increased (p< 0.05, 2 tail Student’s t-test). ± SEM.
Fig. 3
Fig. 3
p-AβPPT724 in i) proteasome-inhibited, and ii) ER-stress-induced human-muscle-culture AβPP+ IBM model. (A) Representative immunoblots of densitometric analysis in (B). (B) Densitometric analysis based on 12 independent experiments of the pAβPPT724 protein bands relative to the actin bands, expressed in arbitrary units, shows that after treatment with Epoxomicin (Epox) pAβPPT724 is significantly increased (6.5 fold, p<0.01, 2 tail Student’s t-test), while the ER stress inducers (Thapsigargin [Tg] and Tunicamycin [Tm]) did not exert this effect. ± SEM.
Fig.4
Fig.4
GSK3β is activated in the epoxomicin-inhibited proteasome in the human-muscle-culture IBM model. (A) Representative immunoblots of densitometric analysis in (B). (B) The ratio of GSK3βY216 to total GSK3β obtained by densitometric analysis of protein bands in 6 independent experiments shows that in proteasome-inhibited cultures, as compared to controls, GSK3βY216 is significantly increased (1.7 fold, p< 0.05, 2 tail Student’s t-test) ± SEM.
Fig.5
Fig.5
Inactive form of GSK3β is increased in LiCl-treated culture-IBM-model. (A) Representative immunoblot of GSK3βSer9 in LiCl-treated and sister-control untreated culture AβPP+ IBM model. (B) Densitometric analysis of GSK3βSer9 bands relative to the actin bands, expressed in arbitrary units, shows that after lithium treatment GSK3βSer9 is significantly increased (2.2, p<0.01, 2 tail, Student’s t-test).
Fig. 6
Fig. 6
Lithium decreases the amount of total AβPP, p-AβPPT724, and Aβ-oligomers. (A) Representative immunoblots of total AβPP in LiCl-treated and control untreated culture AβPP+-IBM-model. (B) Densitometric analysis of the total AβPP bands (130 and115 kDa) relative to the actin bands, expressed in arbitrary units, shows that after lithium treatment AβPP is decreased 30% (p<0.0005, 2 tail); (C) Representative immunoblots of p-AβPPT724 in LiCl-treated and untreated control culture AβPP+ -IBM-model. (D) Densitometric analysis of p-AβPPT724 bands relative to the actin bands, expressed in arbitrary units, shows that after lithium treatment p-AβPPT724 is decreased 50% ( p<0.0001, 2 tail). (E) Representative immunoblots of Aβ oligomers (8 kDa, 12kDa, 16 kDa, 25 kDa bands) in LiCl-treated and control untreated AβPP+-IBM-culture-model. (F) Densitometric analysis of Aβ oligomers bands relative to the actin bands, expressed in arbitrary units, shows that after lithium treatment all Aβ bands calculated together were decreased 25% ( p<0.05, 2 tail Student’s t-test).
Fig.7
Fig.7
Three main proteasome enzyme activities -- peptidyl-glutamyl-peptide hydrolytic (PGPH), chymotrypsin-like (CTL) and trypsin-like (TL) -- in LiCl treated and sister-control untreated culture AβPP+ IBM model. Proteasome activities are expressed per β2 20S proteasome subunit in each culture. After lithium treatment, PGPH activity was increased 2 fold (p<0.005, 2 tail Student’s t-test), CTL activity was increased 1.3 fold (p<0.05, 1 tail Student’s t-test), and TL activity was increased 1.2 fold (p<0.05, 1 tail Student’s t-test).
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