Our Western blot analysis using a novel anti-MetO antibody showed four different patterns among the 31 patients. The anti-MetO antibody recognizes MetO residues depending on their exposure on the protein surface. Previously, it was shown that oxidation of Met to MetO may cause major conformational rearrangements leading to exposure of Met residues that are buried in their native, reduced form [26
]. It is difficult, however, to predict whether this happens in every specific epitope. Thus, the antibody is believed to be specific to the sulfoxide group of MetO [17
], but deciphering the specific binding epitope in a protein of interest will need to be addressed in future studies.
In particular, we found elevated MetO reactivity, measured densitometrically in a 120-kDa band using an anti-MetO antibody (), in persons carrying FAD mutations. This effect appeared to be driven largely by five the FAD mutation carriers in group I, who had high MetO reactivity ( and ). Of these persons, four carried a PSEN1 A431E mutation and one carried an APP V717I mutation. Notably, all non-carriers had low MetO reactivity and the only 3 subjects for whom MetO reactivity was below detectable levels were non-carriers. Thus, the presence of a prominent 120-kDa band in our assay correlated in all cases with an FAD mutation, though the absence of this band did not necessarily indicated the absence of mutation. The findings suggest that high plasma MetO reactivity may be indicative of an ongoing disease process accompanied by oxidative stress, which fails to be mitigated by the Msr system in FAD mutation carriers.
We did not find clear correlations between MetO reactivity and age, adjusted age, or disease status as 4 of the 5 subjects with the highest MetO reactivity levels were young and asymptomatic (CDR score of 0) and one had moderate dementia (CDR score of 2). It is possible that correlations would be found in a larger sample, which was not available for this study.
The strong positive correlation between MetO reactivity and F2-isoprostanes levels support the role of oxidative stress in FAD and suggests that although the pathology occurs in the brain, it is evident also in the periphery in the form of elevated lipid peroxidation (isoprostanes) and protein oxidation (MetO) markers. Unfortunately, F2-isoprostane levels were not available for the 5 subjects for whom MetO levels were highest. We therefore cannot conclude at this point that the correlation found is maintained at such levels.
The positive correlation between MetO reactivity and plasma SOD1 levels is an intriguing finding. SOD1 is a cytosolic protein and its origin in the plasma of the population studied here is uncertain. The correlation found may indicate an increase of SOD1 production and secretion into the plasma as a compensatory response to oxidative stress, or reflect elevated enzyme release due to enhanced cell death. SOD1 is a highly stable protein [27
], suggesting that it may have a long half-life [28
] compared with other cytoplasmic proteins released following cell death. SOD1 plays an important role in preventing mitochondrial-linked cell death [29
]. Accordingly, oxidative stress may prompt cells to respond by upregulating SOD1 as a protective mechanism against oxidative damage [31
]. Failure of this response mechanism may increase the release of SOD1 through oxidative damage to cellular membranes.
The finding of increased MetO reactivity in asymptomatic persons carrying FAD mutations and the correlation between MetO reactivity and other indices of oxidative stress supports an important role for oxidative stress early in the pathogenesis of FAD. As overproduction of Aβ42 is thought to be the critical factor causing the disease in persons with FAD-linked mutations, our findings suggest that though oxidative stress is an early pathologic process in AD, it occurs downstream of the Aβ-induced neuronal injury.