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Keywords:

  • Alzheimer's disease;
  • Amyloid β;
  • apolipoprotein E;
  • behavior;
  • histochemistry;
  • tau

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference
Thumbnail image of graphical abstract

Inheritance of the apolipoprotein E4 (apoE4) genotype has been identified as the major genetic risk factor for late-onset Alzheimer's disease (AD). Studies have shown that the binding between apoE and amyloid-β (Aβ) peptides occurs at residues 244–272 of apoE and residues 12–28 of Aβ. ApoE4 has been implicated in promoting Aβ deposition and impairing clearance of Aβ. We hypothesized that blocking the apoE/Aβ interaction would serve as an effective new approach to AD therapy. We have previously shown that treatment with Aβ12-28P can reduce amyloid plaques in APP/PS1 transgenic (Tg) mice and vascular amyloid in TgSwDI mice with congophilic amyloid angiopathy. In the present study, we investigated whether the Aβ12-28P elicits a therapeutic effect on tau-related pathology in addition to amyloid pathology using old triple transgenic AD mice (3xTg, with PS1M146V, APPSwe and tauP30IL transgenes) with established pathology from the ages of 21 to 26 months. We show that treatment with Aβ12-28P substantially reduces tau pathology both immunohistochemically and biochemically, as well as reducing the amyloid burden and suppressing the activation of astrocytes and microglia. These affects correlate with a behavioral amelioration in the treated Tg mice.

The apolipoprotein E (apoE)/amyloid beta (Aβ) interaction plays an important role in the aggregation and clearance of Aβ. We show that the apoE/Aβ binding blocker, Aβ12-28P, elicits a therapeutic effect on tau related pathology, in addition to amyloid deposition, in 3xTg Alzheimer's disease model mice. These affects correlate with a behavioral amelioration in the treated Tg mice. We believe this presents a novel therapeutic approach for AD.

Abbreviations used
AD

Alzheimer's disease

ApoE4

apolipoprotein E4

amyloid-β

CAA

congophilic amyloid angiopathy

NFTs

neurofibrillary tangles

Tg

transgenic

Alzheimer's disease (AD) is the most common cause of dementia globally (Batsch and Mittelman 2012). The pathological hallmarks of AD are the extracellular accumulations of amyloid-β (Aβ) as plaques and as cerebral amyloid angiopathy (CAA), as well as intracellular neurofibrillary tangles (NFTs) (Nelson et al. 2012). There is extensive evidence for the amyloid cascade hypothesis in which Aβ accumulation and aggregation in the brain are thought to be central events in the pathogenesis of AD (Hardy 2006). More recently it has been recognized that the most toxic forms of aggregated Aβ peptide are soluble oligomeric species (Masters and Selkoe 2012). These mediate neuronal toxicity and also are thought to drive NFT related pathology. NFTs are composed of aggregated, abnormally phosphorylated tau protein. The most toxic forms of aggregated tau are also thought to be oligomeric.

Inheritance of the apolipoprotein E4 (apoE4) allele is the strongest genetic risk factor identified so far for late-onset AD. Carrying an apoE4 allele increases the incidence and decreases the age of onset of amyloid plaques and CAA, as well as enhancing tau pathology (Potter and Wisniewski 2012; Liu et al. 2013). We and others have published a number of studies showing that apoE and Aβ bind in vivo and in vitro, with such binding affecting the conformation of Aβ peptides, highlighting the importance of this interaction for the modulation of aggregation and clearance of Aβ (Potter and Wisniewski 2012; Verghese et al. 2011). We have shown that blocking the Aβ/apoE interaction could constitute a novel treatment for AD by diminishing Aβ deposition both in the form of amyloid plaques and CAA as well as decreasing Aβ toxicity (Sadowski et al. 2004, 2006), work which has been corroborated by others (Hao et al. 2010). Our prior studies showed that treatment with Aβ12-28P in APPK670N/M671L/PS1M146L, and APPK670N/M671L transgenic (Tg) AD mice resulted in a significant reduction of Aβ burden in the brain parenchyma compared to age matched vehicle-treated Tg mice (Sadowski et al. 2004, 2006). Furthermore, behavioral studies showed that treatment with Aβ12-28P prevents memory deficit in APPK670N/M671L Tg mice. Enzyme-Linked Immuno Sorbent Assay (ELISA) measurement of Aβ levels in the brain homogenate revealed a significant reduction in the absolute Aβ levels while the concentrations of the soluble Aβ fraction and Aβ oligomers remained stable in Aβ12-28P treated groups. No signs of toxicity, including systemic amyloidosis or cerebral hemorrhages, were observed in treated animals (Sadowski et al. 2006). The therapeutic effect of Aβ12-28P could not be attributed to an immunization effect, because there were no significant changes of anti-Aβ antibodies in the sera of Aβ12-28P treated mice. Therefore, blocking the Aβ/apoE interaction with Aβ12-28P appears to have a net effect of increasing Aβ clearance in the brain, thus producing a cognitive benefit. To further investigate the therapeutic effect in vivo of apoE/Aβ binding inhibition, we systemic administrated Aβ12-28P in TgSwDI mice. These mice are a model of CAA and develop abundant Aβ deposits in cerebral blood vessels, primarily in the microvasculature beginning at age of 6 months, and have impaired memory in behavioral test (Van Vickle et al. 2008; Xu et al. 2007). We show that this treatment leads to dramatic reductions in CAA, without microhemorrhages, as well as reductions in Aβ oligomers (Yang et al. 2011). One likely mechanism of action of this approach is that blocking Aβ/apoE binding inhibits the oligomerization/fibrillization promoting effects of apoE (apoE4 in particular) on Aβ (Wisniewski and Sadowski 2008; Hao et al. 2010; Koffie et al. 2012). It has been shown that apoE4 colocalizes with Aβ oligomers and increases their levels and toxicity by directing oligomers to synapses (Koffie et al. 2012; Tai et al. 2013). Blocking the interaction between apoE and Aβ has been shown to inhibit the earliest stages of oligomer build up, which occurs intraneuronally (Kuszczyk et al. 2013). In addition, Aβ12-28P likely alters the effects of apoE on Aβ clearance. Evidence strongly suggests that when apoE forms stable complexes with Aβ, Aβ clearance across the blood–brain barrier (BBB) and by cell mediated uptake is significantly reduced compared to free Aβ in an apoE isotype specific manner (Nielsen et al. 2010; Deane et al. 2008; Castellano et al. 2011). Free Aβ rapidly crosses the BBB via LRP1; however apoE-Aβ clearance is markedly lower, with apoE4-Aβ being slower than apoE3-Aβ (Deane et al. 2008; Castellano et al. 2011). Hence, we hypothesize that Aβ12-28P will be particularly beneficial in apoE4 Tg mice/patients; however, it should also be effective with the apoE3 isotype. In the current study we tested whether tau related pathology may be affected. That Aβ12-28P may directly reduce tau related pathology is based on the following observations: ApoE has been shown to bind tau (Strittmatter et al. 1994; Han et al. 1994; Fleming et al. 1996); in particular to phosphorylated tau (Leroy et al. 2010). A number of groups have shown that transgenic mice expressing apoE4 exhibit increased tau phosphorylation (Brecht et al. 2004; Chang et al. 2005; Tesseur et al. 2000; Huang 2011). ApoE expression by neurons (which only occurs under conditions of stress such as the presence of nearby AD related pathology) is associated with increased NFT pathology and mitochondrial dysfunction (Hauser et al. 2011; Chang et al. 2005; Mahley and Huang 2009). A critical region of apoE required for some of these detrimental effects has been shown to be the lipid binding region of amino acids 244–272 (Chang et al. 2005; Mahley and Huang 2009; Bu 2009). The apoE/Aβ binding regions have been mapped on Aβ to be Aβ12-28 (Sadowski et al. 2004, 2006), while on apoE these have been mapped to residues 244–272 (Liu et al. 2011), the same region associated with the potential promoting effects of neuronal apoE on tau related pathology. Hence, in the current study, we tested the hypothesis that Aβ12-28P may reduce tau related pathology in 3xTg mice with both amyloid and tau pathology (Oddo et al. 2003b).

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Synthesis of peptides

The Aβ12-28P and scrambled Aβ12-28P peptides were synthesized at the W.M. Keck Foundation Laboratory (Yale University, New Haven, CT, USA), using D-amino acids and end protection techniques. The synthesized Aβ 12-28P (VHHQKLPFFAEDVGSNK) with substituted valine at position 18 to proline is non-toxic, BBB permeable, and has extended serum half-life (Golabek et al. 1996; Sadowski et al. 2004; Yang et al. 2011). Detailed methods of synthesis and purification were described previously (Golabek et al. 1996; Sadowski et al. 2004; Yang et al. 2011). The scrambled Aβ12-28P peptide (QGKFSDHVNEPHFAVKL) contains the same amino acids composition as the Aβ12-28P peptide but the linear sequence is different. In this study, the scrambled Aβ12-28P peptide serves as an additional control, along with vehicle treated group to show that a specific sequence rather than the amino acid composition is critical for biological function.

Transgenic mice and treatment

The treatment was performed on triple transgenic mouse model of AD (3xTg AD) harboring PS1M146v, APPSwe, and tauP30IL transgenes, which progressively develop amyloid-β (Aβ) plaques and NFTs. The 3xTg AD mice were originally generated by Oddo et al. (Oddo et al. 2003b) and purchased from Jackson Laboratory (Bar Harbor, ME, USA). All mouse care and experimental procedures were compliant with guidelines of animal experimentation and were approved by the Institutional Animal Care and Use Committee at the New York University School of Medicine. Thirty-four animals were divided into three groups and received intraperitoneal (i.p.) injections three times per week for 19 weeks beginning at the age of 21.5 months.: group 1 (n = 12, 6 females and 6 males) was injected with 1 mg of Aβ 12-28P peptide per animal; group 2 (n = 10, 5 females and 5 males) was injected with 1 mg of the scrambled peptide per animal; group 3 (n = 12, six females and six males) was injected with saline alone. All peptide injections were prepared in saline under sterile condition. Both age-matched vehicle treated (saline alone) and the scrambled peptide treated animals were used as controls. During the treatment, veterinary staff monitored animals for any signs of toxicity, such as changes in body weight, physical appearance, and altered behavior. Animals were killed a week after administration of the last treatment dose at the age of 26.5 months.

Locomotor and behavior testing

Before cognitive testing, exploratory locomotor test and accelerating rotarod test which measure motor coordination and balance were performed to verify that any treatment effects observed in the cognitive task could not be confounded by differences in motor abilities similar to prior studies (Asuni et al. 2006; Scholtzova et al. 2009; Yang et al. 2011).

Locomotor activity

A Hamilton-Kinder Smart-frame Photobeam System was used to record mice activity over a designated period of time. Following habituation in a circular open field chamber (70 × 70 cm) for 15 min, each mouse was allowed to explore the environment for 15 min. After each session, the field was cleaned with water and 30% ethanol. Horizontal movements of the mice were automatically recorded by a video camera mounted above the chamber. Results were reported as distance traveled (cm), average and maximum travel velocity (cm/s) and mean resting time (s) of the mouse.

Rotarod

Mice were first habituated in two trials to reach a baseline level of performance, and subsequently tested in three additional trials with 15 min intervene. In each trial, mice were placed on a 3.6 cm diameter rod (Rotarod 7650 accelerating model; Ugo Basile, Biological Research Apparatus, Varese, Italy) with initial speed set at 1.5 rpm that was then raised every 30 s by 0.5 rpm. A soft foam cushion was placed under the rod to prevent injury from falling. The rod was cleaned with water and 30% ethanol after each session. To assess the performance, the speed of the rod was recorded when the mouse fell or inverted (by clinging) from the top of the rotating barrel.

Radial arm maze

Spatial memory was tested using a radial arm maze with eight radial 30-cm long arms originating from the central space, as previously described (Yang et al. 2011; Goni et al. 2010). A well baited with 0.25 mL of 0.1% saccharine solution was placed at the end of each arm. Before testing, mice were deprived of water for 24 h and then their access to water was restricted to 1 h per day for the duration of testing. The task required an animal to enter all arms and drink the saccharine solution until the eight rewards had been consumed. After 3 days of adaptation, mice were subjected to testing for 11 consecutive days. The number of errors (entry into previously visited arms) was recorded during each session. This test was performed during the last month of the experiment while animals were still receiving treatment. The behavioral testing was performed by an individual blinded to the treatment assignment.

Tissue processing and histological studies

Mice were anesthetized with sodium pentobarbital (150 mg/kg, i.p.), perfused transaortically and the brains were immediately removed and processed as previously described (Schmidt et al. 2005; Wadghiri et al. 2013). The left hemisphere was snap-frozen for measurement of Aβ oligomers/aggregates, Aβ peptide and pathological tau levels, whereas the right hemisphere was immersion-fixed in periodate-lysine paraformaldehyde. Following fixation, brains were placed in 2% DMSO/20% glycerol in phosphate-buffered saline (PBS) and stored at 4°C until sectioned. Serial coronal brain sections (40 μm) were cut and stained for immunohistochemical analysis with: (i) a mixture of anti-Aβ monoclonal antibodies 6E10/4G8 (Covance, Princeton, NJ, USA), (ii) anti-tau monoclonal antibodies PHF1 and CP13 (kindly provided by Dr. Peter Davies from Albert Einstein College of Medicine, Bronx, NY, USA), (iii) polyclonal anti-glial fibrillary acidic protein (GFAP) antibody (Dako, Carpinteria, CA, USA), (iv) monoclonal anti-CD11 b (Serotec, Raleigh, NC, USA), and (v) anti-CD45 antibodies (Serotec). To ensure that in Aβ 12-28P treated mice reductions of amyloid detection could not be attributed to epitope masking, two different anti-Aβ monoclonal antibodies 6E10 (epitope Aβ 1-16) and 4G8 (epitope Aβ 17–24) with distinct epitopes were used for amyloid detection (Kim et al. 1990). Tau burden was determined by immunostaining with CP13 (recognizes phosphorylated serine in position 202) and PHF1 (recognizes phosphorylated serine in position 396 and 404) (Otvos et al. 1994) antibodies as previously reported (Boutajangout et al. 2011; Asuni et al. 2007). To exam brain inflammation levels of treated mice, we assessed the degree of astrocytosis with GFAP immunoreactivity and the degree of microgliosis with immunoreactivity to CD11b and CD45 antibodies as previously published (Scholtzova et al. 2009; Yang et al. 2011). GFAP is the principal intermediate filament of mature astrocytes, extensively used as a marker for identifying astrocytes in the CNS (Wisniewski and Goni 2013; Stougaard et al. 2011). CD11b is a protein subunit that forms integrin alpha-M beta-2 (αMβ2) (Rubenstein et al. 2011), while CD45 is a protein tyrosine phosphatase (Kutner et al. 2000); CD11b and CD45 monoclonal antibodies are commonly used for detection of the microglial activation at the earliest and later stages of plaque development, respectively (Morgan et al. 2005). Details of the immunostaining techniques were described previously (Sadowski et al. 2006; Scholtzova et al. 2009; Yang et al. 2011; Chung et al. 2010). For control positive immunostaining, human AD sections were used; negative immunostaining controls were performed on sequential mouse brain sections with omission of the primary antibody. Briefly, free-floating sections were incubated in 0.3% H2O2 for 15 min to block endogenous peroxidase activity, and then incubated in a mouse-on-mouse (MOM) blocking reagent from the MOM immunodetection kit (Vector Laboratories, Burlingame, CA, USA) to block nonspecific binding, followed by incubation in a MOM diluent containing different primary antibodies at 4°C overnight, then reacted with biotinylated anti-mouse IgG (1 : 2000) or anti-rat IgG (1 : 1000) secondary antibody for 1 h for all monoclonal antibodies staining. GFAP staining was performed with a primary antibody diluent containing 0.3% Triton X-100, 0.1% sodium azide, 0.01% bacitracin, 1% bovine serum albumin and 10% normal goat serum in PBS, and a secondary biotinylated goat anti-rabbit antibody (Vector). Antibody staining was revealed with 3, 3′-diaminobenzidine (DAB; Sigma-Aldrich) and nickel ammonium sulfate (Ni; Mallinckrodt, Paris, KY) for intensification.

Quantification of amyloid burden

Amyloid burden were quantified by a Bioquant stereology image analysis system (BIOQUANT Image Analysis Corporation, Nashville, TN, USA), using a random unbiased sampling scheme as previously published (Sadowski et al. 2006; Yang et al. 2011). All measurements were performed by an individual blind to the experimental group assignments of the study. Total Aβ burden (defined as the percentage of test area occupied by Aβ immunoreactivity) was quantified for the cortex, hippocampus and amygdala on coronal plane sections stained with a mixture of anti-Aβ antibodies 6E10/4G8 (data from amygdala were included in the cortex analysis). The staining resulted in black Aβ immunoreactivity due to the intensification with nickel ammonium sulfate and facilitates threshold detection. The cortical area was measured as dorsomedial from the cingulate cortex and extended ventrolaterally to the rhinal fissure within the right hemisphere. Test areas (500 μm × 500 μm) were randomly selected by applying a grid (600 μm × 600 μm) over the traced contour. Eight sections, approximately 80 areas, were analyzed per animal. Hippocampal measurements (400 μm × 400 μm) on a grid (400 μm × 400 μm) were performed similar to the cortical analysis (seven sections, and approximately 85 areas per animal).

Semi-quantitative analysis of tau burden, astrocytosis and microgliosis

The assessment of sections stained with PHF1, CP13, GFAP, CD45, and CD11b antibodies was based on a semi-quantitative analysis, using methods we have previously published (Yang et al. 2011; Scholtzova et al. 2009; Sadowski et al. 2006). Prior to analysis, brains were observed through the microscope and given a rating from 0–4, in increments of 0.5, depending on the degree of pathology and/or the activation stage of the glial cells. The tau burden was analyzed based on the extent of immunostaining with two anti-tau antibodies, PHF1 and CP13. PHF1 was analyzed at 10 × magnification in the hippocampus. Approximately six sections were analyzed per animal. CP13 was analyzed at 10 × magnification for both the cortex and hippocampus. Approximately eight cortical sections and six hippocampal sections were analyzed per animal. The rating was based on number of reactive neuronal bodies and processes. Astrogliosis and microgliosis were analyzed at 5 × and 10 × magnifications respectively in the cortex, hippocampus and thalamus region. Approximately eight cortical sections, six hippocampal sections, and four thalamic sections were analyzed per animal. The rating for astrogliosis was based on the extent of GFAP immuoreactivity (number of GFAP immunoreactive cells and complexity of astrocytic branching) as previously published (Yang et al. 2011; Scholtzova et al. 2009). The assessment of microgliosis was based on the extent of immunoreactivity with CD11b and CD45 antibodies with rating from 0 (few resting microglia) to 4 (numerous ramified/phagocytic microglia) in increments of 0.5. The rating was done by an observer blinded to the treatment status of the mice.

Assessment of levels of Aβ and Aβ aggregates/oligomers in the brain by ELISA

Brains were weighed and homogenized (10% w/v) in tissue homogenization buffer (20 mM Tris base, pH 7.4, 250 mM sucrose, 1 mM EDTA, 1 mM EGTA) with 100 mM phenylmethylsulphonyl fluoride, complete protease inhibitor cocktail and PhosSTOP phosphatase inhibitor cocktail (Roche, Indianapolis, IN, USA) added immediately before homogenization. The extraction of soluble Aβ and total Aβ was according to the method we have published previously (Scholtzova et al. 2009; Goni et al. 2010; Yang et al. 2011). Brain homogenates (200 μL) were mixed with an equal volume of cold 0.4% diethylamine/100 mM NaCl, and subsequently centrifuged at 100 000 × g for 1 h at 4°C. Then the supernatant was neutralized with 1/10 volume of 0.5 M Tris, pH 6.8, flash-frozen on dry ice and stored at −80°C until used as soluble Aβ fraction; meantime, 200 μL of the homogenate was added to 440 μL of cold formic acid (FA) and sonicated for 1 min on ice. Subsequently, 400 μL of this solution was centrifuged at 100 000 × g for 1 h at 4°C and 210 μL of the resulting supernatant was diluted into 4 mL of FA neutralization solution (1 M Tris base, 0.5 M Na2HPO4, 0.05% NaN3), flash-frozen on dry ice and stored at −80°C until used for total Aβ measurement. Soluble and total Aβ levels in the brain were measured by sandwich ELISA which used 6E10 monoclonal antibody as a capture antibody and two detection polyclonal antibodies R162 and R165 specific for Aβ40 and Aβ42, respectively (Scholtzova et al. 2009; Goni et al. 2010; Yang et al. 2011). The assay was performed by an investigator (PM) who was blinded to treatment group assignment. Aggregated/oligomeric Aβ levels were determined by using Human Aggregated Aβ ELISA kit (Invitrogen, Camarillo, CA, USA). This was done by following the manufacturer's instructions. In brief, the levels of aggregated/oligomeric Aβ in each sample were measured against a standard containing aggregated Aβ. Samples diluted in the provided standard diluent buffer were incubated for 2 h at 25°C allowing the Aβ to bind the capture antibody (a monoclonal antibody specific for the N-terminus of human Aβ pre-coated to each well), followed by extensive washing and incubation for 1 h at 25°C with biotin conjugated detection antibody (same as the capture antibody) which binds to the immobilized aggregated Aβ. After removal of excess antibody, horseradish peroxidase-labelled streptavidin was added to incubate for 30 min, followed by washing, tetramethylbenzidine substrate incubation to produce color. The intensity of this colored product is directly proportional to the concentration of aggregated/oligomeric Aβ in the sample. The standards provided a linear curve and the best-fit line determined by linear regression were used to calculate aggregated Aβ concentration in samples.

Western blot analysis of Aβ oligomers and pathological Tau

Brain homogenates were centrifuged at 100 000 × g for 1 h at 4°C. The supernatants were collected and the total protein concentrations were determined by the bicinchoninic acid assay (BCA; Pierce, Rockford, IL, USA). The same amount of protein from each sample, mixed with an equal volume of Tricine sample buffer, was loaded and subjected to overnight electrophoresis on 12.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis under non-reducing conditions. Aβ oligomers were detected by using oligomer-specific A11 polyclonal antibody (Biosource, Camarillo, CA, USA). The specificity of A11 staining was confirmed by stripping and subsequent probing the same membrane with anti-Aβ monoclonal antibodies 6E10. For pathological tau detection, the brain homogenates were centrifuged at 20 800 g for 15 min (4°C) and supernatants were applied to sarkosyl extraction. 30 μL of 11% sarkosyl solution was added to 300 μL supernatant to form a final concentration of 1% sarkosyl and then incubated in the water bath for 1 h at 37°C, followed by centrifuging at 100 000 g for 1 h at 4°C in Beckman TL-100 ultracentrifuge. The high-speed supernatant was collected and used for western blot analysis as (sarkosyl) soluble fraction. Although sarkosyl extraction can dissociate aggregated tau proteins, the end stage of tau pathology characterized as paired helical filaments, are not soluble in sarkosyl (Miller 2011). For the insoluble fraction, the pellet was re-suspended in the same volume of buffer without protease and phosphatase inhibitors, but that contained 1% (v/v) Triton X-100 and 0.25% (w/v) deoxycholate sodium. It was then ultra centrifuged at 50 000 g for 30 min to obtain a detergent extracted supernatant that was analyzed as (sarkosyl) insoluble fraction (Boutajangout et al. 2011). The supernatants from these two fractions were heated at 100°C for 5 min and the same amount of protein was electrophoresed on 12.5% (w/v) polyacrylamide gel overnight. The blots were blocked in 5% nonfat milk with 0.1% Tween-20 in Tris-buffered saline (TBS) and incubated with different anti-tau antibodies (PHF1 and CP13), and monoclonal β-actin antibody (Sigma) overnight. The bounded antibodies to Aβ oligomers and pathological tau were detected by enhanced chemiluminescence (Pierce). Densitometric analyses were performed with NIH Image J software (version 1.34, Bethesda, MD, USA). The levels of pathological tau were normalized relative to β-actin protein.

Statistical analysis

Data from the accelerating rotor rod and locomotor test were analyzed by one-way anova. The data collected from the radial arm maze test was analyzed by two-way anova, and also by one way anova followed by Neuman-Keuls post hoc tests. Differences between the groups in total amyloid burden, fibrillar amyloid burden, levels of extracted Aβ, levels of Aβ oligomers, tau burden, astrogliosis, and microgliosis were analyzed using a one-way anova. All statistical tests were performed using Prism 6.0 (Graphpad, San Diego, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

Behavioral studies

All three groups of 3xTg AD mice receiving Aβ12-28P, the scrambled peptides or vehicle treatment were assessed on both cognitive and sensorimotor tests. No statistical differences were observed between groups in any of the rotarod or locomotor activity parameters measured (Fig. 1). Therefore, the performance on cognitive tests was not confounded by the differences or abnormalities in motor function. In the radial arm maze, Tg mice treated with Aβ12-28P showed a significant different compared to both animals treated with scrambled peptide and vehicle treated mice (Two-way anova: treatment effect, < 0.0001; days effect, = 0.0023; Newman-Keuls post hoc test: vehicle versus Aβ12-28P treated mice < 0.01, scrambled versus Aβ12-28P < 0.001, vehicle versus scrambled not significant) (Fig. 2).

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Figure 1. Locomotor activity. (a) shows in the rotarod test there is no performance difference among all three groups (One-way anova, p = 0.2646); (b) shows no significant differences observed in the locomotor activity test among all three groups in distance traveled (cm) (One-way anova, p = 0.3531), travel velocity: average speed (One-way anova, p = 0.3987), maximum speed (One-way anova, p = 0.0879) and resting time (One way anova, p = 0.4265).

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Figure 2. Working memory improvement in Aβ12-28P treated and control 3xTg mice. This illustrates the result of radial arm maze cognitive testing. The number of errors is plotted versus the days of testing. Two-way repeated measures anova revealed a significant cognition improvement benefit of Aβ 12-28P treatment on 3x Tg Alzheimer's disease (AD) mice: treatment effect, < 0.0001; days effect, = 0.0023; Newman-Keuls post hoc test: vehicle versus Aβ12-28P treated mice < 0.01, scrambled versus Aβ12-28P < 0.001, vehicle versus scrambled not significant.

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Quantification of Aβ burden and tau burden

Total amyloid burden in the regions of cortex, hippocampus, and thalamus were quantified by stereological techniques using random unbiased sampling on the serial sections immunostained with anti-Aβ 6E10/4G8 antibodies (Fig. 3 a–h). Aβ immunostaining showed greater Aβ accumulation in cortical and hippocampal sections of the vehicle and scrambled groups compared to the Aβ12-28P treated mice (a–f). A significant reduction was observed in 6E10/4G8 immunoreactivity in both the cortex and hippocampus in the Aβ12-28P treated Tg mice (= 0.0153 and = 0.0230, respectively) (g and h).

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Figure 3. Treatment with Aβ12-28P decreased cortical and hippocampal amyloid plaque burden in 3xTg mice. Aβ immunostaining (6E10/4G8; 1 : 2000) showed greater Aβ accumulation in cortical and hippocampal sections of the vehicle and scrambled groups compared to the Aβ12-28P treated mice (a–f). There was a significant reduction observed in 6E10/4G8 immunoreactivity in both the cortex and hippocampus in the Aβ12-28P treated mice versus vehicle or scrambled treated mice measured by unbiased stereology (One-way anova, *< 0.05) (g and h).

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To determine the treatment effect of Aβ12-28P on tau pathology, we used a semi-quantitative analysis method to quantify tau burden in the region of cortex, hippocampus, and amygdala on the serial sections from different treatment groups. Two anti-tau antibodies, CP13 which recognizes phosphorylation at serine 202, and PHF1 which recognizes phospho-tau epitopes serine 396 and 404 were applied to the study to exam earlier and end stage tau phosphorylation products, respectively. Sections immunostained with CP13 displayed greater phospho-tau accumulation in cortical, hippocampal, and amygdala regions in both the vehicle and scrambled group compared to Aβ12-28P treated mice (Fig. 4a–i). The reduction of CP13 immunoreactivity observed in the Aβ12-28P treated mice was significant in all three brain regions analyzed compared to vehicle treated Tg mice (Fig. 4j–l, cortex = 0.0038, hippocampus < 0.0001, amygdale < 0.0001). Histological observation of the PHF1 immunoreactivity revealed greater phospho-tau accumulation in hippocampal sections from vehicle and the scrambled groups compared to Aβ12-28P treated Tg mice (Fig. 5a–c). There was a significant reduction observed in PHF1 immunoreactivity in the hippocampus in the Aβ12-28P treated group (= 0.0210, Fig. 5d).

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Figure 4. Treatment with Aβ12-28P decreased cortical, amygdala and hippocampal tau immunoreactivity using CP13 in 3xTg mice. Tau immunostaining using CP13 (1 : 300) displayed greater tau accumulation in cortical, hippocampal, and amygdala sections in both the vehicle and scrambled group compared to Aβ12-28P group (a–i). A significant reduction was observed in the Aβ12-28P treated mice in all three brain regions analyzed compared to vehicle treated mice (j–l) (One-way anova, *< 0.05; **< 0.01; ***< 0.001).

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Figure 5. Treatment with Aβ12-28P decreased hippocampal tau immunoreactivity using PHF1 in 3xTg mice. Histological observation of tau immunostaining (PHF1; 1 : 100) revealed greater tau accumulation in hippocampal sections of the vehicle and scrambled groups compared to the Aβ12-28P group (a–c). There was a significant reduction observed in PHF1 immunoreactivity in the hippocampus in the Aβ12-28P treated mice (= 0.0210, d).

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Neuroinflammatory response after treatment

To evaluate the treatment effect of Aβ12-28P on brain inflammation, we quantitatively examined the extent of gliosis and the activation of microglia in the brain regions of cortex, hippocampus, and thalamus by immunohistochemical staining. Histological observation of astrocytes which were stained with anti-GFAP antibody revealed that there was a greater amount of astrogliosis in the vehicle treated group compared to Aβ12-28P and the scrambled groups in all three brain regions (Fig. 6a–i). More significant reduction of astrogliosis was observed in the cortex and hippocampus brain regions on Aβ12-28P treated mice (Fig. 6j–l). The assessment of the activation of microglia was based on CD45 and CD11b antibody immuno-activities. A reduction in CD45 microgliosis was observed in Aβ12-28P treated mice compared to the vehicle and scrambled groups in all three analyzed regions: cortex, hippocampus and thalamus (Fig. 7 = 0.0018, < 0.0001 and = 0.0035, respectively). The treatment elicited a significant reduction in microglia activity on Aβ12-28P treat mice. Results from the immunohistological analysis of CD45 were in line with the CD11b immunoactivity analysis. There was less CD11b microgliosis in the Aβ12-28P treated group in the cortex, hippocampus, and thalamus (Fig. 8a–i). A significant reduction was observed in all three brain regions analyzed in the Aβ12-28P treated group (< 0.0001, < 0.0001, < 0.0001, respectively (Fig. 8j–l).

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Figure 6. Treatment with Aβ12-28P decreased cortical, hippocampal and thalamic glial fibrillary acidic protein (GFAP) immunoreactivity in 3xTg mice. Histological quantitation of astrocytes (GFAP; 1 : 1000) revealed that there was a greater amount of astrogliosis in the vehicle group compared to the Aβ12-28P and scrambled groups in the cortex, hippocampus, and thalamus (a–i). There was a reduction in astrogliosis in both the Aβ12-28P and scrambled groups; = 0.0016, = 0.0038, and = 0.0080 (j–l) (One-way anova, *< 0.05; **< 0.01).

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Figure 7. Treatment with Aβ12-28P decreased cortical, hippocampal and thalamic CD45 immunoreactivity in 3xTg mice. A reduction in microgliosis using CD45 immunoreactivity was observed in Aβ12-28P treated mice compared to the vehicle and scrambled groups in the three analyzed regions: cortex, hippocampus and thalamus brain region (a-i). The treatment elicited a significant reduction in Aβ12-28P treat mice; (**p = 0.0018, ***p < 0.0001, and, **p = 0.0035, respectively) (j-l).

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Figure 8. Treatment with Aβ12-28P decreased cortical, hippocampal and thalamic CD11b immunoreactivity in 3xTg mice. The quantitation of the CD11b immunoreactivity of CD11b shows there was less microgliosis in the Aβ12-28P treated group in the cortex, hippocampus, and thalamus (a–i). A significant reduction was observed in all three regions analyzed in the Aβ12-28P treated group (***p < 0.0001, j–l).

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Assessment of Aβ levels, Aβ oligomers, Aβ aggregates in the brain

Enzyme-Linked Immuno Sorbent Assay measurements revealed a statistically significant decrease in the levels of soluble (diethylamine extracted) Aβ40 (= 0.0001) and Aβ42 (< 0.0001) after the treatment with Aβ12-28P (Fig. 9, One-way anova followed by Tukey's post hoc test), while no statistical differences were observed between vehicle versus the scrambled group. One-way anova analysis showed no statistical differences were observed among all these three groups in the levels of total (FA extracted) Aβ40 and Aβ42, while there is a strong trend showing a great reduction in the levels of total Aβ42 in Aβ12-28P treated animals. For total Aβ42 the p value for the one way anova is = 0.06; the lack of significance is related to the greater variation in the values in the scrambled group for total Aβ42. Comparing the levels by t-test of Aβ42 in vehicle versus Aβ12-28P and comparing Aβ12-28P to scrambled gives p-values of = 0.01 and = 0.049, respectively.

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Figure 9. Aβ12-28P treatment reduces levels of soluble Aβ in 3xTg Alzheimer's disease (AD) mice. (a) shows the levels of soluble Aβ40 and Aβ42 in the Aβ12-28P treated and control groups. One-way anova analysis revealed a significant reduction in the levels of soluble Aβ40 (= 0.0001) and Aβ42 (< 0.0001) on Aβ12-28P treated mice. Tukey's post hoc test showed compare to vehicle-treated mice, there was a greater decrease of the level of soluble Aβ42 (< 0.001) than soluble Aβ40 (< 0.05) on Aβ12-28P treated mice. No differences in the levels of soluble Aβ40 or Aβ42 were observed between vehicle versus scrambled group (> 0.05). (b) shows the levels of total Aβ40 and Aβ42 in the Aβ12-28P treated and control groups. One-way anova analysis showed no difference in the levels of total Aβ40 or Aβ42 were observed among all three groups. For Aβ42 there was a trend in the Aβ12-28P treated group for reduced levels. For Aβ42 the p-value for the one way anova is = 0.06; the lack of significance is related to the greater variation in the values in the scrambled group for Aβ42. Comparing the levels by t-test of Aβ42 in vehicle versus Aβ12-28P or comparing Aβ12-28P to scrambled gives p-values of = 0.01 and = 0.049, respectively. (One-way anova, *< 0.05; **< 0.01; ***< 0.001).

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The levels of Aβ oligomers in the brain homogenates were analyzed by western blot using the polyclonal A11 oligomer-specific antibody (Fig. 10). Aβ12-28P treatment led to a significant decrease in the levels of A11 reactive oligomers (Approximately 55 kDa) band density (One-way anova followed by Newman-Keuls post hoc test, < 0.0001). Aβ oligomers can, in part, become dissociated when run on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Upadhaya et al. 2012; Watt et al. 2013); hence, we corroborated this treatment associated reduction of oligomers by an ELISA specific to aggregated/oligomeric Aβ, similar to other studies (Kuszczyk et al. 2013; Fukumoto et al. 2010). Two-tail unpaired t-test analysis of this ELISA shows a significant reduce of Aβ aggregates/oligomers in the Aβ12-28P treated group versus vehicle (= 0.0015) or the scrambled peptide (= 0.0061) treated groups, while no difference was observed between the two control groups (vehicle vs. the scrambled, = 0.1495).

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Figure 10. Aβ12-28P treatment markedly decrease the levels of Aβ oligomers (One-way anova,< 0.0001) and Aβ aggregates (One-way anova,= 0.0014) in the brains of 3xTg Alzheimer's disease (AD) mice. (a) shows a representative western blot that was loaded with samples of vehicle, Aβ12-28P, scrambled peptide treated 3xTg AD mice and non-treated 3xTg AD mice and probed with oligomer specific A11 antibody, clearly showing Aβ12-28P treatment markedly reduced the levels of A11 reactive Aβ oligomers (the approximately 55 kDa band). (b) shows graphically the quantitation of the density of the oligomer bands (in arbitrary O.D. values) (***< 0.001). Newman-Keuls post hoc test results: Aβ12-28P versus vehicle < 0.001, Aβ12-28P versus scrambled < 0.001, vehicle versus scrambled no significant difference. (c) shows the result of sandwich Enzyme-Linked Immuno Sorbent Assay (ELISA) measurements of Aβ aggregates. Newman-Keuls post hoc test: Aβ12-28P versus vehicle < 0.05, Aβ12-28P versus scrambled < 0.01, vehicle versus scrambled no significant difference (*< 0.05; **< 0.01).

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Semi-quantification of pathological tau

The pathological tau in the brain was extracted by 1% sarkosyl solution followed by high speed spin to yield a supernatant as sarkosyl soluble fractions. The rest pellet dissolved in 1% (v/v) Triton X-100 and 0.25% (w/v) deoxycholate sodium was analyzed as sarkosyl insoluble fractions. The levels of pathological tau were accessed in both fractions by western blot using monoclonal antibodies CP13 and PHF1. In sarkosyl insoluble fraction, One-way anova analysis revealed that Aβ12-28P treatment reduced the pathological tau CP13 (= 0.0168) and PHF1 (= 0.039) (Fig. 11), but no statistical changes in the levels of pathological tau CP13 or PHF1 were detected between two control groups (vehicle vs. scrambled). In sarkosyl soluble fractions, no statistical differences in the levels of PHF1 or CP13 immunoactivity (pathological tau) were detected between Aβ12-28P treatment group and the control groups (vehicle and the scrambled peptide treated) (Fig. 12).

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Figure 11. Aβ12-28P treatment reduces the level of CP13 and PHF1 pathological tau in the Sarkosyl brain insoluble fractions by western blotting. One-way anova followed by Newman–Keuls post hoc test shows Aβ12-28P treatment reduced the pathological tau CP13 (shown in (a), = 0.0168) and PHF1 (shown in (b), = 0.039) in the sarkosyl insoluble fraction. No statistical changes were observed in neither PHF1 nor CP13 between two control groups (vehicle-treated or scrambled peptide-treated).

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Figure 12. Aβ12-28P treatment did not affect the levels of CP13 and PHF1 pathological tau in the Sarkosyl brain soluble fractions by western blotting. No statistically significant differences in the levels of CP13 (shown in (a), = 0.1322) or PHF1 (shown in (b), = 0.0662) were observed among Aβ12-28P and control in sarkosyl soluble fractions by One-way anova.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

The apoE/Aβ interaction plays a key role in the conformational transformation of soluble Aβ and Aβ deposits in AD brains (Potter and Wisniewski 2012; Liu et al. 2013; Verghese et al. 2011). In prior studies we have shown that treatment with Aβ12-28P in two AD Tg mouse models with primarily amyloid plaque deposition and in one AD model with primarily CAA resulted in a significant reduction of Aβ burden in both brain parenchyma and in brain vessels compared to age matched vehicle-treated Tg mice (Sadowski et al. 2004, 2006; Yang et al. 2011). In the current study we show that this treatment strategy also significantly reduces tau related pathology while also greatly lowering Aβ oligomer levels.

We demonstrate that the administration of Aβ12-28P is able to reverse the cognitive deficits in 3xTg mice, as determined by the radial arm maze behavioral test (< 0.0001 treatment effect), with no significant change in cognitive function in both the vehicle and scrambled Aβ12-28P treatment groups. The cognitive testing was not confounded with any deficits in balance/sensorimotor coordination nor locomotor activity as shown by the results of the accelerating rotor rod and locomotor activity behavioral tests. These results are consistent with our past studies that have shown that treatment with Aβ12-28P has resulted in the prevention of memory deficits based on performance on the radial arm maze (Potter and Wisniewski 2012; Liu et al. 2013; Verghese et al. 2011). We, therefore, have shown that the administration of Aβ12-28P for 19 weeks in vivo can have major cognitive benefits in a model with both Aβ and tau pathology. This behavioral rescue in our study is particularly significant in that treatment was begun when the mice were quite old, 21.5 months. This represents an age when AD related pathology is already advanced; in this model Aβ deposition starts at about 6 months while the tau pathology commences at approximately 12 months (Oddo et al. 2003a). Hence, we began treatment at a stage of AD related pathology that is more equivalent to a patient with early AD; this contrasts with most treatment studies in AD Tg models where the therapeutic trial is begun before or at the time of pathology emergence (Wisniewski and Boutajangout 2010; Zahs and Ashe 2010). This cognitive improvement was coupled with a reduction in soluble Aβ40 and Aβ42 and total Aβ42 levels with no change in the levels of total Aβ40. The reduction of soluble Aβ42 was greater than the reduction of soluble Aβ40. A plausible explanation for not observing any change in the levels of total Aβ40 and the greater effect on soluble Aβ42 could be the distinct biological activity of oligomeric preparations of the two Aβ alloforms. Aβ42 is well known to form a predominantly β-sheet structure and oligomers more easily than Aβ40 (Bitan et al. 2003). It has been shown that apoE binding to Aβ peptides is increased when Aβ is in a more β-sheet conformation (Golabek et al. 1996). This is consistent with an early observation where immunoprecipitation experiments using anti-apoE antibodies in AD brain supernatants show that apoE co-purifies predominantly with dimeric Aβ (Permanne et al. 1997), which later experiments have shown is the predominate toxic, oligomeric species in the CSF and brains of AD patients (Klybin et al. 2008; Cleary et al. 2005). Hence, it is expected that blocking the apoE/Aβ interaction would have greater effect on Aβ42 levels compared to Aβ40.

Significantly, we also observed a reduction in Aβ oligomers, as shown by western blot detection and densitometric analysis, as well as using sandwich ELISA measurements of Aβ aggregates. We also document that by blocking the Aβ/apoE interaction with the administration of Aβ12-28P, we are able to significantly reduce the amyloid burden in both the cortex and hippocampus. Aβ12-28P inhibits the Aβ/apoE interaction by binding to apoE thereby, inhibiting its binding to full length Aβ. Prevention of this binding eradicates apoE's effects on the aggregation state of Aβ (Wisniewski et al. 1994; Sadowski et al. 2006).

Treatment with Aβ12-28P also led to substantial reductions in tau related pathology. Our immunohistochemical analysis of tau pathology was performed with two tau anti-bodies, CP13 and PHF1, which detect different phosphorylation sites. CP13 recognizes phosphorylation at serine 202, while PHF1 recognizes tau phosphorylation at serine 396 and 404 (Boutajangout et al. 2011). CP13 neurons were readily apparent in cortex, hippocampus and amygdala; staining with PHF1, however, was only evident in the hippocampus. Tau pathology is first apparent in the hippocampus of 3xTg mice, particularly in the pyramidal neurons of the CA1 subfield, and then later progresses into cortical structures (Oddo et al. 2003b). Our immunohistochemical findings reveal a significant reduction in all the analyzed regions for both tau markers in the group treated with Aβ12-28P. The administration of Aβ12-28P, therefore, is proven effective in reducing aggregated tau in the brain. Since PHF1 is a later marker compared to CP13, this would explain why PHF1 staining was only observed in the hippocampus. Western blot detection and densitometric analysis of soluble and insoluble tau fractions with both anti-tau antibodies were performed with both anti-tau antibodies. Levels of insoluble tau fractions detected by both CP13 and PHF1 revealed a significant decrease in tau pathology in the group treated with Aβ12-28P; no reduction, however, was observed with either tau antibody for the soluble tau fractions. These biochemical results confirm our immunohistochemical findings and reiterate the reducing effect Aβ12-28P elicits on tau pathology. Currently, while no treatment is available for clearing tau aggregates our findings suggest a novel therapeutic approach to target one of the major hallmarks of AD, NFTs. The progressive development of both amyloid plaque formation and tangles in an age- and region-dependent manner, as seen in 3xTg mice more closely mimics the pathology seen in AD patients. We have shown that the administration of the known Aβ/apoE interaction inhibitor, Aβ12-28P, is useful in ameliorating cognitive deficits, as well as, in reducing amyloid burden and the subsequent development of tangles.

An important concern in administering a treatment that targets Aβ deposition and NFTs is the possibility of increased brain inflammation, ultimately resulting in neuronal dysfunction or death. Our studies show, based on immunohistochemical analysis of GFAP, CD45 and CD11b immuoreactivity, that inflammation is significantly reduced in the cortex, hippocampus and thalamus of Aβ12-28P treated mice. Neuroinflammation, due to an abnormal activation of microglia and astrocytes, is commonly seen in AD brains (Wyss-Coray and Rogers 2012). Microglia are generally recognized as the brain's designated macrophages playing a key role in innate immune and inflammatory responses in an array of neurologic disorders. Recent advances have been made with regard to intermediate states of microglia activation and interaction with Aβ peptides as seen in AD (Morgan et al. 2005; Wyss-Coray and Rogers 2012). The scrambled peptide treatment also reduced GFAP immunoreactivity, but did not alter CD45 or CD11b immunoreactivity compared to vehicle controls. Why the scrambled peptide reduced GFAP immunoreactivity modestly is unclear and may be related to unspecific slight anti-inflammatory effects of giving a peptide by i.p. injection. It can be concluded from these results that treatment with Aβ12-28P reduces neuroinflammation and can be a potential therapy for treating the inflammation seen in AD brains.

At present there is no effective disease-modifying treatment for AD hence, there is an urgent need for better therapeutic interventions (Holtzman et al. 2012). Of the many therapeutic approaches being developed for AD, the greatest promise comes for immunotherapies (Wisniewski and Boutajangout 2010; Lemere and Masliah 2010). However, two large Phase III trials of immunotherapy recently ended without demonstrating any benefit on the primary outcome measure of cognitive benefit (Roher et al. 2013). It is increasingly being recognized that to achieve clinical benefit in AD, it is important to not only target Aβ related pathology but also to reduce tau related disease, as well as reducing vascular amyloid deposition (Panza et al. 2012; Wisniewski 2012). In a prior study we have demonstrated in a vascular amyloid Tg model that Aβ12-28P can reduce CAA effectively (Yang et al. 2011) and it has also been recently shown that Aβ12-28P can reduce intracellular Aβ oligomer accumulation (Kuszczyk et al. 2013). In this study, treatment with Aβ12-28P has been proven useful in ameliorating behavioral deficits, in reducing amyloid burden and tau pathology, as well as, in decreasing the amount of neuroinflammation. Use of the 3xTg model more closely reflects the pathology seen in AD, in particular when the treatment is begun in old mice with existing extensive Aβ and tau pathology. Relatively little effort is being directed at targeting apoE/Aβ interactions as a therapeutic approach for AD currently (Potter and Wisniewski 2012; Liu et al. 2013). Based on behavioral, histological, and biochemical analysis in multiple studies (Sadowski et al. 2006; Yang et al. 2011; Kuszczyk et al. 2013), we suggest that blocking the Aβ/apoE interaction is a novel therapeutic approach in treating AD, which has a greater chance of clinical efficacy as it ameliorates all the major pathological features of AD. Greater effort is need to delineate the multiple roles apoE has in AD pathogenesis, since this appears to be an area that shows therapeutic promise.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. Reference

This manuscript was supported by NIH grants AG20245 and NS073502 and the Alzheimer's Drug Discovery Foundation. None of the authors have any conflicts of interest.

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  6. Acknowledgements
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