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The endopeptidase neprilysin (NEP) is a major amyloid-β (Aβ) degrading enzyme and has been implicated in the pathogenesis of Alzheimer’s disease. Since NEP cleaves substrates other than Aβ, we investigated the potential role of NEP-mediated processing of neuropeptides in the mechanisms of neuroprotection in vivo. Over-expression of NEP at low levels in transgenic (tg) mice affected primarily the levels of neuropeptide Y (NPY) compared to other neuropeptides. Ex vivo and in vivo studies in tg mice and in mice that received lentiviral vector injections showed that NEP cleaved NPY into C-terminal fragments (CTFs), while silencing NEP reduced NPY processing. Immunoblot and mass spectrometry analysis showed that NPY 21-36 and 31-36 were the most abundant fragments generated by NEP activity in vivo. Infusion of these NPY CTFs into the brains of APP tg mice ameliorated the neurodegenerative pathology in this model. Moreover, the amidated NPY CTFs protected human neuronal cultures from the neurotoxic effects of Aβ. This study supports the possibility that the NPY CTFs generated during NEP-mediated proteolysis might exert neuroprotective effects in vivo. This function of NEP represents a unique example of a proteolytic enzyme with dual action—namely, degradation of Aβ as well as processing of NPY.
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder affecting the elderly and is the most common form of dementia (Ashford, 2004). A key mediator of this disease is believed to be amyloid-β (Aβ) peptides, produced by proteolytic processing of the amyloid precursor protein (APP) in the central nervous system (Selkoe, 1994a; Selkoe, 1994b). Recent evidence supports the notion that aggregation of Aβ, resulting in the formation of oligomers rather than fibrils, might be ultimately responsible for the synaptic damage that leads to cognitive dysfunction in patients with AD (Walsh and Selkoe, 2004; Glabe, 2005; Glabe and Kayed, 2006).
Neprilysin (NEP, also known as CD10, EC 220.127.116.11), a zinc metalloendopeptidase (Howell et al., 2005), has been identified as a critical Aβ-degrading enzyme in the brain (Iwata et al., 2000; Iwata et al., 2001). Supporting a role for NEP in AD, previous studies have shown that NEP reduces Aβ levels in vivo (Leissring et al., 2003; Mohajeri et al., 2004; Huang et al., 2006; Farris et al., 2007; Iijima-Ando et al., 2008). These studies are also in agreement with previous experiments showing that viral vector-mediated transfer of NEP reduces the neurodegenerative and amyloid pathology in APP transgenic (tg) mice (Marr et al., 2003; Iwata et al., 2004; Hong et al., 2006; El-Amouri et al., 2008). In patients with AD the levels of NEP in the brain are reduced (Akiyama et al., 2001; Reilly, 2001; Yasojima et al., 2001b; Yasojima et al., 2001a; Caccamo et al., 2005), and a potential genetic linkage is currently being investigated (Sodeyama et al., 2001; Oda et al., 2002; Clarimon et al., 2003; Wood et al., 2007).
Although considerable effort has been focused on investigating the effects of NEP on Aβ pathology, less is known about alternative effects of NEP in the CNS. Previous studies have shown that NEP is capable of cleaving a wide range of neuropeptides, including substance P (SP), enkephalin (ENK) and neuropeptide Y (NPY) (Skidgel and Erdos, 2004). Among them, NPY is of interest because in AD pathology, levels of this neuropeptide are abnormal (Minthon et al., 1990; Ramos et al., 2006) and in APP tg mice the alterations in the NPY network in the hippocampus have been linked to epileptic activity (Palop et al., 2007). NPY is a 36 aa long protein, is one of the most abundant peptide transmitter in the CNS, and has been shown to play a role in appetite regulation (Sokolowski, 2003), behavior (Albers and Ferris, 1984), seizure activity (Vezzani et al., 1999) and memory (Redrobe et al., 1999). NEP-mediated proteolysis of NPY has been traditionally considered a terminal event, however it is possible that in the CNS some of these fragments might have neuroprotective effects relevant to AD. In this context, for the present study we show that C-terminal fragments (CTFs) of NPY derived from NEP processing might have neuroprotective effects in models of AD pathology. Taken together, our studies suggest that NEP might have a unique dual role by processing NPY into neuroactive fragments and reducing amyloid load in the CNS by degrading Aβ.
For these experiments, tg mice expressing high levels of human NEP and APP were utilized. Transgenic mice expressing human NEP under the regulatory control of the platelet-derived growth factor-β (PDGFβ) promoter were generated as previously described (Masliah et al., 2000). These mice were screened by PCR analysis of genomic DNA extracted from tail biopsies and screened for RNA and for levels of protein expression by Western blot. Three lines of mice were generated, and one line displaying the most stable levels of NEP expression was selected for crosses with the APP tg mice as previously described (Rockenstein et al., 2002b). Transgenic lines were maintained by crossing heterozygous tg mice with non tg C57BL/6 x DBA/2 F1 breeders. All mice were heterozygous with respect to the transgene. The APP tg mice express mutated (London V717I and Swedish K670M/N671L) human APP751 under the control of the murine Thy1 promoter (Thy1-hAPP, line 41) (Rockenstein et al., 2001). This tg model was selected because these mice produce high levels of Aβ1-42 and exhibit performance deficits in the water maze, synaptic damage, and plaque formation at an early age (beginning at 3 months) (Rockenstein et al., 2001; Rockenstein et al., 2002a). Additional experiments were performed using homozygous NEP-knockout (KO) mice (generously provided by Dr. Bao Lu, Harvard Medical School, Boston, MA) (Lu et al., 1995). Mice from all lines used were maintained until 6 months of age, followed by biochemical and neuropathological studies.
Briefly as previously described (Veinbergs et al., 2001), in order to evaluate the neuroprotective effects of NPY CTFs, groups of APP tg mice (6 months old) received intraventricular infusions with a cannula implanted into the skull and connected to osmotic minipumps delivering solutions of vehicle alone, amidated NPY CTFs (21-36; 31-36) or non-amidated NPY CTFs (21-36; 31-36). A total of 30 APP tg mice (n= 6 per group) were used for these experiments. Mice were treated for 28 days; the compound was dissolved in 0.9% NaCl/dimethylsulfoxide (90/10) at a concentration of 120 μM. Then, 200 μL of this solution was filled into the osmotic minipump (Alzet®, Charles River Laboratories Inc., Wilmington, MA, USA) ensuring constant delivery (0.2 μL/h). The mini pump was implanted subcutaneously on the back under light anesthesia. An additional group of control non tg (n=6) mice were implanted with minipumps filled with vehicle only. All experiments were approved by the animal subjects committee at the University of California, San Diego (UCSD) and were performed according to NIH recommendations for animal use.
In accordance with NIH guidelines for the humane treatment of animals, mice were euthanized by deep anesthesia with chloral hydrate. Brains were removed and divided sagitally. One hemibrain was post-fixed in phosphate-buffered 4% paraformaldehyde (pH 7.4) at 4°C for 48 hrs and sectioned at 40 μm with a Vibratome 2000 (Leica, Germany), while the other hemibrain was snap frozen and stored at -70°C for RNA and protein analysis.
The proteolytic activity of NEP was measured as previously described (Hemming et al., 2007) using the substrate 3-dansyl-D-Ala-Gly-p-(nitro)-Phe-Gly (DAGNPG; Sigma Pharmaceuticals Limited, Australia). Cell lysates from the neocortex, hippocampus, caudate, and cerebellum were incubated with 50 μM DAGNPG and 1 μM captopril (to inhibit any ACE cleavage of DAGNPG) in a volume of 200 μl at 37°C. Reactions were stopped by heating samples to 100°C for 5 min, followed by centrifugation. The supernatant was diluted into 50 mM Tris (pH 7.4) and fluorescence determined using a Victor2 multilabel plate reader (excitation 342 nm; emission 562 nm).
Western blot analysis was performed essentially as previously described (Rockenstein et al., 2001; Rockenstein et al., 2005a). Briefly, 20 μg per lane of cytosolic and particulate fractions, assayed by the BCA method (Pierce Biotechnology, Rockford, IL), were loaded into 4-12% SDS-PAGE gels and blotted onto polyvinylidene fluoride (PVDF) membranes. Blots were incubated with antibodies against total FL-APP (mouse monoclonal, clone 22C11, 1:500, Chemicon International, Temecula, CA), human APP (mouse monoclonal, 1:500, 8E5 clone, Elan Pharmaceuticals, South San Francisco, CA), Aβ (mouse monoclonal, clone 6E10, 1:1000, Signet Laboratories, Dedham, MA), NEP (mouse monoclonal, clone CD10, 1:1000, Abcam, Cambridge, MA), brain-derived neurotrophic factor (BDNF, mouse monoclonal, clone 35928.11, 1:1000, Calbiochem, San Diego, CA), nerve growth factor (NGF, mouse monoclonal, clone 25623.1, 1:1000, Oncogene, San Diego, CA), neurotrophin-3 (NT3, mouse polyclonal, 1:300, Promega, Madison, WI), NT4 (mouse monoclonal, clone 36507, 1:1000, R&D Systems, Minneapolis, MN), and NPY (mouse polyclonal, 1:1000, Peninsula Laboratories, San Carlos, CA) followed by secondary antibodies tagged with horseradish peroxidase (HRP, 1:5000, Santa Cruz Biotechnology, Inc.) and visualized by enhanced chemiluminescence and analyzed with a Versadoc XL imaging apparatus (BioRad, Hercules, CA). Analysis of actin levels was used as a loading control.
Brain samples from the frontal cortex were homogenized in ice-cold buffer [5 M guanidine-HCl and phosphate-buffered saline (PBS, pH 8.0)] with 1X protease inhibitor cocktail (Calbiochem). Homogenates were then mixed overnight at room temperature and subsequently diluted 10-fold in Dulbecco’s PBS (pH 7.4), containing 5% bovine serum albumin and 0.03% Tween-20. Samples were then centrifuged at 16,000 × g for 20 minutes at 4°C. The resulting supernatants were subjected to quantification with commercially available ELISA kits for NPY (Phoenix Pharmaceuticals, Belmont, CA), SP (Assay Designs, Ann Arbor, MI) and Met-ENK (Peninsula Laboratories).
To confirm the presence of NPY CTFs in mouse brain homogenates by an independent method, briefly as previously described (Medeiros Mdos and Turner, 1996), mass spectrometry was performed by HT Laboratories of San Diego, CA. Samples of non tg and tg mouse brains were normalized to 18 mg/ml and homogenized in 100mM Tris-HCl, pH 7.4, 1.0% Tween-20, 1M Thiorphan, and protease inhibitors (Calbiochem). Peaks matching the NPY 21-36 and 31-36 standards were found in the tissue samples and are depicted in the mass chromatogram given in supplemental Fig. 3G.
To evaluate the integrity of the neuronal structure, briefly as previously described (Rockenstein et al., 2005a; Rockenstein et al., 2005b), blind-coded, 40-μm thick vibratome sections from mouse brains fixed in 4% paraformaldehyde were immunolabeled with the mouse monoclonal antibodies against synaptophysin (synaptic marker, 1:20, Chemicon), microtubule-associated protein-2, (MAP2, dendritic marker, 1:40, Chemicon), NeuN (neuronal marker, 1:1000, Chemicon), or glial fibrillary acidic protein (GFAP, astroglial marker, 1:500, Chemicon) (Mucke et al., 1995). Additional sections were immunostained with the polyclonal antibody against NPY (1:200, Peninsula Laboratories). After overnight incubation with the primary antibodies, sections were incubated with fluorescein isothiocyanate (FITC)-conjugated horse anti-mouse IgG secondary antibody (1:75, Vector Laboratories), transferred to SuperFrost slides (Fisher Scientific, Tustin, CA) and mounted under glass coverslips with anti-fading media (Vector Laboratories). All sections were processed under the same standardized conditions. The immunolabeled blind-coded sections were serially imaged with the laser scanning confocal microscope (LSCM, MRC1024, BioRad) and analyzed with the Image 1.43 program (NIH), as previously described (Toggas et al., 1994; Mucke et al., 1995). For each mouse, a total of three sections were analyzed and for each section, four fields in the frontal cortex and hippocampus were examined. For synaptophysin and MAP2, results were expressed as percent area of the neuropil occupied by immunoreactive terminals and dendrites; for GFAP immmunostaining, levels were expressed as pixel intensity and for NeuN and NPY, the mean neuronal density was estimated using the disector method as previously described (Chana et al., 2003). Briefly, for stereology, immunolabeled sections were counterstained with 1% cresyl violet and analyzed with the optical disector. From each case, four 100-μm-wide fields from at least three sections (180-μm interval) per animal were analyzed and results averaged and expressed as total number per mm3.
To determine the expression levels of NPY, brain homogenates from control and tg mice were homogenized and fractioned as previously described into soluble and insoluble fractions (Kawahara et al., 2008). Briefly, tissues were sonicated in HEPES buffer with 1% Triton-X plus 1X thiorphan, phosphatase and protease inhibitors. Samples were centrifuged for 1 hr at 100,000 rpm in a Beckman TL100 rotor. Samples were run on a 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred onto 0.2 μm PVDF membranes with a 20% MeOH transfer buffer. The membranes were then blocked in 3% BSA and incubated with the rabbit polyclonal antibodies against FL-NPY (Peninsula Laboratories) or NPY CTFs. The antibody against NPY CTFs was prepared at Invitrogen by immunizing rabbits with the peptide corresponding to the amidated 31-36 aa of NPY with an extra Cys added to the N-terminus necessary for conjugation with BSA. Additional analysis was performed with a commercial rabbit polyclonal antibody specific for NPY CTFs (20-36) (Santa Cruz).
Because of the inherent difficulty in western blot detection of peptides as small as the NPY CTFs, some studies were performed utilizing an immunoblot protocol refined for the detection of very small proteins. For this purpose, approximately 0.1 g of tissue was obtained and sonicated in 100mM Tris-HCL pH 7.4, 1% Tween-20, containing protease, phosphatase and thiorphan inhibitors (Calbiochem). The homogenate was then fractionated by centrifugation at 4°C for 1 hour at 100,000 rpm. For western blot analysis, both the soluble and insoluble fractions were separated on 10-20% Tricine gels (Invitrogen) first at 125 volts for 10 minutes, then at 180 volts until the gel ran to completion. Before transfer, a 0.1 μm nitrocellulose membrane was incubated in 0.5% gelatin for 30 min at 37°C, and then allowed to dry for 1 hour at 56°C. The transfer was performed for 15 minutes at 10 volts and then the membrane was removed and placed into an air tight container with towels soaked in 37% formaldehyde for 2.5 hours at 37°C. After this fixation process the membrane was reactivated quickly with ddH2O and PBS. Immunoblots were then probed with antibodies against NPY as described above.
To evaluate the co-localization between APP and NEP and NPY and NEP, double immunocytochemical analysis was performed as previously described (Masliah et al., 2000). For this purpose, vibratome sections were immunolabeled with a monoclonal antibody against NEP (1:10,000, Abcam) detected with the Tyramide Signal Amplification™-Direct (Red) system (1:100, NEN Life Sciences, Boston, MA), and the mouse monoclonal antibody against human APP (1:500, 8E5 clone, Elan Pharmaceuticals) or the rabbit polyclonal antibodies against FL-NPY (Peninsula Laboratories) or NPY CTFs, detected with FITC-conjugated secondary antibodies (1:75, Vector Laboratories) (Masliah et al., 2000). All sections were processed simultaneously under the same conditions and experiments were performed twice to assess reproducibility. Sections were imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss, Germany) with an attached MRC1024 LSCM system (BioRad) (Masliah et al., 2000). To confirm the specificity of primary antibodies, control experiments were performed where sections were incubated overnight in the absence of primary antibody (deleted) or preimmune serum and primary antibody alone.
The relative rate of NPY processing by NEP was determined and the resultant products were visualized by both cell-free and ex vivo processing with homogenates from the tg mouse brains utilizing N-terminal FITC-tagged human NPY (1-36, AnaSpec, San Jose, CA). For the cell-free assays, 200 ng of human recombinant NEP (R&D Systems, Minneapolis, MN) was incubated with 100 mM Tris pH 7.4, 10 μM ZnCl2, and 50 μM FITC-labeled NPY in a final volume of 50 μL. For the ex vivo experiments with tissue homogenates, 1.2 mg of fresh murine brain tissue (from non tg, APP tg, NEP tg and APP/NEP tg mice) was sonicated in lysis buffer (100 mM Tris pH 7.4, and 1% Tween-20), and centrifuged at 5,000 rpm for 5 minutes at 4C. The resulting supernatant was then analyzed by the BCA method (Pierce) to determine protein concentration, after which 40 μg of protein was incubated with 100 mM Tris (pH 7.4) and 50 μM NPY-FITC in a final volume of 50 μL. Thiorphan (1 mM, Calbiochem) was utilized as a NEP-specific protease inhibitor. A time course was then performed for both cell-free and tissue-based assays. Aliquots were taken at varying time points, stopped with an equal volume of 8 M urea, run on 12% SDS-PAGE gels with MES buffer (Invitrogen), and analyzed with a Versadoc XL imaging apparatus (BioRad).
To verify the effects of NEP in cleaving NPY utilizing an alternative system, the ex vivo assay for NPY-FITC was performed with brain homogenates from mice that received intra-hippocampal injections with a lentiviral vector (LV) expressing either NEP, mutant (E585V) inactive NEP (NEP X), or green fluorescent protein (GFP). The effects of silencing NEP were studied by injecting a LV expressing either shRNA (a 19mer construct with a sequence of GCACGTGGTTGAAGACTTG, designed and cloned by Dr. O. Singer, The Salk Institute, La Jolla, CA) for NEP or a control scrambled shRNA. Briefly as previously described (Marr et al., 2003; Singer et al., 2005), 293T cells were transfected with vector and packaging plasmids, and the supernatants were collected and vectors concentrated by centrifugation. The LV titers were estimated by measuring the amount of HIV p24 gag antigen with an ELISA kit (100,000 TU / ng p24, Perkin Elmer Life Science, Boston, MA) or by flow cytometry using an anti-NEP specific antibody (56C6, Research Diagnostics, Inc.).
In total, 24 non tg (n=4 mice per group; 6 months old) (C57) mice were injected with 2 μL of the lentiviral preparations (1.5×109 TU) into the frontal cortex and hippocampus (using a 5 μL Hamilton syringe, 0.25 μL/min). Mice received bilateral injections with either LV control (empty vector), LV-NEP, LV-NEP X, LV-GFP, LV-shRNA NEP, or LV-shRNA scrambled. Four weeks post-injection, mice were sacrificed and the brains removed and prepared for immunoblot and ex vivo NPY-FITC analysis.
To investigate the potential neuroprotective effects of NEP-derived NPY fragments, cultures of primary human neurons were pretreated with NPY fragments followed by Aβ exposure. For this purpose, primary fetal human neurons (generously provided by G. Chana, UCSD) were plated onto a 48-well tissue culture plate at 5 × 105 cells/well in DMEM/F12 media (10% FBS, 1% sodium pyruvate, 0.1% non-essential amino acids, 0.75 g sodium bicarbonate in 500mL). Cells were then incubated for 24 hrs in minimal media containing either FL-NPY (AnaSpec) or amidated or non-amidated NPY CTFs (21-36 and 31-36, Invitrogen) at dilutions ranging from 1 nM-10 μM in 10% DMSO. Following NPY incubation, 10 μM of freshly solubilized Aβ1-42 (American Peptide) was added to each well, followed by fixation with 4% paraformaldehyde and immunocytochemical analysis with antibodies against synaptophysin and MAP2 as described above. In another set of experiments, the primary cultured neurons were pretreated with (S)-N2-[[1-[2-[4-[(R,S)-5,11-Dihydro - 6(6h)-oxodibenz[b,e]azepin-11-yl]-1-piperazinyl]-2-oxoethyl] cyclopentyl] acetyl] -N- [ 2- [1,2-dihydro -3,5 (4H)-dioxo - 1, 2 - diphenyl - 3H-1,2,4-triazol-4-yl]ethyl]-argininamid (BIIE0246, Y2 receptor inhibitor, Tocris Biosciences, Ellisville, MO) or R-N2-(Diphenylacetyl)-N-(4-hydroxyphenyl)-methyl argininamide (BIBP3226, Y1 receptor inhibitor, Sigma Aldrich) or vehicle control at 1 μM for 24 hrs followed by incubation with the amidated NPY 21-36 (10 nM) and challenge with 10 μM of freshly solubilized Aβ1-42 for 24 hrs. Coverslips were prepared in triplicate and analyzed with the MRC1024 LSCM system (BioRad) to determine the levels of synaptophysin immunoreactivity.
Analyses were carried out with the StatView 5.0 program (SAS Institute Inc., Cary, NC). Differences among means were assessed by one-way ANOVA with post-hoc Dunnett’s or Tukey-Kramer tests. All values in the figures are expressed as means ±SEM. Comparisons between 2 groups were done with the unpaired two-tailed Student’s t-test. Correlation studies were carried out by simple linear regression analysis and the null hypothesis was rejected at the 0.05 level.
Since NEP cleaves substrates other than Aβ, it is important to investigate the potential degradative effects of this metalloprotease on neuropeptides and trophic factors. For these studies tg mice expressing NEP under the PDGFβ promoter were generated. These tg mice express NEP approximately 1 to 1.5 fold above endogenous levels (Fig. 1A, B), while NEP KO mice show very low levels of NEP expression (supplemental Fig. 1A-C). The activity assay confirmed similar low levels of NEP activity in NEP KO mice (supplemental Fig. 1D), while in NEP tg mice there was a 50-60% increase in NEP activity in the neocortex and hippocampus, and an approximate 4-fold increase in the caudate, compared to the cerebellum (supplemental Fig. 1D). The increased levels of NEP activity were similar in APP/NEP double tg and NEP single tg mice (Fig. 1C). The NEP tg mice were crossed with APP tg mice because NEP has been shown to play an important role in the pathogenesis of AD and has been considered as a potential therapeutic target. Levels of total (including murine) APP and human APP were comparable between single APP tg mice and APP/NEP double tg mice. Levels of murine APP were comparable between non tg and NEP tg mice (Fig. 1A, B). Double labeling experiments confirmed that, compared to controls (Fig. 1D-F), the transgene-driven human APP and NEP co-localized in neuronal populations in the neocortex (Fig. 1G-I) and hippocampus. To investigate the effects of NEP overexpression on neuropeptides other than Aβ, levels of BDNF, NT3, NT4, SP, Met-ENK and NPY were analyzed by immunoblot. Remarkably, only levels of total FL-NPY were significantly affected in the APP/NEP and NEP tg groups compared to APP and non tg controls (Fig. 2A, B). Levels of the other neurotransmitter peptides and neurotrophic factors were not different among the four groups (Fig. 3A-C). Levels of Met-ENK were slightly elevated in the APP tg mice compared to non tg animals (Fig. 3D). Since previous studies have shown that NEP cleaves NPY at the C-terminus (Medeiros and Turner, 1994), the reduction in the levels of FL-NPY in the total homogenates of the NEP tg might be related to the proteolytic processing of this neuropeptide and the concomitant generation of potentially bioactive fragments rather than being due to decreased NPY expression. In support of this possibility, qRT-PCR showed no differences in the levels of NPY mRNA among the four groups of mice (Fig. 2C).
To search for NPY fragments consistent with NEP processing, immunoblot studies with antibodies against NPY CTFs, mass spectrometry, and ex vivo proteolysis analyses were performed in the brains of NEP tg and APP/NEP tg mice. To begin investigating if NEP promotes the formation of NPY fragments, western blot analysis of synthetic NPY peptides was performed with an antibody generated against the C-terminus of NPY (obtained from Santa Cruz Biotechnology). This antibody recognized the 0.8 kDa and 2 kDa bands corresponding to the 31-36 and 21-36 fragments of NPY (supplemental Fig. 2A). Immunoblot analysis with this NPY CTFs antibody using mouse brain homogenates and gelatin-coated, formaldehyde-treated membranes demonstrated the presence in vivo of the 0.8 kDa and 2 kDa bands corresponding to the NPY CTFs in the insoluble fraction (Fig. 2D). Compared to the non tg and APP tg mice, in the NEP and APP/NEP tg mice levels of immunoreactivity for the lower MW bands corresponding to NPY CTFs were increased in the insoluble fraction (Fig. 2D, E). To confirm the results obtained with the commercial NPY CTFs antibody, we generated a polyclonal antibody with the peptide corresponding to aa 31-36 of NPY. Immunoblot analysis with synthetic NPY peptides showed that this antibody recognized a band at a molecular weight (MW) of 2 kDa consistent with the 21-36 CTF, and a band at approximately 0.8 kDa consistent with the 31-36 NPY CTF (supplemental Fig. 2A). Similar to the commercial NPY CTFs antibody, by immunoblot analysis our antibody (generated with the 31-36 peptide of NPY) detected bands at a MW of 0.8 kDa and 2 kDa in the insoluble fraction of the brain homogenates (supplemental Fig. 3A, B). Consistent with this result, in the NEP and APP/NEP tg mice levels of immunoreactivity with our antibody were increased for the lower MW bands corresponding to NPY CTFs (supplemental Fig. 3A,B).
Immunocytochemical analysis with the commercial NPY CTFs antibody (Fig. 2F-I) showed immunoreactivity associated with neurons similar to those recognized by the antibody against FL-NPY but also immunolabeled a subset of pyramidal neurons (Fig. 2H, I, supplemental Fig. 2B, C). Our NPY CTFs antibody displayed similar patterns of immunoreactivity (supplemental Fig. 2D, supplemental Fig. 3C-F). Compared to non tg and APP tg mouse brains, the antibody from the commercial source (Fig. 2F-I) and our NPY CTFs antibody (supplemental Fig. 3C-F) showed more intense immunoreactivity in the NEP and APP/NEP tg mice. In contrast, in NEP KO mice, levels of NPY CTFs were reduced compared to non tg and NEP tg animals (supplemental Fig. 1E-G, K), while FL-NPY levels were not modified (supplemental Fig. 1H-J, L).
In order to provide additional confirmation of the presence of NPY CTFs in vivo, mass spectrometry analysis was performed with mouse brain homogenates. This study showed that FL-NPY (supplemental Fig. 3G) and two distinct fragments of NPY consistent with NEP processing (21-36 and 31-36, supplemental Fig. 3G) could be detected in the brains of non tg and NEP tg mice.
Further analysis by laser scanning confocal microscopy of double-labeled sections confirmed that NEP was present in neurons displaying NPY CTFs (Fig. 4A-C) and FL-NPY (Fig. 4D-F) immunoreactivity in NEP and APP/NEP tg mice. NPY was present in approximately 8% of the NeuN positive neurons (Fig. 4G-I). Abundant NPY positive axons were identified in the neuropil, and the varicosities of these axons co-localized with the nerve terminal marker, synaptophysin (Fig. 4J-L). Taken together, these studies suggest that NEP mediates the processing of NPY into CTFs that can be detected at higher concentrations in the brains of NEP tg mice.
Since the increased levels of NPY fragments in NEP tg mice and co-localization of NEP and NPY in neurons suggests that these two proteins might interact in the same subcellular compartments, we developed an ex vivo detection system to further investigate the interactions of NEP and NPY and the ability of NEP to generate NPY fragments. For this purpose, FITC-tagged (at the N-terminus site) NPY was incubated with homogenates from non tg and tg mice. After 4 hrs of incubation of the tagged peptide with the brain homogenates, gel electrophoresis and image analysis of the resulting bands were performed. This study showed that levels of FITC-tagged NPY were reduced in all four groups (Fig. 5A). Moreover, brain homogenates cleaved the FITC-tagged NPY, resulting in the generation of two lower molecular-weight bands, one at approximately 3.5 kDa, consistent with the N-terminal 1-30 fragment (corresponding to the C-terminal 31-36) and a fainter and more diffuse band at 2.2 kDa representing the N-terminal 1-20 fragment (corresponding to the C-terminal 21-36) (Fig. 5A, B). The brain homogenates from the NEP and APP/NEP tg mice generated on average 50% more N-terminal NPY fragments compared to APP tg and non tg controls (Fig. 5C-E). The effects of the brain homogenates on NPY cleavage were blocked by the NEP inhibitor thiorphan (Fig. 5A) but not by other protease inhibitors (not shown). Incubation of the FITC-tagged NPY with recombinant human NEP resulted in similar patterns of cleavage that were inhibited by thiorphan (Fig. 5A).
To verify the ex vivo effects of NEP with an alternative model, levels of NPY-FITC cleavage were analyzed in mice that received intra-cerebral injections with a LV expressing NEP or an shRNA to silence NEP (Fig. 6A). Four weeks post-infection, the levels of NEP expression in the area of the injection were analyzed by immunoblot. Whereas with LV-NEP, levels of expression were increased by 45% (Fig. 6B), with LV-NEP shRNA, there was a 90% decrease (Fig. 6B). When NPY-FITC was incubated with homogenates from the brains of mice that received LV-NEP, levels of FL-NPY were reduced (Fig. 6C), while levels of NPY CTFs were increased (Fig. 6D). In contrast, NPY-FITC processing was reduced when incubated with brain homogenates from mice that were injected with LV-NEP shRNA (Fig. 6E, F). No significant effects were observed when NPY-FITC was incubated with brain samples from mice inoculated with a mutant, inactive NEP (LV-NEP X) (Fig. 6C, D) or with a scrambled LV-shRNA (Fig. 6E, F). Together, these studies indicate that most of the NEP activity in the mouse brain cleaves NPY between aa 20 and 21, resulting in the generation of the 21-36 and 31-36 aa CTFs.
To investigate if the NPY CTFs generated by NEP processing might be neuroprotective or represent a pathway to terminate the effects of NPY in the CNS, amidated and non-amidated peptides of 21-36 and 31-36 NPY CTFs (Fig. 7A) were infused into the brains of non tg and APP tg mice. After 4 weeks, the brains of mice were analyzed with antibodies against NPY CTFs and MAP2. Confocal microscopy showed that compared to vehicle controls (Fig. 7B, C), mice that were infused with NPY 21-36 (Fig. 7D, E) and 31-36 (not shown) peptides displayed increased levels of NPY CTFs immunoreactivity in the neocortex and hippocampus. Compared to vehicle-infused non tg controls (Fig. 7F, J), vehicle-infused APP tg mice displayed a significant decrease in the area occupied by MAP2-immunoreactive dendrites in the neocortex (Fig. 7G, J). In contrast, infusion of amidated NPY 21-36 or NPY 31-36 ameliorated the neurodegenerative pathology in the APP tg mice (Fig. 7H-J). However, infusion of the non-amidated NPY CTFs did not revert the alterations in MAP2 immunoreactivity in the APP tg mice (Fig. 7J).
To confirm if the NPY CTFs generated by NEP processing might be neuroprotective, primary human cortical neurons were pre-treated with amidated and non-amidated FL-NPY, NPY 21-36, NPY 31-36, a scrambled peptide, or vehicle alone (DMSO) for 24 hrs followed by challenge with Aβ1-42 (Fig. 8). Laser scanning confocal analysis of cells double-immunolabeled with antibodies against synaptophysin and MAP2 showed that compared to vehicle-treated cells (Fig. 8A, E), Aβ1-42 treatment alone resulted in an average 40% decrease in synaptophysin immunoreactivity after 24 hrs of exposure (Fig. 8B, E). In contrast, pre-treatment with the amidated NPY peptides protected neurons from the Aβ1-42-mediated decrease in synaptophysin immunoreactivity compared to controls (Fig. 8C, E). The amidated FL-NPY had similar neuroprotective effects to the 21-36 and 31-36 NPY CTFs. No significant differences were noted between amidated 21-36, 31-36 or FL-NPY. The scrambled peptide (not shown) and the non-amidated fragments had no protective effects (Fig. 8D, E). To verify the specificity of the effects of the NPY fragments, primary neuronal cells were pre-treated with a Y1 (BIBP3226) or Y2 receptor (BIIE0246) inhibitor followed by NPY and Aβ exposure. This study showed that the Y2 receptor inhibitor was able to block the protective effects of the amidated 21-36 NPY CTF in primary neuronal cells challenged with Aβ1-42 (Fig. 8F). In contrast, the Y1 inhibitor and a control had no effects in blocking the neuroprotective effects of the 21-36 NPY CTF (Fig. 8F).
Together, the in vitro and in vivo studies support the contention that NPY CTFs resulting from NEP activity have neuroprotective abilities in AD-related models.
Most studies on NEP activity in AD models have focused on the ability of NEP to degrade Aβ (Iwata et al., 2001; Leissring et al., 2003; Mohajeri et al., 2004; Carter et al., 2006; Huang et al., 2006; Farris et al., 2007; El-Amouri et al., 2008; Iijima-Ando et al., 2008), however NEP is known to cleave other neuropeptides and processing of these alternative substrates might contribute to the neuroprotective effects. For this reason we investigated the effects of NEP on neurotrophic factors and neuropeptides. Our study showed that expression of NEP at moderate levels in tg mice and in crosses with an APP tg model results in increased generation of NPY CTFs that displayed neuroprotective activity both in primary neurons and APP tg mice.
The mature NPY protein, one of the most abundant neuropeptides in the CNS, is a 36 aa protein that possesses an amidated C-terminal residue (Medeiros Mdos and Turner, 1996; Silva et al., 2005). The FL-NPY can be cleaved by dipeptidyl peptidase IV and aminopeptidase P resulting in NPY 3-36 and NPY 2-36, respectively, and these fragments are agonists of the Y2/Y5 receptors (Silva et al., 2003). Although considerable attention has been devoted to the NPY 2-36 and NPY 3-36 because of their ability to regulate food intake (Naveilhan et al., 1999), less is known about the intriguing role of alternative, shorter NPY CTFs (21-36, 31-36).
In the present study we show that NPY CTFs consistent with NEP processing can be found in the brains of NEP and NEP/APP tg mice. This is in agreement with other in vitro analysis that NEP can generate such CTF products (Medeiros Mdos and Turner, 1996). FL-NPY can be cleaved by NEP at four different places in the C-terminal region; the primary sites are between Tyr20-Tyr21 and Leu30-Ile31. This cleavage results in the production of the N-terminal fragments of NPY 1-20 and NPY 1-30 and the complementary C-terminal portions of NPY 21-36 and NPY 31-36. In general, it has been thought that NEP hydrolyzes and terminates the activity of a variety of neuropeptides, including enkephalins, tachykinins, brain natriuretic peptides and somatostatin (Skidgel and Erdos, 2004); however, the present study suggests that the NPY CTFs resulting from NEP processing might play an active role in neuroprotection. This hypothesis is consistent with a previous report that has proposed that such fragments might bind the Y receptors (Kaga et al., 2001) and that NPY protects hippocampal neurons from glutamate excitotoxicity in a receptor-dependent manner (Silva et al., 2003). Previous studies have shown that binding of NPY to the Y receptors protects CA1 and binding to Y1, Y2 and Y5 protects CA3 and the dentate gyrus (Silva et al., 2003). The direct detection and binding to the Y receptors by the putative C-terminal NPY fragments derived from NEP processing awaits future investigation.
NPY fragments consistent with NEP processing have been also detected by MALDI-mass spectrometry in the CSF of control and AD patients (Nilsson et al., 2001); and here we confirmed their presence in the brains of non tg and tg mice by mass spectrometry. However, it has been difficult to detect these products in the brain. For this reason, we generated a new NPY CTFs antibody (with a coupled 21-36 peptide) and used FITC-tagged FL-NPY to show ex vivo that NEP in brain homogenates generates NPY fragments that, by infusion into APP tg mice and in neuronal cultures, provide protection from the toxic effects of Aβ. The difficulty in directly detecting NPY fragments in the brain might be related to the possibility that these products are generated at low levels in vivo and that they have short half-lives. Thus, isolating and characterizing the bioactivity of these fragments will require more detailed investigation. Nonetheless, it is reasonable to postulate that NPY CTFs might be neuroactive, since the intact C-terminus is necessary for binding to Y2 whereas the N-terminus is necessary for binding to the Y1 receptor. Moreover, while Y1 is mostly postsynaptic and has been linked to food intake and anxiety (Silva et al., 2005), the Y2 receptor is mainly presynaptic and activation of the Y2 receptor in the hippocampus has been implicated in learning and memory (Redrobe et al., 2004). Thus it is possible that rather than simply representing terminal products of hydrolysis, the amidated shorter NPY CTFs generated by NEP might protect from neurotoxicity by activating Y2 receptors in the hippocampus. In the present study we showed that direct delivery of NPY CTFs is neuroprotective in a mouse model of AD. Consistent with these findings, a recent study showed that adeno-associated virus (AAV)-mediated expression of NPY CTFs is neuroprotective in a rat seizure model (Foti et al., 2007).
This is also of interest because a recent study by Palop et al (Palop et al., 2007) showed that abnormalities in the NPY network in the hippocampus of APP tg mice might be responsible for the seizure activity that is characteristic in these mice and that has also been reported in patients with advanced AD (Silva et al., 2005; Amatniek et al., 2006). This suggests that therapy with NEP or alternatively with NPY CTFs might play a role in remodeling and controlling seizure activity in the APP tg model. Future studies will be necessary to investigate this possibility.
NPY is co-localized with somatostatin and GABA in interneurons in the cerebral cortex and subcortical white matter (Jinno and Kosaka, 2003). Moreover, we have shown that NPY also co-localizes with NEP in the brains of tg mice. During the aging process (Cha et al., 1997; Cadacio et al., 2003) and in AD, NPY neurons are susceptible to degeneration and previous studies have shown decreased levels of these neuropeptide-positive neurons in the neocortex and hippocampus (Chan-Palay et al., 1985; Davies et al., 1990). In APPxPS1 tg mice, the levels of NPY mRNA and the numbers of NPY/somatostatin interneurons are significantly reduced (Ramos et al., 2006). In PDAPP (Diez et al., 2000) and APP23 tg mouse lines, NPY fibers in the hippocampus are prominent (Palop et al., 2007) and dystrophic neurites around plaques show abundant NPY, galanin, ENK and cholecystokinin immunoreactivity (Diez et al., 2003).
Finally, our study also showed that at moderate levels NEP overexpression had no adverse effects on neurotrophic factors and other neurotransmitter pathways. NEP did not alter the expression or proteolysis of BDNF, NGF, NT4, NT3, or other NEP substrates, such as SP. This finding is important because, based on its anti-amyloidogenic and neuroprotective effects, NEP has been considered as a potential therapeutic target for AD. In conclusion, this study suggests that NEP might also have beneficial effects by generating protective neuropeptides. This function of NEP represents a unique example of a proteolytic enzyme with dual action—namely, degradation of Aβ as well as processing of NPY.
Characterization of NEP expression and activity in various brain regions in tg and KO mouse models. A-C, Patterns of NEP expression in the brains of non tg, NEP tg and NEP KO mice. D, Levels of NEP activity in the neocortex, hippocampus, caudate and cerebellum of non tg, NEP tg and NEP KO mice (n=3 mice per group; 6 months old). E-G, The NPY CTFs antibody was custom generated (Invitrogen) with a 31-36 peptide of NPY. This antibody immunostained non-pyramidal neurons and some pyramidal neurons in the frontal cortex of non tg and NEP tg mice, and detected reduced immunoreactivity in the brains of NEP KO mice. H-J, The antibody against FL-NPY recognized non-pyramidal neurons in the neocortex of non tg, NEP tg, and NEP KO mice. K, Semi-quantitative analysis of levels of NPY CTFs in the frontal cortex of non tg, NEP tg and NEP KO mice. J, Semi-quantitative analysis of levels of FL-NPY in the frontal cortex of non tg, NEP tg and NEP KO mice. (*p < 0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett’s; n=3 mice per group; 6 months old). Scale bars, (A-C) 100 μm, (E-G, H-I) 40 μm.
Characterization of the polyclonal antibodies against NPY CTFs. A, Representative immunoblot with the antibodies against FL-NPY and NPY CTFs. Lanes were loaded with FL (1-36), NPY CTFs (21-36; 31-36), scrambled peptide or mouse brain homogenate. The rabbit polyclonal NPY CTFs (1) antibody was generated with a 20-36 peptide of NPY (Santa Cruz). The rabbit polyclonal NPY CTFs (2) antibody was custom generated (Invitrogen) with a 31-36 peptide of NPY. These two antibodies preferentially recognize the NPY CTFs. B, The antibody against FL-NPY recognizes non-pyramidal neurons in the neocortex. C, D, The antibodies against NPY CTFs immunostained non-pyramidal neurons and some pyramidal neurons. E-G, Only background staining was observed in control mouse brain sections reacted with the pre-immune serum or the peptide-adsorbed antibodies. Scale bar, 40 μm.
Detection of NPY fragments in NEP and APP/NEP tg mice with an NPY CTFs antibody and by mass spectrometry analysis. A, Representative immunoblot analysis of levels of NPY CTFs utilizing an antibody raised with a peptide corresponding to the NPY 31-36 region. The 1.6 kDa band represents the 21-36 fragment and the 0.8-kDa band the 31-36 fragment. B, Increased immunoblot levels of NPY CTFs immunoreactivity in APP/NEP and NEP tg mice compared to controls. C-F, Patterns of NPY CTFs immunoreactivity comparing non tg and tg mice. Immunoreactivity was associated with some interneurons and pyramidal neurons in the neocortex. More intense immunostaining was observed in the APP/NEP and NEP tg mice compared to non tg controls. G, Representative mass chromatograms from homogenates of brains from NEP tg mice showing FL-NPY 1-36, 21-36 fragment of NPY, and 31-36 fragment of NPY. NL = neutral loss; m/z = mass to charge ratio (*p < 0.05 compared to non tg controls by one-way ANOVA with post-hoc Dunnett’s; n=6 mice per group; 6 months old). Scale bar, 40 μm.
This work was supported by NIH Grants, AG10435, AG022074, AG18440, and AG5131.