Expression and functional profiling of neprilysin, insulin-degrading enzyme, and endothelin-converting enzyme in prospectively studied elderly and Alzheimer's brain

Abstract

The brain steady state level of β-amyloid (Aβ) is determined by the balance between its production and removal, the latter through egress across blood and CSF barriers as well as Aβ degradation. The major Aβ-degrading enzymes are neprilysin (NEP), insulin-degrading enzyme (IDE), and endothelin-converting enzyme (ECE-1). Although evidence suggests that NEP is down-regulated in Alzheimer's disease (AD), the role of IDE and ECE in the Aβ accumulation in aging and dementia remains less certain. In this study, we examined mRNA and protein expression, as well as biological activity of NEP, IDE, and ECE-1 in human frontal cortex by real-time RT-PCR for mRNA, immunoblotting for protein, and highly sensitive and specific fluorescence assays for activity. The relationships between Aβ-degrading enzymes and pathologic measures and clinical features were also assessed. The results showed that NEP mRNA, protein level, and activity were decreased in AD compared with normal controls with no cognitive impairment (NCI). In contrast, IDE activity was unchanged, but there was higher expression of IDE mRNA, indicating a possible compensatory reaction because of deficits in activity. ECE-1 expression in AD brain showed no significant difference compared with age-matched controls. Correlation analyses suggested that NEP expression was correlated with Aβ accumulation and clinical diagnosis, being lower in AD than in no cognitive impairment. In contrast, neither IDE nor ECE-1 correlated with Aβ or clinical diagnosis. These findings provide additional support for NEP as the major protease involved in Aβ degradation and suggest its possible therapeutic targeting in AD.

Figure 1. NEP, IDE and ECE levels in frontal cortex of NCI, MCI and AD patients

(A): Alterations of NEP, IDE and ECE mRNA expression in frontal cortex of NCI, MCI and AD patients. Relative NEP, IDE and ECE mRNA levels in three groups were detected by qRT-PCR using the comparative cycle threshold method, assigning a value of 1 to the NCI group. NEP mRNA levels were normalized to β-actin in each sample and IDE and ECE levels were normalized to S26 in each sample. Bars show mean values and standard error of mean (n=10 in each group). Decrease NEP mRNA level was observed in AD group compared with NCI group. Increase IDE and ECE mRNA level in AD group compared with NCI group. (B): NEP protein levels are significantly decreased in AD patient, while IDE and ECE protein expression stay steady in three groups. Western blot analysis on brain frontal cortex were performed, the actin bands confirm an equal loading of all samples. Data shown are representative of all brain biopsies studied. The densitometry of western blots showed the down regulation of NEP protein in AD group compared with NCI (p<0.05). NEP protein level in MCI group tend to decrease, but not statistically significant. IDE and ECE protein level in three groups did not changed significantly. (C): NEP activity in AD frontal cortex is significantly decreased while IDE and ECE activity remains unchanged. Specific NEP and ECE activity in brain homogenate from all cases were measured based on high sensitive fluorogenic analysis, specific IDE activity is measured by using immunocapture fluorometric assay. Assays were repeated on 3 separate occasions. Bar show mean values and standard error of mean of 9 cases in AD group and 10 cases in NCI, MCI group, respectively. *:compare with NCI group, p<0.05

Figure 2

Neuronal loss makes limited contribution to NEP protein down-regulation. Synaptophysin, pre-synaptic marker in neuron, was measured by western blot (fig 2A). AD and MCI group has very slightly decline synaptophysin expression compare to NCI, the difference did not reach statistic significance. NEP protein levels were significantly decreased in AD cases by normalizing with synaptophysin (fig 2B). *: compare to NCI, p<0.05.

Figure 3

Western blot analysis conform Aβ deposition in AD patients. Total Aβ protein content in frontal cortex homogenate was detected by full length Aβ antibody in three groups. As a reference protein, β-actin indicates equal loading of all samples. Results show Aβ increases dramatically in AD group compared with NCI. **: p<0.01 as compared with NCI group.

Figure 4

Correlation of NEP protein level and Aβ accumulation. The negative correlation between NEP protein and Aβ accumulation was observed (r2=0.37, p=0.0004) when combined all the tree groups together suggesting that NEP is a key factor in Aβ clearance during AD development. However, when correlation analysis were performed in sub-group, the positive correlation between NEP protein level and Aβ content was found in AD group (r2=0.26, p=0.13). These results implied that the capacity of NEP clearance in AD development is not unlimited.

Figure 5

Correlation of NEP enzymatic activity and AD clinical parameters. The positive correlation between NEP activity and MMSE, NEP activity and Global z-score were observed when all cases were pooled together, indicating that NEP activity may be an essential factor in AD clinical physiological features development.