Effects of nonsteroidal anti-inflammatory drugs on amyloid-beta pathology in mouse skeletal muscle

Abstract

Sporadic inclusion body myositis (sIBM) is a common age-related inflammatory myopathy characterized by the presence of intracellular inclusions that contain the amyloid-beta (Abeta) peptide, a derivative of the amyloid precursor protein (APP). Abeta is believed to cause Alzheimer's disease (AD), suggesting that a link may exist between the two diseases. If AD and sIBM are linked, then treatments that lower Abeta in brain may prove useful for sIBM. To test this hypothesis, transgenic mice that overexpress APP in skeletal muscle were treated for 6 months with a variety of nonsteroidal anti-inflammatory drugs (NSAIDs; naproxen, ibuprofen, carprofen or R-flurbiprofen), a subset of which reduce Abeta in brain and cultured cells. Only ibuprofen lowered Abeta in muscle, and this was not accompanied by corresponding improvements in phenotype. These results indicate that the effects of NSAIDs in the brain may be different from other tissues and that Abeta alone cannot account for skeletal muscle dysfunction in these mice.

Fig. 1

Evaluation of effects of NSAIDs on cultured H4 neuroglioma (15x) cells. Control levels of Aβ42 are indicated by the solid line, and the error range (s.e.m.) by dotted lines on either side. Aβ42 was selectively reduced by ibuprofen, R-flurbiprofen, and carprofen treatment; naproxen was ineffective (Dunnett's test; * = p<0.05, ** = p<0.01). There were no differences detected between sources or formulations of ibuprofen or naproxen.

Fig. 2

Wire suspension ability in T7A6 mice. Normal T7A6 mice show a gradual decline in motor function, starting at ~15 months of age, whereas the WT mice do not [T7A6, r = 0.28, p<0.005, n = 23 (11F, 12M); WT, r = 0.09, p<0.35, n = 20 (9F, 11M)]. This is qualitatively similar to the rota-rod deficit seen at the same end-point, ~18 months of age (inset, * = p<0.02).

Fig. 3

Skeletal Muscle Aβ42 in NSAID treated mice. T7A6 transgenic mice had more Aβ42 in their quadriceps as compared to wild type littermate controls. Ibuprofen was the only NSAID that was effective at lowering Aβ42 (* = p<0.05, Dunnett's test). Number of subjects / group: Control (WT), n = 8M / 9F; Control (TG), n = 7M / 8F; Ibuprofen, n = 9M / 9F; Naproxen, n = 7M / 8F; Carprofen, n = 7M / 9F; R-Flurbiprofen, n = 7M / 11F.

Fig. 4

COX activity in treated mice. NSAID treatment was effective at reducing total COX activity in skeletal muscle (p<0.02). Ibuprofen, naproxen and R-flurbiprofen were equally effective at these dosages (* = p<0.05, Dunnett's test).

Fig. 5

APP and Aβ in skeletal muscle. Equal quantities of protein (100 μg) from SDS extracted quadriceps were separated by SDS-PAGE. (A) Expression of full length APP (antibody 22C11 against the N-terminus of APP). Lysate (10 μg) from H4 neuroglioma cells over expressing human APP was run as a marker control. T7A6 mice over express human APP (~1.3x) as compared to wild type littermates. NSAID treatment did not alter the expression of APP protein. (B) Oligomeric Aβ (antibodies 6E10 + 4G8). Synthetically prepared oligomeric Aβ (3 pmol) and SDS extract from an AD case (~100 μg protein) were run as positive controls (note the appearance of monomeric Aβ slightly above the 3 kDa marker). Neither monomeric nor oligomeric Aβ was detectable in skeletal muscle from T7A6 mice.

Fig. 6

Histopathology in T7A6 mice (magnification, 200x). Sections were treated with Hematoxylin and Eosin (H&E; top panel), Engel-Gomori trichrome stain (middle panel) and antibody 4G8 against Aβ (bottom panel). Histopathology in the T7A6 mice was relatively mild, with H&E and Engel-Gomori stains showing infrequent centric nuclei, and occasional irregular fibers. 4G8 immunoreactivity was sparse, and consisted of small areas of diffusely immunoreactive fibers, and rare punctate inclusions in isolated fibers.

Fig. 7

Estimated amyloid load, as determined from 4G8 immunoreactivity. Positive pixels are expressed as a percentage of the total number of pixels in the section. Consistent with the immunoassay data, ibuprofen was the only NSAID effective at lowering Aβ immunoreactivity in T7A6 muscle (* = p<0.04).

Fig. 8

Motor performance in T7A6 mice. NSAIDs did not affect either (A) rota-rod or (B) wire suspension performance at the end of the study. There were no significant differences between individual NSAIDs.