Peripheral Delivery of a CNS Targeted, Metalo-Protease Reduces Aβ Toxicity in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD), an incurable, progressive neurodegenerative disorder, is the most common form of dementia. Therapeutic options have been elusive due to the inability to deliver proteins across the blood-brain barrier (BBB). In order to improve the therapeutic potential for AD, we utilized a promising new approach for delivery of proteins across the BBB. We generated a lentivirus vector expressing the amyloid β-degrading enzyme, neprilysin, fused to the ApoB transport domain and delivered this by intra-peritoneal injection to amyloid protein precursor (APP) transgenic model of AD. Treated mice had reduced levels of Aβ, reduced plaques and increased synaptic density in the CNS. Furthermore, mice treated with the neprilysin targeting the CNS had a reversal of memory deficits. Thus, the addition of the ApoB transport domain to the secreted neprilysin generated a non-invasive therapeutic approach that may be a potential treatment in patients with AD.


Introduction
Alzheimer's disease (AD) is an incurable progressive neurodegenerative disorder affecting over 10 million people in the US alone [1]. This neurological disorder is characterized by widespread neurodegeneration throughout the association cortex and limbic system, deposition of Ab in the neuropil and around the blood vessels, and formation of neurofibrillary tangles [2]. In spite of the considerable progress towards better understanding the pathogenesis of AD, no effective therapeutic approaches are currently available. A fundamental problem toward the goal of developing new therapies for AD has been the difficulty in crossing the blood brain barrier (BBB) [3].
Experimental treatments for AD include reducing the synthesis or aggregation of Ab or increasing the clearance of Ab. Recently, progress has been made towards identifying endopeptidases, which directly degrade Ab and play an important role in the homeostatic control of this peptide. Among them, Neprilysin (NEP, also known as CD10, EC 3.4.24.11)-a zinc metalloendopeptidase-has been identified as a critical Ab-degrading enzyme in the brain [4,5,6]. Neprilysin has been shown to degrade Ab monomers; however, the ability of NEP to degrade Ab-oligomers is controversial while some groups have reported that this endopeptidase breaks down oligomers [7,8], others have not seen such effects [9,10]. Neprilysin levels are reduced in the brains of AD patients and a potential genetic linkage is currently being investigated (reviewed in [11]). We and other groups have shown that overexpression of NEP by gene transfer with viral vectors [12,13,14], transgenesis [15], or induction [16,17,18,19,20] resulted in a reduction in amyloid pathology.
Viral vector gene delivery of NEP via stereotactic injection into the CNS has proven to be a viable approach to treating the small brain of mice or rats; however, scaling up to the size of the human brain would require numerous injections that would make these treatments undesirable. An alternative approach for delivery of a therapeutic protein to the CNS is by transport across the BBB.
Recently, a novel approach was developed for delivering therapeutic proteins to neurons of the CNS by targeting passage across the BBB [21]. Fusion of the Apolipoprotein B (ApoB) lowdensity lipoprotein (LDL) receptor-binding domain to a targeted protein allows active transport of the protein across the BBB to the CNS. The fusion proteins can be taken up by neurons and astrocytes across the whole brain. To investigate the potential therapeutic value of a secreted NEP targeted to the CNS, we generated lentivirus vectors expressing either the wildtype NEP, a secreted form of the NEP or a secreted form of the NEP fused with the LDL-receptor binding domain of ApoB and injected them intra-peritoneally in an amyloid protein precursor (APP) transgenic (tg) model of AD-like pathology. We found that ApoBSec-NEP was efficiently trafficked into the CNS and reduced levels of Ab and synaptic alterations in the brains of mice. In addition, we observed improvements in learning and memory just 1 month after vector delivery. These results suggest a novel, and improved approach for delivery of an Ab degrading enzyme and other neuroactive peptides to the CNS for treatment of AD.

Results
The apoB-secreted neprilysin fusion protein is active and functions similarly to endogenous neprilysin To determine if the fusion ApoBSecNEP protein and SecNEP variant proteins were secreted and active, lentiviruses were produced and used to infect 293T cells. Infected cell lysates and conditioned media were collected 72 hours after virus infection and analyzed by western blot. The NEP antibody recognized a band at ,100 kDa in the lysates of the 293T cells infected with the LV-NEP, LV-SecNEP and the LV-ApoBSecNEP ( Figure S1B). This band was not observed in uninfected 293T cells or cells infected with the LV-ApoBGFP virus indicating that NEP is not endogenously produced by the 293T cells. Conditioned media contained the secreted NEP reacting band at 100 kDa only in cultures infected with the LV-SecNEP and LV-ApoBSecNEP viruses whereas the media from control cells or cells infected with the LV-NEP did not contain NEP ( Figure S1C).
In order to determine if the vectors expressing the fusion NEP constructs were enzymatically active, cell lysates or conditioned media samples were incubated with an N-terminus FITC labeled Ab 42 for 24 hours. In this system, NEP activity is detected by the presence of a 2 kDa band corresponding to the N-terminal cleavage product of Ab. Lysates of infected 293T cells showed increased NEP activity from all the NEP vectors tested with the wildtype NEP displaying a 6 fold increase in Ab cleavage compared to uninfected 293T cells ( Figure 1A). Of particular interest, the SecNEP and ApoBSecNEP proteins were present at reduced levels in the lysates of the infected cells, however the NEP activity was as efficient as wild type NEP with the SecNEP and 50% higher with the ApoBSecNEP ( Figure 1C) Conditioned media from infected cells was subjected to the same N-terminus FITC labeled Ab cleavage assay ( Figure 1B). Low levels of Ab cleavage activity were observed from uninfected and LV-NEP infected conditioned media, however as expected, NEP activity was present in the conditioned media of cells infected with either the LV-SecNEP or LV-ApoBSecNEP vectors. Ab cleavage was detected at levels comparable to cell lysates only in those samples that had the secreted NEP ( Figure 1D). The Ab cleavage was specific to the presence of NEP as the addition of thiorphan, a NEP specific inhibitor, blocked the formation of the Ab cleavage product ( Figure 1A,B).
To determine if the fusion NEP constructs were active in a cell based system, differentiated adult rat neural progenitor cells (NPCs) exposed to Ab were utilized. In this system, differentiated NPCs treated for 24 hours with Ab showed reduced levels of btubulin immunoreactivity and increased activated caspase 3 levels indicating Ab induced toxicity (Figure 2A-C). To examine whether the recombinant NEP could protect the differentiated NPCs cells from the Ab induced toxicity, cells were infected with the LV-NEP, LV-SecNEP or LV-ApoBSecNEP and then challenged with Ab (10 nM) for 24 hours. Cells that expressed either the wildtype NEP or either of the secreted NEP proteins had increased b-tubulin immunoreactivity and showed no increase in In conclusion, the lentiviral vectors containing SecNEP and fusion ApoBSecNEP express the NEP protein and secrete the protein into the supernatent of infected cells. In addition, the fusion NEP constructs are active at cleaving the Ab 42 protein in a similar manner to the wildtype NEP and can protect differentiated NPCs from an extracellular challenge of Ab. The next step was to determine if the ApoBSecNEP protein traffics into the brain of APP tg mice and if it reduces the pathology and deficits associated with amyloid production.

ApoBSecNEP traffics into the CNS of APP tg mice and accumulates in neurons in the hipoocampus and neocortex
To determine whether these vectors might be effective at reducing the levels of Ab in vivo, we delivered 1610 9 tdu of lentivirus vector to 6 month old APP tg mice via a single intraperitoneal injection. One month after injection, mice were sacrificed and brains were removed for analysis of Ab and NEP. In order to verify that the ApoBSecNEP molecule trafficked into the CNS, immunohistochemical analysis was performed. One month after lentivirus delivery in the periphery, widespread NEP uptake was detected only in those mice that had received the LV-ApoBSecNEP injections ( Figure 3D-F) whereas only low levels of NEP were observed in those mice that received LV-SecNep ( Figure 3A-C). Neprilysin was particularly concentrated in the hippocampus and the lower layers of the cortex and could be identified in both the soma and axon of neurons. Consistent with these results and our previous studies [21], control experiments utilizing a lentiviral vector expressing the ApoB-GFP fusion protein confirmed that at 30 days after intra-peritoneal injections, GFP accumulation was detected in the hippocampus ( Figure S2).
Analysis of the levels of NEP immunoreactivity ( Figure 3G) and enzyme activity ( Figure 3H) showed almost 10 fold more enzyme in the brains of mice that had received the ApoBSecNEP compared to those that received SecNEP or control animals. Interestingly, the levels of NEP enzyme that accumulated in the CNS of the APP tg mice was significantly greater than in the brains of nontg mice. Western blot analysis from the CNS confirmed the immunohistochemical results ( Figure S3).
Closer examination of the sections from mice that received the LV-ApoBSecNEP showed recombinant protein throughout the hippocampus. In particular, NEP co-localized with NeuN and MAP2 positive neurons in the CA1 region and dentate gyrus of the hippocampus ( Figure 4). Intracellular recombinant protein accumulated in the neuronal soma with some extending into the axons (Figures 3E, 4A-C).

ApoBSecNEP reduces the levels of Ab in APP tg mice
Previous studies have shown that NEP preferentially degrades monomeric forms of Ab rather than fibrils [9,10]. Thus to evaluate the effects of ApoBSecNEP on Ab, we dissected the hippocampus of the mice treated with the lentiviral vectors and analyzed by immunoblot. Compared to APP tg mice treated with LV-control, LV-NEP or LV-secNEP, treatment with LV-ApoBSecNEP resulted in a 60% reduction in the levels of Ab monomer ( Figure 5A,B). In addition, the levels of an Ab immunoreactive band right above the 4 kDa monomer was noted to be significantly reduced with the LV-ApoBSecNEP. The identity of this band at approximately 6 kDa is unclear but might represent a posttranscriptionally modified version of Ab that is sensitive to NEP proteolysis. In contrast, no significant effects were observed in the levels of full length APP ( Figure 5A,C).
When the effects of the LV treatments were analyzed by immunocytochemistry, one month followed intra-peritoneal de- livery of the LV-ApoBSecNEP, mice had a 70% reduction in the load of Ab immunoreactive plaques in the neocortex and hippocampus compared to APP tg mice treated with either LVcontrol and LV-NEP ( Figure 5D-H; 5I-L). The APP tg mice treated with LV-secNEP displayed a 20% reduction in the load of Ab immunoreactive plaques in the hippocampus ( Figure 5H).
Since NEP favors the cleavage of monomers rather than fibrils, the reduction in the number of plaques might be driven in part by the change in ratio between monomeric and aggregated Ab, however other factors might be involved including increased clearing by microglia. Consistent with this possibility, double labeling and confocal microscopy showed that compared to APP tg mice treated with LV-control, in mice treated with LV-ApoBsecNEP macrophage/microglial cells embedded in the plaques displayed abundant NEP immunoreactivity ( Figure 6). Higher resolution and double labeling analysis confirmed that the cells associated with the plaques were immmunostained with antibodies against macrophage markers such as CD68 and CD11b and contained abundant aggregated Ab immunoreactive material ( Figure S4). Taken together, these results suggest that the ApoBSecNEP might also be taken up by macrophages and activate these cells to promote amyloid clearance.
In addition to the effects on extracellular Ab, NEP might modify AD-like pathology by reducing the accumulation of intracellular APP metabolites such as Ab [22,23]. Increasingly, intracellular Ab is being recognized as deleterious to the neuron as much or more than extracellular Ab [23,24,25]. Detection of intracellular Ab by immunocytochemistry is difficult because of the cross-reactivity of most antibodies with APP. However, some studies have suggested that use of antibodies against the amino terminal of Ab might be helpful [23,24,25]. Immunocytochemical analysis with the monoclonal antibody against Ab 1-16 (clone 82E1) showed that compared to APP tg mice treated with LV-control, LV-NEP or LV-secNEP, mice treated with LV-ApoBSecNEP displayed a 60% reduction in the levels of intracellular APP/Ab immunoreactivity ( Figure 5M-Q).

ApoBSecNEP ameliorates the alterations in synaptic markers in APP tg mice
It has been proposed that Ab neurotoxicity targets the synapses [7,26,27]. To determine if delivery of the ApoBSecNEP could correct the synaptic alterations in these mice, immunohistochemical and western blot analysis were performed. Mice that received the LV-control, LV-NEP or LV-SecNEP had significantly decreased levels of the post-synaptic marker, PSD 95, and the pre-synaptic marker, SNAP25, comparable to untreated APP tg mice ( Figure 7A-C, E-G). Whereas mice that received the LV-ApoBSecNEP showed levels of both synaptic proteins comparable to nontg mice ( Figure 7D,H). Similarly, delivery of the ApoBSecNEP to nontg mice did not appear to affect synaptic protein levels indicating that accumulation of the soluble neprilysin did not have a detrimental effect ( Figure 7I,J). Consistent with the immunocytochemical analysis, western blot analysis confirmed that, compared to APP tg mice that received the LV-control, LV-NEP or LV-SecNEP, mice that were treated with LV-ApoBSec-NEP displayed levels of PSD 95 and SNAP25 comparable to nontg controls ( Figure 7K-M).

ApoBSecNEP reverse memory deficits in APP tg mice
The alterations in synaptic markers in APP tg mice have been associated with memory and learning deficits beginning at 3-6 months of age as measured in the Morris water maze [28]. To determine if delivery of the ApoBSecNEP could ameliorate these deficits, APP tg mice were tested one month after intraperitoneal delivery of either the LV-control, LV-SecNEP or LV-ApoBSec-NEP vectors. During the training period of the test, all groups of mice performed similarly at locating the visible platform after 3 days of testing. In the subsequent days of testing (d4-7) with the platform submerged nontg mice located the platform in the water pool, with progressively shorter path distances over time ( Figure 8A). Consistently, linear regression analysis showed a significant negative slope when plotting distance over time ( Figure 8B). Compared to nontg controls, APP tg mice that received the LV-control did not show improvements in the spatial learning with the submerged platform and the path distance in fact increased over time ( Figure 8A) with a positive slope for the linear regression analysis ( Figure 8C). Similarly, the APP tg treated with LV-secNEP displayed poor performance taking longer distances to locate the hidden platform for days 4-6, however at day 7 displayed a trend toward an improvement ( Figure 8A). However, the linear regression analysis was not significant ( Figure 8D). In contrast, APP tg mice that received the LV-ApoBSecNEP vector were able to learn the location of the hidden platform significantly faster and at a rate similar to nontg mice ( Figure 8A). In concordance, linear regression analysis showed a significant negative slope when plotting distance over time ( Figure 8E). Taken together, these results suggest that delivery of the ApoBSecNEP protein to the CNS of APP tg mice has the capability of ameliorating the synaptic and memory deficits

Discussion
Development of new approaches for the delivery of neuroactive peptides and compounds across the BBB is of fundamental importance for advancing the therapeutics of AD. For the present study, we showed that the ApoBSecNEP fusion protein was efficiently produced from the lentiviral vector in peripheral organs, trafficked into the CNS and reduced levels of Ab and synaptic alterations in the brains of mice. In contrast to previous methods for delivery of soluble NEP [29,30], this study utilized a BBB targeting peptide fused to the soluble protein for transport to the CNS. We had previously shown that we could deliver a recombinant protein across the CNS through the use of the LDL-R binding domain of ApoB [21], however, this is the first report showing that this approach can be used in a therapeutic manner to treat a neurodegenerative disease.
Utilizing this unique approach, intra-peritoneal delivery of the lentivirus vector results in primarily transduction of the liver and spleen [21,31], with the liver acting as a depot organ for the production and secretion of the recombinant protein. In this study, the ApoBSecNEP expressed in the liver was delivered to the blood stream from where it traffics to the CNS. The ApoB LDL-R binding domain on the recombinant protein specifically facilitates transport across the BBB since the SecNEP protein (lacking apoB) was not present in the CNS following similar vector delivery and did not mitigated the neuropathogy in the APP tg mice. Delivery to the CNS by this route resulted in widespread uptake as evidenced by the immunoreactivity across areas of the CNS including the hippocampus and the neocortex, that are prominently affected in AD [2,32]. Moreover, our studies showed that the fusion protein displayed, both in vitro and in vivo enzymatic activity comparable to the wildtype secNEP.
This activity included cleavage of an artificial specific substrate, an FITC-tagged Ab 1-42 and the human Ab produced in the brains from the transgene. Upon examination of the Ab species in the CNS, we observed a significant reduction in the monomer Ab but not full length APP. This is consistent with previous studies showing that NEP favors the degradation of monomeric Ab over fibrillar species. The effects of the ApoBsecNEP vector on the levels of Ab oligomers were difficult to determine by western blot given the proximity of the molecular weight with C-terminus fragments of APP. Future studies utilizing SELDI-TOF mass spectrometry analysis will be needed to identify with greater precision trimers and other Ab multimers in tissues as has been recently done by Crouch et al [33]. However, since the synaptic pathology in AD and in APP tg mice has been associated with the progressive accumulation of Ab oligomers [34,35,36,37,38], then it is remarkable that we observed a recovery in the synaptic and behavioral deficits in the APP tg mice treated with ApoBsecNEP. One possible explanation is that even through the levels of existing oligomers were not affected, because of the reduction in the monomers, the progressive generation of more oligomers was decreased or it is also possible that reducing excessive levels of Ab 1-42 might be beneficial. For example, some studies have shown that in contrast to Ab 1-40, monomeric Ab 1-42 could reduce synaptic function and LTP via glutamate receptors [39,40,41]. An alternative possibility as shown in this study is that the beneficial effects of ApoBsecNEP might be associated with a decrease in intra-cellular Ab. A growing number of studies have shown that accumulation of intracellular Ab could be deleterious [23,24,25,42,43] by triggering aberrant signaling via CREB [44]. Supporting this possibility, previous studies have shown that NEP ameliorates the deficits in the fly [22] and rodent models [23] by diminishing Ab from the intracellular compartment.
Although ApoBsecNEP was located in the soma and dendrites of neurons, it is not clear if the reduced intracellular Ab may be driven by this source of NEP. It is also plausible that extracellular ApoBsecNEP is capable of degrading Ab and this shifts the equilibrium of Ab from the intracellular space to the extracellular space thus reducing the intracellular accumulation of Ab indirectly.
In addition to the effects on monomeric and intracellular Ab, we observed that of ApoBsecNEP trafficking into the CNS reduced the accumulation of amyloid plaques in the APPtg mice. Given that NEP does not degrade Ab fibrils alternative possibilities were investigated. Remarkably, the present study showed that ApoBsecNEP is also taken up by macrophages/microglia around the plaques and that these cells contained Ab immunoreactive material, suggesting that NEP activates the macrophages/ microglia to clear the Ab in the plaques. It's not clear what drives microglial cells to the site of plaques in mice treated with the ApoBSecNEP; however, the increased accumulation of NEP with the plaques may be the signal for increased microglial activation. Previous studies have shown that monocyte derived macrophages are capable of taking up Ab and degrading Ab fibrils, however, fibrillar Ab degradation in macrophages was sensitive to lysosomal and insulin degrading enzyme inhibitors but insensitive to proteasomal and neprilysin inhibitors [45]. Therefore, it is unlikely ApoBsecNEP detected in macrophages was directly involved in the degradation of Ab fibrils but rather it might have increased the cleavage of neuropeptide substrates that promote leukocyte migration [46]. NEP has previously been shown to be expressed by leukocytes and process peptides involved in migration such bacterial chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (fMLP). Inhibition of CD10/NEP on the surface of human neutrophils (PMNs) in vitro inhibits migration toward this chemotaxin, suggesting that enzymatic inactivation by NEP regulates the neutrophil response to fMLP [47].
The accumulation of ApoBSecNEP in the CNS of APPtg mice was significantly higher than in the non-tg controls. Since the levels of the targeted receptor on the BBB namely a member of the LDL receptor family was not different between control and tg mice then it is possible that other mechanisms might have been involved. One possibility is that the rate of accumulation of the recombinant protein in the CNS might depend on the Ab burden in CNS. Increased trafficking of ApoBsecNEP into the CNS of APP tg mice might raise questions as to the potential for toxicity. In addition to peptides involved in chemoattraction, NEP has been shown to cleave neuroactive peptides such as metenkephalin, substance P and neuropepetide Y. We have previously shown that regulated expression of NEP in the CNS is neuroprotective and does not result in a deleterious degradation of neuropeptides or trophic factors [30,48]. Moreover, the present study showed that mice treated with the ApoBsecNEP displayed improved behavioral performance in water maze and amelioration of the synaptic pathology. Taken together these results suggest that targeted delivery of NEP across the BBB may be a viable therapy for AD. Although the studies here utilize the lentivirus vector for delivery of the gene for the recombinant protein to the mouse liver, it is likely that delivery of the ApoBSecNEP protein by intravenous injection would have similar effects. Thus, the ApoBSecNEP may be a feasible noninvasive therapeutic option for reducing the functional deficits and the accumulation of Ab in AD.

Ethics Statement
All experiments described were carried out in strict accordance with good animal practice according to NIH recommendations, and all procedures for animal use were approved by the Institutional Animal Care and Use Committee at the University of California at San Diego (UCSD) under protocol #S07221.

Lentivirus vector production
The pre-pro trypsin secretory signal was cloned to the 59 of a vector expressing the human secreted neprilysin (aa 52-750). The vector was designated LV-SecNEP. The ApoB LDL-R binding domain (aa 3371-3409) [21] was cloned with the secreted neprilysin between the secretory signal and the coding sequence for the human neprilysin producing LV-ApoBSecNEP ( Figure S1A). As a control for transported proteins, a virus was generated as described above with eGFP, pre-pro trypsin secretory signal and the ApoB LDL-R binding domain (pLV-ApoBGFP). Lentiviruses expressing NEP, SecNEP, ApoBSecNEP, GFP or ApoBGFP were generated essentially as described [49]. An empty lentiviral vector (LV-control) was utilized for comparison purposes and as a control. Titers were determined by p24 ELISA assay (Perkin Elmer) as described [49].

Neprilysin activity assays
The relative rate of Ab processing by NEP was determined by using an N-terminal FITC-tagged human Ab (Ab 42 ; rPeptide). For cell-free assays, 20 mg of cell lysates, 20 ml conditioned cell media or 100 ng of human recombinant NEP (R&D Systems) was incubated with 100 mM Tris, pH 7.4, 10 mM ZnCl 2 , and 33 mM FITC-labeled Ab in a final volume of 50 ml. Thiorphan (1 mM; Calbiochem) was used as a NEP-specific protease inhibitor. Aliquots were taken at 0 and 24 hours, 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 (Bio-Rad). The proteolytic activity of NEP was measured as previously described [29] using the substrate 3dansyl-D-Ala-Gly-p-(nitro)-Phe-Gly (DAGNPG; Sigma). Cell lysate was incubated with 50 mM DAGNPG and 1 mM captopril (to inhibit ACE cleavage of DAGNPG) in a volume of 200 ml at 37uC. Reactions were stopped by heating samples to 100uC for 5 min, then centrifuging. 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).

Lentiviral infection of neuronal progenitor cells and challenge with Ab
Adult rat hippocampal neural progenitor cells (NPCs) (Chemicon) were grown as previously described [50]. Briefly, NPCs were grown in DMEM/Ham's F-12 medium containing B27 supplements without Vitamin A (Gibco). Cells were plated onto glass coverslips and differentiated in DMEM/F12 medium containing N2 supplements (Gibco) for 4 days. The cells were then infected with LV-control, LV-NEP, LV-SecNEP or LV-ApoBSecNEP and after 24 hrs challenged with 10 nM Ab 1-42 (American Peptide) for 24 hours. One set of cells were grown in plates for analysis of NEP activity and protein levels in lysates and conditioned media and the other set were grown on coverslips and fixed in 4% PFA for immunohistochemistry. For this purpose, coverslips were immunolabeled with antibodies against NEP (Abcam), neuron specific tubulin-III (Millipore Corporation) and activated caspase-3 (Cell Signaling) followed by secondary antibodies tagged with tyramide red or FITC and imaged with the laser scanning confocal microscope. Images were analyzed with the NIH Image J program to assess levels of pixel intensity.

Lentivirus injections into APP tg mice
The APP tg mice used in these studies express mutated human (h)APP751 under the control of the mThy-1 promoter (mThy1-hAPP751; line 41). These tg mice are unique in that, compared to other tg models, amyloid plaques are found in the cortex beginning at 3 months and in the hippocampus at 4 months of age [51]. In addition, the mThy1-hAPP751 mice show learning and memory deficit in the Morris water maze beginning at 6 months of age [28].
Following NIH guidelines for the humane treatment of animals, briefly, as described [21], APP tg mice or nontg control littermates (age 6 months) were injected intra-peritoneally with either the LVcontrol, LV-NEP, LV-SecNEP or LV-ApoBSecNEP (1610 9 transducing units (tdu)) in a total volume of 200 ml. For each group 8 mice were included for a total of 32 nontg and 32 APP tg mice. For control experiments of the recombinant hybrid protein trafficking into the CNS a subgroup of nontg (n = 4 each per group) and APP tg mice (n = 4 each per groups) were injected intra-peritoneally with LV-GFP or LV-ApoBGFP.

Water maze testing
As previously described [28], in order to evaluate the functional effects of LV-ApoBSecNEP treatment in mice, groups of APP tg animals were tested in the water maze. For this purpose, a pool (diameter 180 cm) was filled with opaque water (24uC) and mice were first trained to locate a visible platform (days 1-3) and then a submerged hidden platform (days 4-7) in three daily trials 2-3 min apart. Mice that failed to find the hidden platform within 90 seconds were placed on it for 30 seconds. The same platform location was used for all sessions and all mice. The starting point at which each mouse was placed into the water was changed randomly between two alternative entry points located at a similar distance from the platform. On day 8, another visible platform trial was performed to exclude differences in motivation and fatigue. Time to reach the platform (latency), path length, and swim speed were recorded with a Noldus Instruments EthoVision video tracking system (San Diego Instruments) set to analyze two samples per second.

Animal maintenance and tissue processing
Four weeks after the lentiviral intra-peritoneal injections, mice were tested in the water maze and then anesthetized, perfused with cold saline, and their brains removed. The left hemibrain was frozen in isopentane cooled in a Histobath (Shandon Lipshaw, Pittsburgh, PA) and was used for western blot analysis. The right hemibrain was immersion-fixed in 4% PFA in PBS, pH 7.4. Fixed hemibrains were serially sectioned at 40 mm with a vibratome (Leica) for immunocytochemical and LSCM analysis.

Tissue and cell fractionation and western blot analysis
Samples were homogenized and separated into cytosolic (soluble) and membrane (insoluble) fractions as described [23,38]. Briefly, mouse brain samples (0. . The samples were centrifuged at 1,0006 g for 10 minutes at 4uC. Supernatants were retained and placed into appropriate ultracentrifuge tubes and the pellets were re-homogenized in 0.3 ml of Buffer A and re-centrifuged at 1,0006 g for 10 minutes at 4uC. The second supernatant was collected and combined with the first supernatant and centrifuged at 100,0006 g for one hour at 4uC. This final supernatant was collected to serve as the cytosolic fraction and the remaining pellet was resuspended in 0.2 ml of buffer and re-homogenized; this was the membrane fraction. Protein concentrations were quantified by BCA (Pierce). Levels of APP and Ab immunoreactivity were determined in brain homogenates by western blot as described previously [38,52] utilizing the membrane fractions. Antibodies against full-length (FL) APP (mouse monoclonal, clone 22C11, Chemicon), Ab -82E1 (IBL International) and NEP (mouse monoclonal, clone 56C6, Abcam) followed by secondary antibodies tagged with horseradish peroxidase (HRP) (Santa Cruz Biotechnology) were used and then visualized by enhanced chemiluminescence and analyzed with a Versadoc XL imaging apparatus (Bio-Rad). Analyses of actin levels were used as a loading control.

Analysis of Ab and plaque load by immunocytochemistry
To evaluate the amyloid plaque load, briefly as previously described [28], vibratome sections were incubated overnight at 4uC with the mouse monoclonal antibody against Ab (clone 82E1, IBL international) followed by fluorescein isothiocyanate (FITC)conjugated anti-mouse IgG (Vector Laboratories). The FITClabeled sections were imaged with the laser scanning confocal microscope (LSCM, MRC1024, BioRad) as described previously [27]. Digitized images were analyzed with the NIH Image 1.43 program to determine the percent area of the neuropil occupied by Ab-immunoreactive deposits in the frontal cortex and hippocampus. To evaluate effects on intracellular Ab, sections were immunolabeled with the mouse monoclonal antibody against Ab (clone 4G8; Senetek) and with the antibody against the Nterminus of Ab (aa 1-16; clone 82E1; Immunobiological Llaboratorie Co.) followed by incubation with secondary biotinylated anti-mouse IgG and then ABC and DAB. Sections were transferred to SuperFrost slides (Fisher Scientific) and mounted under glass coverslips with anti-fading media (Vector Laboratories). All sections were processed under the same standardized conditions. Three immunolabeled sections were analyzed per mouse and the average of individual measurements was used to calculate group means.

Analysis of NEP expression, double immunolabeling and neurodegeneration
To verify the expression levels of NEP, vibratome sections were immunolabeled with a monoclonal antibody against NEP (CD10, Abcam) and detected with the Tyramide Signal Amplification TM -Direct (Red) system (NEN Life Sciences,). To evaluate the colocalization of NEP, double immunocytochemical analysis was performed as previously described [53]. For this purpose, vibratome sections were immunolabeled with a monoclonal antibody against NEP (Abcam) detected with the Tyramide Signal Amplification TM -Direct (Red) system (NEN Life Sciences) and the mouse monoclonal antibodies against NeuN (Millipore), MAP2 (Millipore) and Ab (clone 82E1) detected with FITCconjugated secondary antibodies (Vector Laboratories) [53]. Additional co-localization studies were performed with antibodies against Ab (clone 82E1) and microglia/macrophage markers Iba-1 (Wako), CD68 (Abcam) and CD11b (Abcam). 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) with an attached MRC1024 LSCM system (BioRad) [53]. 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 integrity of the neuronal structure was evaluated as previously described [28,54]; briefly, blind-coded, 40 mm thick vibratome sections from mouse brains fixed in 4% paraformaldehyde were immunolabeled with the mouse monoclonal antibodies against SNAP25 (synaptic marker, Millipore) and PSD95 (postsynaptic marker, Abcam). After overnight incubation, sections were incubated with the Tyramide Signal Amplification TM -Direct (Red) system (NEN Life Sciences) transferred to SuperFrost slides (Fisher Scientific) and mounted under glass coverslips with antifading media (Vector Laboratories). All sections were processed under the same standardized conditions. The immunolabeled blind-coded sections were serially imaged with the LSCM (MRC1024, BioRad) and analyzed with the Image 1.43 program (NIH), as previously described [55]. For each mouse, a total of 3 sections were analyzed and for each section, 4 fields in the frontal cortex and hippocampus were examined. Results were expressed as percent area of the neuropil occupied.

Statistical analyses
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 6SEM. Comparisons between 2 groups were done with the unpaired two-tailed Student's ttest. Correlation studies were carried out by simple regression analysis and the null hypothesis was rejected at the 0.05 level.  Figure S4 Co-localization of Ab with macrophage/microglial cell markers in APP tg mice treated with LV-ApoBSecNEP. For these studies vibratome sections from APP tg mice were double labeled with antibodies against Ab (green), or the microglial markers, Iba1 (red) or CD11b (red) and analyzed with the laser scanning confocal microscope. DAPI (blue) was used to visualize nuclei. Images are from plaques distributed in the hippocampus. (A-F) Double labeling analysis with antibodies against Ab and Iba1 in mice treated with LV-control or LV-ApoBSecNEP respectively. (G-L) Double labeling analysis with antibodies against Ab and CD11b in mice treated with LV-control or LV-ApoBSecNEP respectively. Arrows indicate areas of colocalization. Scale bar = 50 mm. (TIF)