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,1
* Protein Studies Program, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma, USA;
Department of Pathology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, USA; and
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma, USA
1Correspondence: Protein Studies Program, Oklahoma Medical Research Foundation, 825 N.E. 13th St., Oklahoma City, OK 73104, USA. E-mail: jordan-tang{at}omrf.ouhsc.edu; wanpin-chang{at}omrf.ouhsc.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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35%) in the immunized mice as compared to controls. Immunized mice also showed better cognitive performance than controls in both cohorts. Brain histological analyses found no evidence of T cell/microglia/astrocyte activation in the immunized mice, suggesting the absence of inflammatory responses. These results suggest that memapsin 2 immunization in Tg2576 was effective in reducing Aß production and improving cognitive function and that the current approach warrants further investigation as a therapy for AD.—Chang, W.-P., Downs, D., Huang, X.-P., Da, H., Fung, K. M., Tang, J. Amyloid-beta reduction by memepsin 2 (beta-secretase) immunization.
Key Words: immunotherapy • Alzheimer’s disease • BACE1
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Amyloid ß, a peptide of 40 or 42 residues, is generated from ß-amyloid precursor protein (APP) by the action of two proteases, memapsin 2 (ß-secretase, BACE1) and 
-secretase. Aß is cleared by further proteolysis or by being transported out of the brain. Evidence supports the contention that an increase of Aß production, such as in the Swedish mutation of APP (APPsw), enhances the risk of AD (3)
. Therefore, a major approach to develop a treatment for AD based on therapeutic interventions is designed to reduce Aß production. The obvious therapeutic targets in this approach are the two proteases mentioned above. The development of an inhibitor for -secretase has been actively pursued, and some advanced drug candidates have reached clinical trials (4)
. However, this protease has many ancillary physiological functions; thus, its inhibition has resulted in a number of toxic side effects (5)
. Segregating -secretase inhibition from the undesirable side effects remains a major challenge. A recent study (6)
even suggested that -secretase inhibition would aggravate instead of ameliorate neurodegeneration and dementia.
Since the identification of memapsin 2 by several groups 6 yr ago (7
8
9
10
11)
, AD therapy targeted at this protease has been actively studied. Most of the effort in this area has been directed toward the development of an inhibitor of this protease. Despite significant progress (12
, 13)
, there is not a memapsin 2 inhibitor drug candidate in clinical trial at this time, owing in part to the difficulty of designing potent and selective memapsin 2 inhibitors small enough to penetrate the blood-brain barrier (BBB).
We have explored the idea that neutralizing antibodies against memapsin 2, generated from the immunization with the protease itself, may be enlisted to reduce Aß production. The basis of such an approach is schematically shown in Fig. 1
. A fraction of anti-memapsin 2 (anti-M2) antibodies resulting from immunization may penetrate BBB and bind to memapsin 2 located on the surface of neurons. When the complex of memapsin 2 and antibody is endocytosed (14
, 15)
, the cleavage of APP by memapsin 2 is interfered with by the bound antibody resulting in a decrease of Aß production. This therapeutic scheme is appealing for the following reasons. 1) A fraction of anti-M2 antibodies is expected to penetrate the BBB, as it is well known that IgG molecules cross the BBB (16
17
18)
. 2) Endosomes are a well-established site for memapsin 2 activity and the generation of Aß (19
20
21)
; thus, the inhibition of memapsin 2 activity should significantly affect Aß production. 3) Antibody-mediated inhibition of memapsin 2 should render good selectivity. 4) A reduction of memapsin 2 activity in mice is well tolerated, as indicated by the fact that memapsin 2 knock-out mice have only a few minor phenotypes (22
23
24
25
26)
. A more complex phenotype manifested by the memapsin 2-deficient mice (27)
appears to associate mainly with the developmental phase of young mice and may be related to the role of memapsin 2 in regulating peripheral nerve myelination (28)
, which functions exclusively in young animals. Therefore, for a therapy designed to partially reduce memapsin 2 activity in AD treatment, effective management of these phenotypes appears likely.
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It should be also noted that immunotherapy for AD has been studied using Aß (29)
or a fragment of APP (30)
as the immunogen. In Aß immunization, anti-Aß antibodies have been shown to induce the activation of T cells and microglia (31)
, which directly participate in plaque clearing. Unfortunately, that same mechanism may have played a role in the adverse responses in the AN1792 clinical trial (32)
. Memapsin 2 immunization is based on a completely different principle in which anti-M2 antibody functions as an inhibitor of the protease. As a result, the activation of immune cells should be minimal without losing effectiveness in Aß reduction. We report here evidence that supports the feasibility of memapsin 2 immunotherapy. The main steps of the proposed mechanism were shown to be operative in cellular models. Furthermore, in proof-of-concept studies, immunization of Tg2576 transgenic AD mice resulted in a reduction of Aß in the plasma and brain, a reduction of plaque formation, and strikingly, an improvement of cognitive performance.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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with minor modification (7)
. Goat anti-memapsin 2 ectodomain antibodies (anti-M2) (8)
were purified by affinity chromatography using purified memapsin 2 ectodomain coupled to Affi-Gel-10 agarose (Pierce, Rockford, IL, USA). Cultured CHO cells stably transfected with APP Swedish and London variant mutations were incubated with purified anti-M2 antibodies in a range of 0.03 to 3 µg/ml. Cultured supernatants after overnight incubation were collected and subjected to Aß40 sandwich ELISA (Invitrogen, Carlsbad, CA, USA). From the inhibition curve, IC50 and Ki were estimated.
Antibody titer, specificity, and inhibition assays
Anti-M2 antibody specificity was assayed by ELISA against several human aspartic proteases including memapsins 2 and 1, cathepsins D and E, gastricsin, renin, pepsin, and a negative control, ß-amylase. Antibody titers were determined by measuring optical density values from a 4-nitrophenyl phosphate disodium salt hexahydrate (PNPP) substrate in microtiter plates (Sigma, St. Louis, MO, USA). Inhibition of protease activity by anti-M2 antibodies was determined for human memapsin 2 and cathepsin D in the presence of stoichiometric amounts of antibody using a fluorogenic assay at pH 6.0 (34)
. For -secretase assay, a plasmid expressing substrate APP fragments C99 or C83 with a c-myc marker (35)
was transiently transfected separately into HEK-293 (i.e., 293/APPsw/M2) and N2a (i.e., N2a/APPsw) cells. Six hours posttransfection, 1 µg/ml of control antibodies (goat IgG, Sigma) or anti-M2 antibodies was added to the culture medium. The -secretase inhibitor L485 (Calbiochem, San Diego, CA, USA) was added (1 µg/ml) to control for the inhibition of -secretase activity. After overnight incubation, cell lysates were harvested for Western blotting using anti-c-myc (Invitrogen) as the probe for ß- and 
-secretase cleaved APP derivatives, C99 and C83, respectively.
Cellular uptake of anti-M2 antibodies
HEK-293 cells with and without double transfection of APPsw and memapsin 2 (i.e., 293 and 293/APPsw/M2) were separately incubated with 200 µg/ml of Alexa 488 conjugated anti-M2 antibodies at 37°C for a period from 1 to 80 min. At the end of incubation, cells were treated with 10 µg/ml of trypsin (Sigma) for 20 min on ice to remove the cell surface proteins. Control cells were incubated without trypsin. Fluorescence intensity of Alexa 488 was measured using flow cytometry. The percentages of internalized and surface bound antibodies were calculated at each time point for each cell line. For subcellular localization studies, HeLa cells with or without double transfection of APPsw and memapsin 2 (i.e., HeLa and HeLa/APPsw/M2) were separately incubated with 196.5 µg/ml of Alexa 488 conjugated anti-M2 antibodies or 400 µg/ml of fluorescence labeled control antibodies (i.e., Alexa 488 conjugated donkey anti-rabbit IgG, H+L) at 37°C for 2 h. Confocal fluorescence microscopy (Leica TCS NT) was used to examine the subcellular distribution of fluorescence. Markers specific for subcellular compartments were used for colocalization studies of anti-M2 antibodies. The procedures were essentially those described previously (36)
. Briefly, HeLa and HeLa/APPsw/M2 cells were separately incubated with 32 µg/ml of Alexa 488 conjugated anti-M2 antibodies at 37°C for 2 h. After fixation and permeabilization, cells were immunostained with the indicated primary antibodies including Bip for the endoplasmic reticulum, Lamp-1 for lysosomes, p230 for Golgi, CI-MPR for late endosomes, MAB1560 for APP, and EEA1 for early endosomes. Markers were visualized using Cy3-conjugated secondary antibodies.
BBB penetration of anti-M2 antibodies
Ten-month-old Tg2576 transgenic mice (Taconic Farms, Hudson, NY, USA; n=2 mice per group) and wild-type FvB/N mice (n=2 mice per group, data not shown) were intravenously injected with 500 µl of 4 µM of Alexa 488 conjugated goat anti-M2 antibodies, Alexa 488 conjugated goat anti-human IgG antibodies, or PBS buffer (the latter 2 were the negative controls). Blood and cerebrospinal fluid (CSF) were harvested after 2 h, and this was followed by brain collection. Fluorescence intensity and antibody titer in plasma and CSF were measured by fluorometer and memapsin 2 ELISA, respectively. Briefly for memapsin 2 ELISA, serial sample dilutions were added to microtiter plates coated with memapsin 2 (1.25 µg/ml) for a 2 h incubation, followed by indirect immunodetection with alkaline phosphatase-conjugated anti-goat IgG and PNPP substrate (Sigma). One hemisphere of the brain was incubated in OCT/PBS for 2 days followed by embedding in tissue freezing medium (TFM; Triangle Biomedical Sciences, Durham, NC, USA). Brain sections (5 µm) were collected using a Leica CM3050 research cryostat, fixed, and stained with Alexa 488 conjugated donkey anti-goat IgG (H+L) antibodies (Molecular Probes, Eugene, OR, USA) and nuclei stained with DAPI (Sigma).
Immunization studies
Tg2576 mice were subcutaneously injected with memapsin 2 ectodomain or PBS (control) emulsified with adjuvant according to the following protocol. The first injection employed Freund’s complete adjuvant, which was followed by four injections using Freund’s incomplete adjuvant. The remaining injections were without adjuvant. Mice of either 4 wk (young cohort) or 10 months of age (adult cohort) received 6 or 50 µg of memapsin 2, respectively, in a 100 µl injectate. The duration and frequency of immunization are indicated in
Fig. 4
b, e. Blood was sampled from the saphenous vein, and plasma was separated by centrifugation at 1000 g. Concentrations of plasma Aß40 and Aß42 were determined by sandwich ELISA (Invitrogen), and titers of anti-M2 antibodies were measured by memapsin 2 ELISA (see above). At the end of studies, animals were anesthetized by isofluorane, they were intracardially perfused with PBS for 2 min, and their brains were harvested. One hemisphere was stored in liquid nitrogen followed by a sequential extraction of Aß by homogenization on ice using a 3-[(3-cholamidopropyl)dimethylammonic]-1-propane sulfonate (CHAPS) solution followed by guanidine-HCl buffer (37)
. Concentrations of Aß40 and Aß42 were determined by sandwich ELISA (Invitrogen). Western blots to visualize Aß40 and Aß42 (10–20% gradient gel from Invitrogen and urea-SDS-PAGE; ref 38
, 39
) from samples of brain homogenates immunoprecipitated by anti-Aß17–24 antibody (MAB1561 from Chemicon, Temecula, CA, USA) were also performed. The other hemisphere of the brain was fixed for 2 days in 10% buffered formalin and sectioned into 5 µm slices for immunohistochemical staining using the same MAB1561 antibody for amyloid plaques, anti-CD3
(AnaSpec, San Jose, CA, USA) for T-cells, anti-astroglial fibrillary acidic protein (anit-GFAP; Zymed, Burlingame, CA, USA) for astrocytes, and anti-CD68 and anti-CD163 (Biocare Medical, Concord, CA, USA) for microglia. Species-specific HRP-conjugated anti-immunoglobulin antibodies (Vector Laboratories, Burlingame, CA, USA) were used for indirect immunodetection. Routine histochemistry was also performed using hematoxylin and eosin (H&E), thioflavin S (AnaSpec), and leucocyte common antigen (LCA) (anti-CD45; Biocare Medical).
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Cognitive studies
Cognitive performance of experimental mice was evaluated in the spatial reference memory version of the Morris water maze (MWM; ref 40
). All MWM tests were performed in a 160 cm in diameter pool, with an escape platform submerged 1 cm underwater (11 cm in diameter). The surface of the water (23.8±0.6°C) in the pool was made opaque by the addition of nonfat milk powder. The pool was situated in a separate testing room fitted with several distant spatial cues. The first test was performed over 3 days, with the escape platform located 30 cm from the wall of the pool in the target quadrant. Each mouse was given four daily 60 s training trials with an intertrial interval time (ITI) of 5 min. During each trial, a mouse was released into the water facing the wall of the pool from randomly chosen quadrants. To evaluate the development of spatial memory, all mice were given probe trials 24 h after the end of day 3 training. During probe trials, which were administered as the first trial of the day, the escape platform was removed from the pool and the mice were allowed to search the pool uninterrupted for 60 s. The crossing times in each quadrant were recorded, and the Annulus-crossing index (ACI) was calculated (see Data analysis). The reference-memory test was followed by a cued (visible platform) version of MWM (4 trials/day, 2 days) in which an escape platform was marked by a 15-cm-high black and white pole, and the extramaze cues were removed. The visible platform test was intended as a vision test for the subjects and was similar between both groups in all tests (data not shown). The latency during each trial was recorded by a video tracking system.
Data analysis
In the cognitive studies, the time to reach and climb the submerged hidden platform was recorded (latency time). The latency measurements from four daily training trials were averaged for each mouse in both cued and reference-memory MWM tests. The spatial memory for the platform location during probe trials was evaluated by the dwelling time in each quadrant of the pool and the ACI. The ACI represents the number of crosses over the platform site in a quadrant that contained the escape platform (target quadrant) adjusted for crosses over platform sites in alternative quadrants. For all data, Student’s t test and analysis of variance (ANOVA) were used for statistical analysis. Correlations were analyzed by linear regression (Pearson’s R). The critical P value was set to 0.05 for all statistical analyses. All values reported are mean ± SEM.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Inhibition of Aß production by anti-M2 antibodies in cultured cells
The mechanism in Fig. 1
predicts that the addition of anti-M2 antibodies to cultured cells would inhibit Aß production. Figure 2
a shows that the presence of anti-M2 antibodies in the culture medium of Aß-producing CHO cells reduced Aß40 levels in a dosage-dependent manner. The inhibition curve gives rise to an IC50 of 0.09 µg/ml, equivalent to an apparent inhibition constant (Ki) of 10–9 M. This value is within the common range for protein antigen-antibody associations.
Cell surface bound anti-M2 antibodies are endocytosed to endosomes
We observed that Alexa 488 conjugated anti-M2 antibodies were rapidly taken up by HeLa cells stably transfected with APPsw and memapsin 2 (Fig. 2b
). Cytometric analyses showed that >80% of surface bound anti-M2 antibodies were endocytosed within 1 min. In control parental cells with only basal levels of endogenous memapsin 2, minimal surface antibody accumulation and endocytosis were observed (Fig. 2b
, open circles). Subcellular colocalization studies from confocal images established that anti-M2 antibodies were primarily internalized to early endosomes (Figs. 2c, d
, top) where they colocalized with memapsin 2 and APP (Fig. 2d
, middle) but not with a marker of endoplasmic reticulum (Fig. 2d
, bottom). The endosomal localization of anti-M2 antibodies is consistent with the Aß inhibition data described above.
Absence of cytotoxicity of anti-M2 antibodies
As a therapy to a chronic disease, it is important to demonstrate that anti-M2 antibodies do not significantly harm neuronal cells. To this end, increasing amounts of anti-M2 antibodies were added to cultured human and murine neuroblastoma cells. We detected no change in either cell number or cell morphology on the addition of anti-M2 antibodies up to 1 µg/ml over a 4 day period (data not shown).
Assessment of selectivity of anti-M2 antibodies against human aspartic proteases
Since aspartic proteases share some structural motifs, we studied the selectivity of anti-M2 antibodies in cross-reactivity and in enzymatic inhibition. No inhibition of human pepsin, gastricsin, cathepsin D, cathepsin E, renin, memapsin 1 (BACE-2), and -secretase was observed from the addition of anti-M2 antibodies up to 1 µg/ml (data not shown). Using an ELISA assay for memapsin 2, we observed no detectable cross immunoreactivity against the same aspartic proteases above except memapsin 1 (BACE-2), which reacted at 10% of the level as compared to memapsin 2.
In vivo demonstration of BBB penetration by anti-M2 antibodies
The ability of anti-M2 antibodies to cross the BBB was demonstrated in transgenic mice Tg2576. Animals were injected intravenously with either fluorescently labeled anti-M2 antibodies or controls such as fluorescently labeled anti-IgG or PBS. The animals were under observation for 6 h after injection. All three groups of mice had similar appearance and activities. No adverse physical reaction was observed for mice from anti-M2 antibody infusion. These mice had normal posture (e.g., no hunching), normal cage and gait movements, no piloerection or porphyrin staining around the eyes, and no signs of discomfort, stress, or behavior changes. Two hours after injection, blood, CSF, and brain were harvested. Fluorescence intensity and antibody titer were determined for plasma and CSF samples by fluorometer and memapsin 2 ELISA, respectively. Specific fluorescence and anti-M2 antibody titer were observed in both plasma and CSF (Fig. 3
a). The distribution of fluorescently labeled antibodies in the brain tissues of the injected mice was observed from the brain sections directly and after specifically enhanced by immunohistochemical staining using an anti-Alexa 488 antibody. Although these results are basically the same, we discuss below only the results from enhanced images because these images had better intensely. Anti-M2 antibodies were detected in the cortex and hippocampus of the mice that received anti-M2 antibodies but not in mice treated with labeled unrelated IgG antibodies or PBS (Fig. 3b
). Antibody staining was specifically enriched in the cingulate area of cortex and the CA2 area of hippocampus, consistent with other studies localizing memapsin 2 to these areas (41)
. These results suggest that injected antibodies had crossed the BBB and bound to memapsin 2 expressing cells in the brain. In the mice that received the anti-M2 antibodies, the ratio of antibody titers present in CSF and plasma is 3%; the ratio calculated from the fluorescence intensity is 1% (Fig. 3a
), comparable with 2% observed in a control mouse model (42)
. In the group that received the unrelated pan IgG antibodies, the ratio calculated from the fluorescence intensity is 0.4%, comparable to a typical BBB penetration of immunoglobulins in humans (43)
. Nevertheless, the 2.5-fold increase of BBB penetration by anti-M2 antibodies over pan IgG antibodies from the peripheral administration could be a result of mass action from higher concentration of memapsin 2 expressed in the brain cells. We have carried out experiments using wild-type FvB/N mice and obtained similar results (data not shown).
In vitro and In vivo data shown above suggested that the basic mechanism of immunization of memapsin 2 is operative, including inhibition of Aß production, penetration of the BBB, and localization to the site of memapsin 2 activity in specific areas of the brain. We then studied this approach in an AD mouse model.
Memapsin 2 immunization in young AD mice
To assess memapsin 2 immunization on various therapeutic markers in AD mice, we carried out three separate experiments in young Tg2576 mice starting at 4 wk of age. Results from one of the experiments are described below. During the course of 14 months, the anti-M2 antibody titers as well as the levels of Aß40 and Aß42 in the plasma were monitored. Finally, cognitive performance was assessed at the end. After the conclusion of experiments, amyloid load in the brain was analyzed.
Plasma anti-M2 antibodies and Aß levels
Figure 4
a shows that plasma Aß40 of the immunized group started to drop below the control group near the third immunization. This point coincided well with the rise of anti-M2 antibody titer (Fig. 4b
). The differences in both Aß40, ranging from 30 to 50%, and antibody titer were statistically significant throughout the duration of the experiment (P<0.05). The reduction of Aß40 in the memapsin 2 immunized mice was positively correlated with the anti-M2 antibody titer (Fig. 4c
, R2=0.096, P=0.028). We have analyzed plasma Aß42 to be 5% of the combined Aß40 and Aß42 and observed a significant reduction in the immunized group (results not shown), whereas the ratio of Aß40 over Aß42 in all the experiments remained nearly constant at 23 to 1 (i.e., 22.9±0.9). We concluded that the effect on the reduction of Aß40 over Aß42 from memapsin 2 immunization is very similar; therefore, the Aß ratio remained unchanged. The results in two other series of experiments had a similar titer and reduction of Aß.
Cognitive performance
At the end of the 14 month immunization period, cognitive function was tested for the mice in a reference-memory version of the Morris water maze, an evaluation of hippocampus-dependent learning. We observed that immunized Tg2576 animals learned faster during the first three training sessions as compared with adjuvant/PBS-only control mice (Fig. 5
a) with significantly different slopes over 3 days (immunized, –10.6; control, –2.3, P<0.01). The mice were tested for memory bias for the platform location on day 4, 24 h after the last training session. The means of the ACI (Fig. 5b
) of the immunized group showed >2-fold improvement over the control, indicating that the immunized group had better retention (P=0.027). The difference between the two groups was also seen in the analysis of the percentage of time spent in the target quadrant (where the hidden platform had been located) where the immunized mice had a significantly higher value (P=0.02; Fig. 5c
). Moreover, the immunized mice spent significantly less time in the opposite quadrant than the control mice in both cohorts (P=0.04). These results suggest that the memapsin 2 immunized mice have better cognitive performance than PBS immunized mice in the young cohort.
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Brain amyloid load and Aß
Brain amyloid load and the contents of Aß40 and Aß42 were determined at the end of immunization study (mouse age: 15 months) for two of the three experiments. Using immunohistochemical staining, we observed a reduced amyloid load in immunized mice as compared with the control mice. Both total plaque numbers and plaque area in the immunized mice were lower than those in control mice by 35% (P=0.012 and 0.017, respectively; Fig. 6
a). In the cortex of the immunized mice, both plaque number and plaque area were lower (P=0.019 and 0.006, respectively). In the hippocampus of the immunized mice, the plaque area was significantly lower (P=0.056) but the lowering of plaque number was not statistically significant (P=0.15; Fig. 6a
), which may be attributed to larger but fewer plaques in this brain region. Amyloid-ß was extracted from one of the two brain hemispheres, and the content of Aß40 and Aß42 was assessed. Brain Aß42 was significantly lower in the immunized group (ranging from 30 to 50%; Fig. 6b
) as compared with the control group in both the CHAPS-buffer extract (loosely deposited Aß, P=0.0029) and the guanidine-buffer extract (hardened Aß deposit, P=0.037). The lowering of Aß40 (by 20%) was statistically significant in the CHAPS-buffer extract (P=0.0075). Although Aß40 in the guanidine extract was lowered by immunization, it was not statistically significant (P=0.32). The decrease of more Aß42 than Aß40 was confirmed by urea-SDS-PAGE/Western blot of mouse brain extracts. Choosing six pairs of characteristic samples from ELISA data, we have confirmed the larger reduction in Aß42 than in Aß40 with a typical pair shown in Fig. 6c
.
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Brain T cell, microglia, and astrocyte
Since antibody is involved in this therapeutic mechanism, we analyzed for evidence of T cell and microglia infiltration in the brain as indicators of a potential inflammatory autoimmune response. No obvious differences in T cell and microglial staining between the brains of immunized and control mice were seen (Fig. 7
a, b), indicating the absence of significant T cell/microglial response as a result of immunization with memapsin 2. An elevated astrocyte staining specifically located around the amyloid plaques was seen in control mice (Fig. 7c
, PBS). Astrocyte staining from immunized mice was significantly less prominent (Fig. 7c
, M2). These observations suggest that memapsin 2 immunization did not induce T cell/microglia/astrocyte activation but may have reduced the already existing inflammatory reactions near the plaques in the aged AD mouse brain. Along with the results from the H&E and LCA stainings of brain sections (data not shown), we observed no mononuclear, small inflammatory cells in the brain (including the leptomeninges overlying region), which are characteristic of the meningoencephalitis seen in some of the Aß immunized AD mice (44)
.
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The other two series of immunization experiments produced similar results in the reduction of plasma Aß and brain amyloid load (results not shown) but were not tested for cognitive function and autoimmune parameters. Together these data demonstrate that immunization of AD mice with memapsin 2 starting at a young age produces positive responses in therapeutic markers, which included a 35–40% reduction in the level of Aß in the plasma and brain. The reduction of the more insoluble and pathological Aß42 in the brain is particularly significant.
Memapsin 2 immunization in adult AD mice
Since immunization of memapsin 2 in young mice produced beneficial responses, we carried out memapsin 2 immunization in adult AD mice to simulate immunotherapy for the late-onset AD. The aim of this experiment was to answer the question if memapsin 2 immunization in older AD mice would produce an equivalent antibody response and other favorable therapeutic markers as in young mice. Tg2576 (10 months of age) were immunized with memapsin 2 ectodomain over a period of 6 months using a similar protocol as in the immunization of young mice. We analyzed all the parameters discussed above in the young AD mice studies and observed similar responses as described below.
Plasma anti-M2 antibody titer increased over 3 months (Fig. 4e
) to reach the level comparable in the immunization of young mice. Plasma Aß40 levels of the immunized group were lower than that of the PBS control group during the first 3 months but achieved 30% (P<0.05) only after the plasma anti-M2 antibody titer had reached maximum (Fig. 4d
). As observed in the experiments with young AD mice, there was also a positive correlation between the percentage reduction of Aß40 and the anti-M2 antibody titer (Fig. 4f
; R2=0.258; P=0.045) and the ratio of plasma Aß40 and Aß42 remained at 23:1 (results not shown). These observations indicate that older AD mice can respond positively to memapsin 2 immunization to produce anti-M2 antibodies and Aß reduction albeit at a slower rate.
In the cognitive tests, there was a separation of latency times of the two groups (Fig. 5d
), as in the case of young mice, with two slightly but significantly different slopes (i.e.,–3.0 vs. –2.1 for immunized and control mice, respectively, P=0.012). We observed a large difference of ACI values of the two groups (Fig. 5e
; P=0,004). The analysis for residence time in quadrants produced a pattern (Fig. 5f
) similar to that from the young mice (Fig. 5c
). There is a clearer bias for the immunized group in the target quadrant (P=0.01) and avoidance of both the adjacent right quadrant (P=0.03) and the opposite quadrant (P=0.01). Together, these results demonstrate that adult mice immunized with memapsin 2 performed better in the cognitive tests than PBS immunized mice.
The brain amyloid load of the immunized group was consistently lower than the control group in both plaque numbers and plaque areas (Fig. 8
a). Statistical significance was observed in the difference of total plaque area (P=0.036), plaque area in the cortex (P=0.039), and both plaque number and area in the hippocampus (P=0.027 and 0.007, respectively). Although the brain Aß40 and Aß42 were reduced in the immunized group, they were not statistically significant (Fig. 8b
).
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As in the experiments of young AD mice, the brain T cells and microglia are essentially the same in the immunized and control older AD mice (Fig. 7)
. There was also a reduction of astrocytes in the immunized group. These results taken together support the conclusion that memapsin 2 immunization in adult AD mice produced similar beneficial responses in pathogenic parameters as seen in the immunization of young mice.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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35 to 40% in young immunized mice (Figs. 4
and 6)
and up to a similar level in the adult immunized mice (Figs. 4
and 8)
. A partial Aß reduction in this case is expected since Aß is known to be generated from both secretory (19
, 45)
and endocytic (19
20
21)
pathways, but only the latter is subjected to the inhibition by memapsin 2 antibodies (Fig. 1)
. The relative contribution of these two pathways on the production of Aß in vivo has not been clearly defined. A significant fraction of cellular memapsin 2 and APP is found in the Golgi network (46
, 47)
. However, reticulon proteins that inhibit memapsin 2 activity are also present there (48)
. Recent genetic evidence linking incidence of AD to SorL1, a receptor that regulates APP recycling in the endocytic pathway, suggests the importance of the endocytic pathway in Aß production and AD pathogenesis (49)
. An Aß reduction up to 40% observed here appears consistent with this view. Judging from the IC50 value (10–9 M) for inhibition of cellular Aß production, a complete abolishment of Aß production from memapsin 2 immunization also seems unlikely. However, from the therapeutic view point, the level of Aß reduction observed here appears significant as the immunization significantly reduced the brain amyloid load in AD mice (Figs. 6
and 8)
and improved cognitive performance (Fig. 5)
. Such benefits would constitute a worthy clinical treatment for an Aß reduction therapy.
The reduction of Aß in immunized AD mice is apparently derived from the presence of neutralizing anti-M2 antibodies. We have shown that the key steps in this therapeutic scheme (Fig. 1)
are operative in cultured cells, including the binding of anti-M2 antibodies to cell-surface memapsin 2, the internalization of anti-M2 from cell surface to endosomes, and the inhibition of Aß production. We have also demonstrated in mice that anti-M2 antibodies can penetrate the BBB and enter into regions of the brain that express ß-secretase activity. Additional support is found in the immunization experiments. We observed a statistically significant correlation of antibody titer and plasma Aß (Fig. 4c, f
). Plasma Aß is a convenient monitoring tool to follow the progression of immunization experiments and a good indicator for brain Aß for the following reasons. In Tg2576 mice, Aß is expressed specifically in the brain (50)
and the efflux of Aß from the brain to plasma is also rapid (51)
. Also, in a previous study, we demonstrated a good correlation between the plasma and brain Aß in this mouse model (37)
. Finally, a similar decrease in Aß in the brain of immunized mice (Fig. 6)
as compared to that in the plasma (Fig. 4)
confirmed that plasma Aß closely reflects brain Aß. The fact that the ratio of Aß40/Aß42 remained near constant suggests that the reduction of Aß40 and Aß42 as consequence of immunization is about the same. This is expected since the Aß40/Aß42 ratio is determined by -secretase activity that is not subjected to inhibition in the current scheme.
Memapsin 2 immunization studies on young mice were primarily designed to examine the changes of important pathogenic parameters for AD in a cohort of mice destined to develop disease. This study can also be viewed as vaccination against AD in young animals. Although a clear benefit in Aß reduction and cognitive improvement was observed, the value of preventive vaccination of memapsin 2 in human populations would require more supporting data. To assure the greatest effect in the young mouse experiments, repeated immunizations throughout the course of the study were built into the design (Fig. 4a, b
). Though intended as a proof of concept and not optimized pharmacologically, it is recognized that the frequency of inoculation used in the study is not practical as a clinical procedure. A much reduced immunization frequency employing clinically acceptable adjuvants needs be explored in future studies to establish the feasibility.
The experiments in older mice were intended to simulate therapeutic immunization at the early stages of AD in humans, since amyloid plaques are present in the brain of 10-month-old Tg2576 mice, the age of the cohort at the start of the experiment. Although many of the parameters, such as plasma and brain Aß and cognitive function, responded in a similar manner as in the younger mice, there were clear differences. Whereas the antibody titers in young mice rose to the maximum in around 6 wk, the time required in older mice was more than twice as long (Fig. 4)
. As a result, the time for plasma Aß to reach a statistically significant difference was also longer in older mice. This relative delay, apparently due to a weaker immune response in older animals, would not likely be a serious obstacle in the immunotherapy of a slow-developing disease as AD. In addition, the difference in plasma Aß between the immunized and control groups of the older mice, although statistically significant, is not as pronounced as a whole as in young mice (Fig. 4)
and the differences in brain Aß40 and Aß42 were not statistically significant in the older mice (Fig. 8b
) in contrast with those in young mice (Fig. 6b
). The Aß data in older mice could be influenced by a larger amyloid deposit pool that already existed in the brain of older AD mice that provides a higher background value in the brain and in the plasma by efflux. Despite these differences, observed trends in plasma Aß, brain plaque load, and cognitive performance support a further exploration in the development of memapsin 2 immunization as a therapeutic procedure.
The results presented here confirm in an animal model the attractive features of memapsin 2 immunization as both a potential preventive as well as treatment for AD. There are perhaps two issues that need additional consideration. The first issue is that we observed a cross-reactivity of anti-M2 antibodies against memapsin 1 (BACE-2), suggesting a potential selectivity problem. If we assume that the activities of memapsins 1 and 2 are reduced in vivo proportional to the ratio of their measured titers (data not shown), the 35% reduction in memapsin 2 activity (i.e., 35% based on the Aß reduction) predicts memapsin 1 activity in vivo to be barely affected, at only 3.5% inhibition. Since gene deletion of memapsin 1 in mice does not produce a detectable phenotype (27)
, a small reduction of its activity in memapsin 2 immunization is unlikely to produce adverse side effects, especially when used as a therapeutic vaccination. The second issue is the safety of immunizing with human memapsin 2, which avoids producing what are inflammatory autoimmune responses. This is particularly important in view of the adverse inflammatory response observed in some patients in the clinical trial of Aß immunization (32)
. We observed no detectable inflammatory autoimmune response of T cells, microglia, and astrocytes (Fig. 7)
from immunizing AD mice with human memapsin 2 ectodomain. In fact, astrocytes are significantly reduced by the immunization, possibly a secondary effect of plaque reduction in the brain. In the published literature, there is only a 6-amino acid difference in the memapsin 2 (ectodomain) sequence between mice and humans. Nonetheless, titers were sufficient to reduce Aß production without creating inflammatory autoimmune responses. Therefore, the absence of adverse response in the current studies is encouraging for further exploration of this therapeutic approach.
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ACKNOWLEDGMENTS |
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Received for publication December 29, 2006. Accepted for publication April 5, 2007.
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