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Am J Transl Res 2013;5(6):608-621
Original Article
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, Chunsheng Wei, Wei Huang, David A Bennett, Dennis W Dickson, Rui Wang, Dengshun Wang
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China; Rush Alzheimer’s Disease Center, Rush University Medical Center,
Chicago, IL, USA; Department of Pathology (Neuropathology) and Neuroscience, Mayo Clinic College of Medicine,
Jacksonville, FL 32224, USA; Department of Pathology and Laboratory Medicine, School of Medicine and Public
Health, University of Wisconsin, Madison, Wisconsin, USA. Equal contributors.
Received August 8, 2013; Accepted August 27, 2013; Epub September 25, 2013; Published September 30, 2013
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. (AJTR1308003).
Keywords: Alzheimer’s disease, amyloid-β, Aβ degrading enzymes, neprilysin, subcellular compartments, Aβ
clearance
Address correspondence to: Dr. Rui Wang, Shanghai Key Laboratory of New Drug Design, School of Pharmacy,
East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Tel: +86-21-
64250823; Fax: +86-21-64250823; E-mail: ruiwang@ecust.edu.cn; Dr. Dengshun Wang, 1300 University Avenue,
Madison, WI 53706, USA. Tel: +1-608-262-9825; Fax: +1-608-265-3301; E-mail: dwang6@wisc.edu
Original Article
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, Chunsheng Wei, Wei Huang, David A Bennett, Dennis W Dickson, Rui Wang, Dengshun Wang
Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China; Rush Alzheimer’s Disease Center, Rush University Medical Center,
Chicago, IL, USA; Department of Pathology (Neuropathology) and Neuroscience, Mayo Clinic College of Medicine,
Jacksonville, FL 32224, USA; Department of Pathology and Laboratory Medicine, School of Medicine and Public
Health, University of Wisconsin, Madison, Wisconsin, USA. Equal contributors.
Received August 8, 2013; Accepted August 27, 2013; Epub September 25, 2013; Published September 30, 2013
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. (AJTR1308003).
Keywords: Alzheimer’s disease, amyloid-β, Aβ degrading enzymes, neprilysin, subcellular compartments, Aβ
clearance
Address correspondence to: Dr. Rui Wang, Shanghai Key Laboratory of New Drug Design, School of Pharmacy,
East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. Tel: +86-21-
64250823; Fax: +86-21-64250823; E-mail: ruiwang@ecust.edu.cn; Dr. Dengshun Wang, 1300 University Avenue,
Madison, WI 53706, USA. Tel: +1-608-262-9825; Fax: +1-608-265-3301; E-mail: dwang6@wisc.edu
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