Our results suggest that MCI subjects may exhibit a sexually dimorphic difference in NEP2 and NEP mRNA expression levels (higher in males, lower in females) relative to their NI counterparts (). The higher levels in MCI males may represent an adaptive response to increased Aβ. Conversely, the lower levels in MCI females may contribute to the development of AD and increased female susceptibility to AD [17
]. NEP expression has been shown to be sex hormone responsive [18
] and the same may be true of NEP2. However, it is difficult to predict how sex hormones would affect NEP expression in men and women as both androgen and estrogen stimulate NEP expression and because of other potential complications such as hormone replacement therapy. As far as we know, no other study has compared NEP expression between men and women. Furthermore, regulation of NEP2 expression is more difficult to predict as the regulatory elements have yet to be characterized. It is perhaps unlikely that these gender differences can be explained by sex hormones alone as, based on androgen/estrogen levels, lower NEP/NEP2 levels would have been predicted in postmenopausal women. The finding that NEP2 and NEP mRNA expression in AD subjects was similar to that of NI subjects was unexpected. Yet changes in mRNA expression may not necessarily correlate with enzymatic activity due to alternate splicing, translational, and post-translational modifications. Our previous work showed that only one of the three known NEP2 splice forms (β) degraded Aβ [4
] and our qRT-PCR primers detected all isoforms. This makes it difficult to determine from our qRT-PCR analysis alone if these changes in expression are directly involved in AD pathogenesis. Specific qRT-PCR primers could differentiate between the active NEP2-β and inactive NEP2-γ forms because of the large missing exon in NEP2-γ. However, exclusive qRT-PCR analysis of NEP2-β is complicated by the fact it shares all the same sequences with the active-site-lacking NEP2-δ form (with the exception of a 29 base pair frameshift insertion in NEP2-δ), regardless of other potential complications from yet unidentified splice forms [3
]. However, since these “global” expression changes occur in MCI, they may be very useful as expression markers predicting risk of AD. In addition to splicing, oxidation can also reduce endopeptidase activity and previous studies have found that NEP and IDE were found in a more oxidized and inactive state in AD [24
]. In support of this, we found that NEP2 enzymatic activity was reduced in association with AD (). Furthermore, analysis independent of diagnostic classification showed that higher NEP2 activity was associated with better cognition. Finally, we cannot rule out that NEP2 expression is altered in general in neurodegenerative diseases as we have not yet included control specimens from other diseases such as Parkinson’s disease and frontotemporal dementia.
Our NEP mRNA expression data is at odds with previous studies showing reduced expression in association with AD and/or aging [3
]. One possible explanation for this discrepancy is that these studies used tissues from different sources, species, disease states, and brain regions. In addition, our study used a quantitative real-time PCR approach while most previous studies did not (to date only 6 of approximately 20 NEP expression studies used PCR analysis of cDNA comparing human NI to AD). Most of these previous studies used semiquantitative endpoint PCR [27
] with the former study supporting our findings [27
]. It should also be noted that other studies have also found no change or increased NEP in association with AD by immunohistochemistry and activity assay [12
]; therefore, our finding of unchanged or increased NEP mRNA in AD is not without precedence. As we did not assay NEP activity in this study, we cannot directly compare our findings with NEP activity data from other studies. Since NEP2 activity has not previously been analyzed, we have no precedent for comparison. However, for NEP mRNA expression in the MFG in particular, we expected reduced NEP expression in AD as previous research using specimens from the same study had demonstrated [16
]. Yet our research shows NEP expression in the MFG is not significantly changed in MCI and AD (), suggesting more work must be done for clarification. Regional expression comparisons () showed the highest NEP2 and NEP mRNA levels in the caudate, with NEP mRNA also high the cerebellum, correlating with relative resistance to Aβ pathology. This is contrary to NEP2 expression observed in rats as little to no NEP2 was found in the caudate by in-situ hybridization [34
]. However, significant (but relatively lower) levels of NEP2 were found by reverse-transcription-PCR in the rat caudate [35
In summary, this work is significant because we are the first to measure NEP2 expression and activity in relation to MCI and AD. We found that NEP2 expression and activity was altered in association with MCI. Furthermore, if these changes can also be detected in easily accessible fluids (i.e., blood or cerebrospinal fluid) NEP2 could be used as a preclinical marker for AD. Finally, our analysis of NEP2 activity is functionally relevant to the etiology of AD.