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. 2013 Sep 25;5(6):608-21.
eCollection 2013.

Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer's disease patients, transgenic mice and cultured human neuronal cells

Affiliations

Distinct subcellular patterns of neprilysin protein and activity in the brains of Alzheimer's disease patients, transgenic mice and cultured human neuronal cells

Li Zhou et al. Am J Transl Res. .

Abstract

We investigated the subcellular distribution of NEP protein and activity in brains of human individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI) and AD dementia, as well as double transgenic mice and human neuronal cell line treated with Aβ and 4-hydroxy-2-nonenal (HNE). Total cortical neuronal-related NEP was significantly increased in MCI compared to NCI brains. NeuN was decreased in both MCI and AD, consistent with neuronal loss occurring in MCI and AD. Negative relationship between NEP protein and NeuN in MCI brains, and positive correlation between NEP and pan-cadherin in NCI and MCI brains, suggesting the increased NEP expression in NCI and MCI might be due to membrane associated NEP in non-neuronal cells. In subcellular extracts, NEP protein decreased in cytoplasmic fractions in MCI and AD, but increased in membrane fractions, with a significant increase in the membrane/cytoplasmic ratio of NEP protein in AD brains. By contrast, NEP activity was decreased in AD. Similar results were observed in AD-mimic transgenic mice. Studies of SH-SY5Y neuroblastoma showed an up-regulation of NEP protein in the cytoplasmic compartment induced by HNE and Aβ; however, NEP activity decreased in cytoplasmic fractions. Activity of NEP in membrane fractions increased at 48 hours and then significantly decreased after treatment with HNE and Aβ. The cytoplasmic/membrane ratio of NEP protein increased at 24 hours and then decreased in both HNE and Aβ treated cells. Both HNE and Aβ up-regulate NEP expression, but NEP enzyme activity did not show the same increase, possibly indicating immature cytoplasmic NEP is less active than membrane associated NEP. These observations indicate that modulation of NEP protein levels and its subcellular location influence the net proteolytic activity and this complex association might participate in deficiency of Aβ degradation that is associated with amyloid deposition in AD.

Keywords: Alzheimer’s disease; Aβ clearance; Aβ degrading enzymes; amyloid-β; neprilysin; subcellular compartments.

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Figures

Figure 1
Figure 1
The relative levels of NEP, NeuN and Pan-cadherin in NCI, MCI and AD cortex. Brain slides from NCI, MCI and AD cortex were immunostained for NEP, NeuN and pan-cadherin followed by quantitative analysis using Art Imaging Systems VectraTM. A: The representative photos from the Nuance analysis. B: NEP; C: NeuN; D: Pan-cadherin. The data are expressed as mean ± SEM. ***p < 0.01 vs NCI control.
Figure 2
Figure 2
Correlation analysis of NEP/NeuN and NEP/pan-cadherin pixel density. A significant negative correlation between NEP and NeuN was observed only in MCI brains (R2 = 0.3271, P < 0.0001). Positive correlation between NEP and pan-cadherin was observed in both NCI (R2 = 0.096, P = 0.002) and MCI (R2 = 0.3836, P < 0.0001).
Figure 3
Figure 3
Alterations of NEP membrane/cytoplasmic ratio for protein and enzymatic activity in NCI, MCI and AD. The subcellular fractions of cortical tissues from NCI, MCI and AD brains were prepared for NEP ELISA and activity assays. A: Cytoplasmic and membrane NEP protein levels; B: Cytoplasmic and membrane NEP activity; C: The membrane/cytoplasmic ratio of NEP protein and activity. The data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs NCI control.
Figure 4
Figure 4
Thioflavine-S staining of brain slices from different months of APP/PS1 and WT mice. A-E: Represents the sections of WT and APP/PS1 transgenic mice of 3, 6, 9 and 12 month, respectively. F: Total fluorescent area measured and quantified by Image J analysis for each group.
Figure 5
Figure 5
Alteration of cortical NEP levels in APP/PS1 transgenic mice. Brain slides from C57 WT mice and various ages of transgenic APP/PS1 were immunostained for NEP, NeuN and pan-cadherin following by quantitative analysis using Art Imaging Systems NuanceTM. A: Total positive pixel area for NEP, NeuN and pan-cadherin in mice at 3, 6, 9 and 12 months of age. B: Total NEP positive area; C: Membrane/cytoplasmic NEP positive area; D: NEP membrane/cytoplasm ratio. The data are expressed as mean ± SEM. *p < 0.05, **p < 0.01 vs WT control.
Figure 6
Figure 6
Alteration of cytoplasmic and membrane NEP protein and activity in HNE and Aβ1-42 treated SH-SY5Y cells. Different concentrations of HNE or Aβ were added to the cultures 24-72 h before harvest. The membrane and cytoplasm fractionations were extracted for the determination of NEP protein and activity. A and C: Cytoplasmic NEP protein and activity, respectively; B and D: Membrane NEP protein and activity, respectively. E and F: The membrane/cytoplasmic ration of NEP protein and activity.

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