Letters to the Editor

Apolipoprotein E immunoreactive deposits in inclusion-body muscle diseases

Apolipoprotein E (APOE) is a 299-aminoacid protein encoded by the APOE gene. Three common polymorphisms in the APOE gene, ɛ2, ɛ3, and ɛ4, result in a single aminoacid change in the APOE protein. APOE ɛ2, ɛ3, and ɛ4 alleles strongly alter, in a dose-dependent manner, the likelihood of developing Alzheimer's disease and cerebral amyloid angiopathy. In particular, APOE ɛ4 is associated with increased risk for Alzheimer's disease whereas APOE ɛ2 is associated with decreased risk. The effects of APOE genotype on risk of these diseases are likely to be mediated by differential effects of APOE on amyloid-β accumulation in the brain and its vasculature. Response to treatment for Alzheimer's disease might differ according to APOE genotype. Because convincing evidence ties the APOE genotype to risk of Alzheimer's disease and cerebral amyloid angiopathy, APOE has been studied in other neurological diseases. APOE ɛ4 is associated with poor outcome after traumatic brain injury and brain haemorrhage, although the mechanisms underlying these associations are unclear. The possibility that APOE has a role in these and other neurological diseases has been of great interest, but convincing associations have not yet emerged.

Inclusion body myositis (IBM) is an acquired inflammatory myopathy in which a wide range of proteins is deposited within the cytoplasm of muscle fibres. To explore the possibility that this deposition occurs due to uncontrolled protein production arising from a defect at the nuclear level, we studied muscle samples from IBM and control subjects using two-dimensional gel electrophoresis. Twenty-seven gels from five controls and 24 gels from six patients with IBM were exhaustively compared using image analysis software and visual inspection. We found significant intra- and inter-subject variability in the number of protein spots on the gels. From 2272 to 4522 spots were found in different control gels, and from 2821 to 4153 spots in the IBM gels. No unique spots were identified in the IBM gels. When viewed with other work, the results of this study suggest that widespread, uncontrolled activation of genes is unlikely to be a component of the pathogenesis in IBM.

Previous studies have differed as to whether APOE ε4 is a susceptibility factor for developing sporadic inclusion body myositis (sIBM), with a positive association being found only in an Australian cohort of cases. We have now re-examined this in a larger cohort of 57 sIBM cases and have also carried out a meta-analysis of all the published studies looking for evidence of a risk association or effect of APOE alleles on disease expression. Our findings argue against a specific role for any APOE alleles in conferring susceptibility to sIBM but have demonstrated a non-significant trend towards an earlier age-of-onset in patients with the ε2 allele.

The accumulation of tubular aggregates in type II skeletal muscle fibres and fibrillo–granular inclusions in hippocampal protoplasmic astrocytes are characteristic lesions of apolipoprotein E deficient mice. Moreover these inclusions reacted immunocytochemically with an antibody specific to fragment 17–24 of the published sequence of Alzheimer’s amyloid peptide. In an effort to evaluate the role of apolipoprotein E in the formation of these abnormal structures, we examined the tibialis anterior muscle and the hippocampus of several groups of animals including: (i) apolipoprotein E “knockout” mice which had been whole body irradiated with 1200 rads and bone marrow replenished with apolipoprotein E sufficient marrow; and (ii) three transgenic murine strains that had been genetically engineered to express either human apolipoprotein E2, E3 or E4 protein on an apoE deficient background. The results of this study showed that the presence of murine apolipoprotein E (even in subnormal levels in the serum) in irradiated bone marrow replenished mice and in all three (E2, E3 or E4) human apoE transgenic strains was sufficient to prevent the aggregation of sarcoplasmic tubules in the tibialis anterior type II muscle fibres. Similarly apolipoprotein E “knockout” bone marrow replenished mice and all three transgenic strains expressing the different human apolipoprotein E alleles reduced the number of the astrocytic inclusions in the hippocampus to levels not significantly different to those observed in control C57Bl6J animals.

The data obtained in this study indicate that neurological and neuromuscular abnormalities found in apoE deficient mice are reversed when apoE protein is replaced in the circulation, either by bone marrow transplantation of normal apoE sufficient marrow, or by gene therapy with the apoE gene, albeit of human origin and irrespective of the allele used.

Inclusion body myopathy is a progressive muscle disorder characterized by nuclear and cytoplasmic inclusions and vacuolation of muscle fibers. Affected muscle fibers contain deposits of congophilic amyloid, amyloid-β immunoreactive filaments, and paired helical filaments, all of which are pathological hallmarks of Alzheimer's disease in brain. Accumulations of amyloid-β and its precursor are thought to play important roles in the pathogenesis of both inclusion body myopathy and Alzheimer's disease. Overexpression of mutant forms of β protein precursor in transgenic mice by neuron-specific promoters has been reported to cause amyloid deposits in the brain. Here we report that overexpression in transgenic mice of the signal plus 99-amino acid carboxyl-terminal sequences of β protein precursor, under the control of a cytomegalovirus enhancer/β-actin promoter, resulted in vacuolation and increasing accumulation of the 4-kd amyloid-β and the carboxyl-terminus in skeletal muscle fibers during aging. These deposits in transgenic muscle only rarely showed Congo red birefringence. Thus, overexpression of part of β protein precursor in transgenic mice led to development of some of the characteristic features of inclusion body myopathy. These mice may be a useful model of inclusion body myopathy, which shares a number of pathological markers with Alzheimer's disease.

Inclusion body myositis (IBM) is the most common muscle disease in the elderly. Amyloid-β protein (Aβ) has been shown to accumulate abnormally in the vacuolated fibers and to localize to amyloid-like fibrils in muscles from IBM patients. We studied the skeletal muscles from a line of transgenic mice over-expressing the carboxyl-terminal 99 amino acids (C99) of the β-amyloid precursor protein (βPP) with a substitution of lysine-612 to valine (K612V), intended to abolish α-secretase recognition and to preserve the Aβ domain of C99. The majority (87%) of the 24-month-old transgenic mice showed myopathic changes, and approximately one-third of them had degenerating fibers with sarcoplasmic vacuoles and thioflavin-S-positive deposits. Ultrastructurally, the inclusions were aggregates of short thin amyloid-like fibrils, 6 to 8 nm in diameter. These features are similar to those of human IBM. Immunocytochemistry using an antibody against Aβ showed membranous staining in most muscle fibers of transgenic mice, as well as granular or vacuolar cytoplasmic staining in the atrophic fibers. Western blots showed a high level of accumulation of carboxyl-terminal fragments of βPP in the muscles of the transgenic mice with the most severe IBM-like lesions. The expression of IBM-like lesions was age dependent. These transgenic mice provide a model for the study of IBM and for the peripheral expression of a key element in the pathogenesis of Alzheimer disease.