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NEUROPHARMACOLOGY
Departments of Neurology (Y.M., A.J.G., L.M., C.H.-F., H.-F.L., L.F., P.S.A.), Biochemistry (L.L.), and Psychology (B.R.W.), Georgetown University Medical Center, Washington, District of Columbia; Adams Super Center for Brain Studies (I.G.), Elton Laboratory for Molecular Neuroendocrinology, Department of Human Molecular Genetics and Biochemistry, Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel (Y.J., I.G.); Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Columbia University, New York, New York (E.P., O.A.); and Alzheimer's Disease Cooperative Study, Department of Neurosciences, University of California San Diego, School of Medicine, San Diego, California (P.S.A.)
Received for publication
August 20, 2007
Accepted
January 15, 2008.
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Abstract |
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), and a recent transgenic mouse study suggests that the pathogenic processes leading to cognitive impairment occur before the formation of classic tangles (Roberson et al., 2007). Tau is a neuronal microtubule-associated protein, and phosphorylation of tau regulates its ability to promote microtubule assembly (Lindwall and Cole, 1984). Abnormal hyperphosphorylation is thought to interfere with the normal biological function of tau by reducing its ability to bind to and stabilize microtubules (Bramblett et al., 1993; Alonso et al., 1994). Treatment with lithium chloride, a glycogen synthase kinase-3 inhibitor, reduced tau phosphorylation and prevented or reversed several aspects of tauopathy in mice (Noble et al., 2005; Engel et al., 2006; Plattner et al., 2006) and flies (Mudher et al., 2004). Nevertheless, it is not clear whether inhibitor-mediated reduction of phosphotau improved tau binding to microtubules in these studies. Paclitaxel (Taxol), a tubulin binding agent and United States Food and Drug Administration-approved potent anticancer drug, restored fast axonal transport and motor dysfunction in a tau transgenic mouse in vivo (Zhang et al., 2005). However, paclitaxel is not neuron-specific, has the capacity to inhibit microtubule dynamics, has limited brain bioavailability, and presents side effects including inhibition of axonal transport associated with peripheral neuropathy (Lee and Swain, 2006). Thus, more specific, brain-penetrable microtubule-interacting drugs are required (Michaelis, 2006).
The octapeptide NAPVSIPQ (NAP) preferentially interacts with neuronal and glial tubulin, promotes microtubule assembly, influences microtubule dynamics in postmitotic cells, and reduces phosphorylated tau in vitro at femtomolar concentrations (Divinski et al., 2004, 2006; Gozes and Divinski, 2004). Peripheral administration of NAP has shown significant efficacy in various in vivo models and penetration into the brain at therapeutic levels (Gozes et al., 2005). Phase I clinical trials indicate that intranasally administered NAP is well tolerated and produces blood levels of the peptide expected, on the basis of animal studies, to reflect therapeutic concentrations in brain (Allon Therapeutics Inc., personal communication).
We have shown that chronic intranasal NAP treatment in a triple transgenic mouse model with AD-type Aβ and tau pathology (Oddo et al., 2003) reduced Aβ accumulation and tau hyperphosphorylation when administered at an early pathological stage (9–12 months of age) (Matsuoka et al., 2007), Here, we extend these studies to examine the effect of increased NAP dosage on tau pathology and cognitive function in the same mouse model at a later stage of disease (12–18 months of age).
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Materials and Methods |
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). Mice were bred and maintained at the Georgetown University animal facility. NAP was synthesized as previously reported (Bassan et al., 1999) and was dissolved in 7.5 mg of NaCl, 1.7 mg of citric acid monohydrate, 3.0 mg of Na2PO4 2H2O, and 0.2 mg of benzalkonium chloride solution (50%), per 1 ml of United States Pharmacopeia sterile water (Alcalay et al., 2004). Mice were treated with NAP for 3 or 6 months starting from 12 months of age and sacrificed at 15 or 18 months of age, respectively. We used 12 mice each (six female and six male mice) at 12.1 ± 0.3 (vehicle) and 12.1 ± 0.2 (NAP) months of age for 3-month treatment and 12 mice (five male and seven female) at 11.6 ± 0.4 (vehicle) and 13 mice (five male and eight female) at 11.6 ± 0.4 (NAP) months of age for 6-month treatment. Mice were intranasally treated with either NAP (2 µg NAP/mouse/day, 2.5 µl for each nostril, 5.0 µl/mouse/day) or vehicle (2.5 µl for each nostril, 5.0 µl/mouse/day) daily from Monday through Friday between 10:00 AM and 2:00 PM. We monitored overall health status on a daily basis during the entire treatment period.
Mice were sacrificed by cervical dislocation, and the brain was quickly isolated after the olfactory bulb and cerebellum were discarded. Each hemibrain was snap-frozen in dry ice or immersion fixed in 4% paraformaldehyde in 100 mM phosphate buffer for biochemical and histochemical studies, respectively.
Biochemical Analysis. Levels of phosphotau were analyzed by immunoblotting. The gel system used allowed the comparison of 26 samples simultaneously, and all samples from two groups were run on one gel and compared within the gel. The brain was homogenized in a 5-fold volume of 50 mM Tris-HCl buffer, pH 7.4, consisting 1% Nonidet P40, 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethyl-sulfonyl fluoride, and protease inhibitor cocktail (crude brain homogenate). The homogenate was centrifuged at 20,000g for 20 min at 4°C, and the supernatant was used as a crude tau fraction. The crude tau fraction was heat-treated at 100°C for 5 min, centrifuged at 20,000g for 20 min at room temperature, and the supernatant was used as heat-stable soluble tau fraction. The crude tau fraction was treated with sarkosyl (1% final concentration) for 30 min at room temperature and ultracentrifuged at 100,000g for 1 h at 4 °C. The pellet contained the sarkosyl-insoluble tau fraction.
Samples were solubilized in sample buffer, Tris-HCl buffer, pH 7.0, consisting of 10% glycerol, 5% 2-mercaptoethanol, 2.3% SDS and protease and phosphatase inhibitor cocktail, and run on a 4 to 15% gradient SDS-polyacrylamide gel electrophoresis gel (Criterion; Bio-Rad, Hercules, CA). Proteins were transferred to polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membrane was probed with one of the primary antibodies. For total tau, we used clone tau46, which binds to an epitope within the phosphorylation-independent region of tau (1.2 µg of IgG/ml; Sigma-Aldrich, St. Louis, MO) (Kosik et al., 1988). For phosphorylated tau, we used clones AT8, AT180, and CP13, which detect phosphorylated tau at Ser202/Thr205 (Mercken et al., 1992), Thr231 (Greenberg and Davies, 1990), and Ser202, respectively (AT8 and AT180, 1 µg IgG/ml were obtained from Pierce Biotechnology, Rockford, IL; CP13, 1:200 dilution, was a gift from Dr. Peter Davies, Albert Einstein College of Medicine, New York, NY). The primary antibodies were detected by a horseradish peroxidase-coupled anti-mouse IgG antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA), and visualized with a chemiluminescence kit (Pierce Biotechnology). The protein bands were densitometrically analyzed (Quantity One; Bio-Rad). Because the tau expression level in transgenic mice is not completely consistent among animals, we standardized levels of phosphorylated tau by total tau level; i.e., densitometric measures of phosphorylated tau bands (antibodies AT8, AT180, and CP13) were divided by densitometric measures of total tau bands (antibody tau46).
For Aβ quantification, the crude brain homogenate was centrifuged at 135,000g for 1 h at 4 °C, and the supernatant was used to determine the levels of soluble Aβ. To determine the levels of total Aβ, the crude brain homogenate was mixed with formic acid to yield 70% final concentration, sonicated, ultracentrifuged at 135,000g for 45 min, and the supernatant was used. Levels of Aβ 1-40 and 1-42 were quantified using enzyme-linked immunosorbent assays developed by our group (Horikoshi et al., 2004). In brief, a 96-well plate was coated with either Aβ 40- or 42-specific antibody (clones 1A10 and 1C3, respectively; IBL, Takasaki, Gunma, Japan), then standards and samples were incubated overnight. The plate was then incubated with a horseradish peroxidase-coupled N terminus end-specific antibody (clone 82E1; IBL) and visualized using tetramethylbenzidine as a substrate (Pierce Biotechnology).
Histochemical Analysis. After incubation with 15% sucrose containing phosphate buffer for cryoprotection, 30-µm-thick sagittal sections were prepared using a cryostat. Sections were treated with 0.3% hydrogen peroxide in 10 mM phosphate-buffered saline, pH 7.4, containing 0.3% Triton X-100 for 1 h and then incubated with 1% normal goat serum for 1 h to block nonspecific binding. Sections were incubated with primary antibodies for phosphorylated tau (AT8 and AT180, 1 µg IgG/ml; CP13, 1:200 dilution) and Aβ (82E1, 1 µg IgG/ml) (Horikoshi et al., 2004) overnight at 4°C. Primary antibodies were detected by biotinylated anti-mouse IgG (1 µg IgG/ml in phosphate-buffered saline, pH 7.4, containing 0.3% Triton X-100; Vector Laboratories, Burlingame, CA) for 2 h at room temperature and then visualized using a kit (Vector stain ABC kit; Vector Laboratories). For semiquantitative analysis, we used three hippocampal sections from each mouse; the first section at the Bregma –1.46 mm (Franklin and Paxinos, 1997) and the 10th and the 20th sections from the first section (section thickness, 30 µm; i.e., 10th and 20th sections are at 0.3 and 0.6 mm toward the back from the first section). The number of phosphorylated tau-bearing pyramidal neurons in the CA1 region and the number of Aβ plaques in the hippocampal formation were counted using a 20x objective lens. Slides were coded, and the code was broken after all counts were completed.
Behavioral Analysis. The Morris water maze (MWM) (Morris et al., 1982) is a behavioral procedure designed to test spatial learning and memory by measurements of the time required to find a hidden platform in a pool (defined as latency). A pool (160-cm diameter and 50 cm high) was filled with white-colored water (1 liter of Crayola Premier Tempera white paint; Crayola, Easton, PA), and water temperature was maintained at 24°C. Visual cues were placed around the pool, and a hidden platform (15-cm diameter) was placed at the fixed location. Two daily training sessions, 120 s each, were performed during 5 consecutive days. On the fifth day, immediately after the second daily training, the platform was removed from the pool, and the time spent by the mice in the pool's quarter where the platform used to be was recorded (for a maximal period of 120 s) was termed probe test. Morris water maze was analyzed using video-tracking software (Actimetrics, Wilmette, IL).
To control for animal's visual abilities, we tested the animals in the visible platform version of the MWM. In this variant of the task, the platform with very easily recognizable object was placed into the pool. Mice were released into the water, and time taken to find the platform was recorded (maximum 60 s). Animals that did not climb on the platform during this test were excluded from the statistical analysis as animals with visual defects.
Olfactory Habituation/Dishabituation Task. This test is designed to determine whether a mouse can remember and distinguish between odors (Sánchez-Andrade et al., 2005). The odors were presented on a suspended cotton swab to the test mouse placed into the clean cage with fresh shavings. Each mouse was tested during three consecutive 2-min periods for each odor, with 2-min intervals between presentations. The time that the mouse smelled the swab was recorded (beginning whenever the animal oriented its nostrils toward the cotton swab, within 2 cm or less). The time period measured for water sniffing gave a baseline level comparable with the time period spent in sniffing other odors. After three trials (first block) with the odorless swab (water), the same three trials were repeated (second block) with the swab dipped in diluted vanilla (1:33; McCormick, Hunt Valley, MD). The increase in sniffing time represented the smell sensitivity of the mouse. Mice exhibiting impairment in smell sensitivity were excluded from the test. In general, mice habituate very quickly to odors, and sniffing time declines when the mouse is exposed several times to the same odor. When a new odor is introduced, there is an increase in sniffing time, indicating that the animal can discriminate between odors. After the second block of trials, the swab was dipped in almond extract (1:100; McCormick) and presented to the animals (third block). Increase in sniffing time in the first trial of the third block represented the ability to distinguish between odors and/or the ability to remember the previous odor.
Statistical Analysis. Statistical significance was determined by Student's t test (SPSS, Inc., Chicago, IL).
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), were housed at Georgetown University. Because different mouse colonies may show different rates of progression of tau and Aβ pathology, we examined the progression of pathology using mice at 3, 6, 9, 12, 16, 20, and 23 months of age (n = 6 at each age, age variance ±0.0–0.4 months of age depending on the time points) and found slower progression of pathology in our mouse colony (C. Hirata-Fukae, H.-F. Li, H.-S. Hoe, A. J. Gray, S. S. Minami, K. Hamada, F. Hua, H. Tsukagoshi-Nagai, Y. Horikoshi-Sakuraba, M. Mughal, et al., unpublished data) compared with the original 3xTg-AD mouse colony (Oddo et al., 2003). In brief, at the time we started treatment, 12 months of age, extracellular Aβ plaques were initially detected in the subiculum, and intraneuronal Aβ was seen in the cerebral cortex. Phosphorylated tau was detected in both heat-stable soluble and sarkosyl-insoluble tau fractions, and phosphorylated tau-bearing neurons were seen in the hippocampus and cerebral cortex. Levels of Aβ load, plaques, phosphorylated tau, and phosphorylated tau-bearing neurons increased with aging, and more advanced pathology was seen at the end points of the study; i.e., 15 and 18 months of age. In our colony, thioflavin T-positive mature neurofibrillary tangles were initially detected at 20 months of age. During the treatment period, we monitored the overall health status of the mice in each treatment group. We assigned 12 mice to vehicle treatment and 12 mice to NAP treatment groups for the 3-month treatment period (12–15 months). In this experiment, all mice survived throughout the entire testing period, showing no abnormalities and maintaining their body weights. In the 6-month treatment period (12–18 months), 12 and 13 mice were assigned to the vehicle and NAP treatment groups, respectively. During the treatment, two mice in the vehicle-treated group and one mouse in the NAP-treated group died. These mice died without any change in the overall health status.
After 3 months of intranasal NAP treatment, the level of phosphorylated tau was significantly reduced in the brains of 15-month-old mice (Fig. 1, A–C): 74% at Ser202/Thr205 (P < 0.001), 74% at Thr231 (P < 0.001), and 43% at Ser202 (P < 0.001) in the crude tau fraction (Fig. 1, D–F). In particular, after NAP treatment the number of phosphorylated tau-bearing hippocampal pyramidal neurons was significantly reduced (Fig. 1, G–I versus J–L). Semiquantitative analysis indicated that the number of phosphorylated tau-bearing neurons was significantly reduced after 3 months of NAP treatment: AT8, 53 ± 13% (P < 0.01); AT180, 63 ± 10% (P < 0.01); and CP13, 59 ± 18% (P < 0.01). Both biochemical and histological analysis detected significant NAP-associated reduction at all tau phosphorylation sites that were examined in the current study. Note that our colony of triple transgenic mice shows a slower progression of pathology than the original colony from which it was derived (Oddo et al., 2003), and no mature thioflavin T-positive neurofibrillary tangles were evident at 15 or 18 months of age (C. Hirata-Fukae, H.-F. Li, H.-S. Hoe, A. J. Gray, S. S. Minami, K. Hamada, F. Hua, H. Tsukagoshi-Nagai, Y. Horikoshi-Sakuraba, M. Mughal et al., unpublished data).
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We examined the levels of soluble and total Aβ using specific enzyme-linked immunosorbent assays. In contrast to our previous results with younger mice that showed statistically significant reduction in Aβ1-42 upon NAP treatment (Matsuoka et al., 2007), in these experiments, the NAPassociated reduction in Aβ did not reach statistical significance in older mice. Results after 3 months of NAP treatment were as follows: soluble Aβ 1-40, 80 ± 17% (P = 0.67); soluble Aβ 1-42, 75 ± 16% (P = 0.72); total Aβ, 1-40, 96 ± 15% (P = 0.89); and total Aβ 1-42, 86 ± 13% (P = 0.73), compared with the vehicle-treated mice. We detected a few extracellular Aβ plaques in the hippocampus and intraneuronal Aβ in the cerebral cortex in untreated and vehicle-treated control mice (data not shown); however, the paucity of plaques did not allow for precise determination of a possible effect of NAP treatment (data not shown).
Six-month daily NAP treatment resulted in significant cognitive enhancement in 18-month-old mice as measured in the MWM and in the habituation/dishabituation test. In the MWM hidden platform probe test, NAP-treated mice remembered the hidden platform location (rescue point) after a 5-day training period, spending significantly more time in the quadrant where the platform was originally placed (P < 0.05; Fig. 3A) and crossing the platform location more frequently compared with vehicle-treated mice (P < 0.05; Fig. 3B).
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In the habituation/dishabituation test that exposes the mice to water followed by two different odorants, each presented three consecutive times, both NAPand vehicletreated mice showed an apparent response to the first odorant (Fig. 3D1), and the arithmetic difference between first odorant and third water investigation time (i.e., Fig. 3, D1–C3; P = 0.435) was not statistically significant between the tested groups. In contrast, in response to the second odorant (Fig. 3E1), only the NAP-treated mice showed significantly more interest compared with their interest in the previous odorant (Fig. 3, E1–D3; P < 0.05), indicating significantly increased olfactory recognition memory as a consequence of NAP treatment.
Six months of NAP treatment increased levels of heatstable soluble tau (Fig. 4A), whereas sarkosyl-insoluble, presumably abnormal, tau was unchanged (Fig. 4B). Quantitative analysis of the protein bands indicated that the NAP-related cognitive benefit after 6 months of treatment was associated with an increase of soluble tau; heat-stable soluble tau was significantly increased (by 
62%; P < 0.05) (Fig. 4C), but sarkosyl-insoluble tau was unchanged (Fig. 4D), implicating a significant change in tau solubility as a consequence of NAP treatment.
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An apparent decrease in tau phosphorylation at Ser202/Thr205 (35%; P = 0.067) was found in heat-stable soluble tau fraction (Fig. 4, E and F) in the NAP-treated mice compared with vehicle-treated mice. All other phosphorylation sites examined in this study were unchanged (P > 0.10). Histological examination detected no changes in phosphorylated tau-bearing hippocampal neurons (data not shown). Histological examination is thought to represent the crude (total) tau fraction. Because the biochemical method did not detect significant changes in the phosphorylation levels of tau in the crude fraction, both biochemical and histological examinations were in agreement.
In contrast to results obtained with NAP treatment during the early pathological stage (9–12 months) that reduced Aβ content (Matsuoka et al., 2007), and in agreement with results obtained after a shorter treatment at later stages (12–15 months, above), the levels of Aβ were not affected by 6 months of NAP treatment (12–18 months). Results for NAP-treated mice compared with the vehicle-treated mice were as follows: soluble Aβ 1-40, 95 ± 13% (P = 0.86); soluble Aβ 1-42, 93 ± 17% (P = 0.76); total Aβ 1-40, 106 ± 13% (P = 0.89); and total Aβ 1-42, 87 ± 19% (P = 0.81). As expected from the biochemical data, we did not find a difference in histologically detected Aβ plaques between the vehicle- and NAP-treated mice after 6 months of treatment (data not shown).
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We previously tested a lower dose of NAP (0.5 µg/day/mouse) at an earlier pathological stage in the 3xTg-AD model (9–12 months of age) and found a significant 40% reduction of phosphorylated tau and 20% reduction of Aβ load after NAP treatment (Matsuoka et al., 2007). Although the detailed mechanism is unknown, NAP is thought to modulate tau phosphorylation through interaction with neuronal and glial tubulin. As the pathology gradually increases with aging in the 3xTg AD model, we assumed that a higher dose of NAP might be required to alter tau pathology and behavioral outcomes in mice with more advanced pathology. For this reason, we used a higher NAP dose in the current studies, 2 µg/day/mouse.
A chronic 3-month treatment (12–15 months of age) with the higher NAP dose significantly reduced the levels of phosphorylated tau and sarkosyl-insoluble abnormal tau. The most robust reduction was detected in Ser202/Thr205, a phosphorylation site on tau that was observed to respond to NAP treatment in previous studies (Gozes and Divinski, 2004; Matsuoka et al., 2007; Vulih-Shultzman et al., 2007). The reduction in tau phosphorylation at Ser202/Thr205 was maintained (although to a lower degree) also after the longer 6 months of treatment, under conditions of worsening pathology and increased sample variability (12–18 months of age). A recent study investigated the degree of tau phosphorylation in the cerebrospinal fluid of AD patients. Levels of phosphorylated tau at Ser202/Thr205 (AT8 site) were significantly higher in AD patients compared with the nondemented control group, suggesting that tau phosphorylation at Ser202/Thr205 is associated with the presence of dementia in human AD cases (Maccioni et al., 2006). Tau phosphorylation at Ser202/Thr205 antagonizes the tau-mediated nucleation of tubulin (Hashiguchi et al., 2002), probably resulting in a paucity of microtubules leading to neuronal dysfunction and cognitive decline.
Although a direct head-to-head comparison is necessary to demonstrate dose dependence, our experience across these two sets of studies is consistent with a NAP dose-related activity. This assumption is further corroborated by our preliminary study that indicates that 30 µg NAP/day/mouse may have a greater effect on the behavioral outcomes when compared head-to-head with a 2 µg/day/mouse regimen of treatment (data not shown).
The 2 µg/day/mouse NAP regimen used here, chronically applied for 6 months in the aged 3xTg-AD mice, increased cognitive function compared with vehicle treatment. These results extend and broaden previous studies using short-term treatment with NAP in other AD-related animal models. Thus, NAP enhanced spatial learning and memory in apolipoprotein E-deficient mice [apolipoprotein E is the most important risk factor for AD (Bassan et al., 1999), and control apolipoprotein E-deficient mice show increased tau pathology (Gordon et al., 1996)]. Similar findings were also observed in rats exposed to the cholinotoxin, AF64A (Gozes et al., 2000), which is associated with neuronal cell death and hence microtubule disruption.
Although there is a general consensus that reduction of hyperphosphorylated and conformationally abnormal tau will be beneficial, prior studies have not provided precise evidence linking specific forms of tau to cognitive dysfunction. More than 30 phosphorylation sites on tau have been identified (Ahlijanian et al., 2000; Spittaels et al., 2000), but the importance of each phosphorylation site is unclear. The current study may implicate Ser202/Thr205 as important for tau/microtubule involvement in cognitive functions and further suggests that the amount of soluble, presumably functional tau versus insoluble, presumably functionally impaired tau could be a useful index to evaluate the potency of tau therapeutic agents in tau models of cognitive deficits.
Although the mechanisms are not fully understood, there is evidence that the Aβ and tau pathways influence each other, and reduction of Aβ may be associated with reduced levels of phosphorylated tau in vivo (Oddo et al., 2004). NAP treatment reduced phosphorylated tau levels and changed tau distribution throughout the course of the development of the pathology, without significantly altering Aβ load at later pathological states. Because cognitive performance was also improved in NAP-treated mice and was associated with increased soluble tau, the current results suggest a correlation between tau pathology and cognitive function and support previous data that reduction of phosphorylated tau without altering Aβ load through genetic manipulation may lead to improvement of cognitive impairment (SantaCruz et al., 2005). The results further imply that a significant pharmacological inhibition of tau pathology offers effective therapeutic benefits as exemplified by cognitive enhancement.
The NAP amino acid structure (NAPVSIPQ) has beta sheet breaker characteristics, and NAP interferes with Aβ aggregation in vitro (Ashur-Fabian et al., 2003). In our previous study using 3xTg-AD mice at an earlier pathological stage (9–12 months of age), we found that 3-month intranasal treatment significantly reduced the Aβ load (Matsuoka et al., 2007), suggesting, as indicated above, temporal effect of NAP on both the Aβ and the tau pathological hallmarks of AD at the early stages of the disease.
We administered NAP by intranasal drops. It should be noted that pharmacokinetic studies indicate that NAP enters the central nervous system through the blood, even with intranasal administration, i.e., NAP is found in the cerebrospinal fluid concurrently with being found in the plasma regardless of the administration route (e.g., Gozes et al., 2005; Morimoto et al., 2006). Measurements of NAP were conducted by a tandem liquid chromatography mass spectroscopy method. In addition, preliminary data suggest beneficial effects of NAP on the habituation/dishabituation task when administered by i.p. injection (I. Gozes and R. Pikman, unpublished data).
Initial memory formation and its short-term retention require the function of the olfactory bulb, the piriform and entorhinal cortices, and the hippocampus. It is thus suggested that NAP treatment increased olfactory memory function rather than smell sensitivity (which may not be impaired in the tested 3xTg-AD mice). Normal control mice, young and aged (Frick et al., 2000), behave in a similar manner to NAP-treated transgenic mice (data not shown). It is interesting to note that AD patients frequently have odor recognition pathology (Wilson et al., 2007). Furthermore, deficits in olfactory detection thresholds and identification occur early in AD, before clinical symptoms are fully developed and decline further over the course of the disease. High detection thresholds, together with impaired identification, may be useful as an early indicator of AD (Djordjevic et al., 2007) in addition to being responsive to NAP administration. It is interesting to note that anosmia has been related to microtubule dysfunction (Kulaga et al., 2004).
Previous studies have shown that NAP treatment transiently increases the amount of nonphosphosphorylated tau in a time course paralleling apparent increased microtubule stabilization (Gozes and Divinski, 2004) and that NAP treatment reduced tau hyperphosphorylation and increased cognitive function in mice lacking its parent protein, activity-dependent neuroprotective protein (Vulih-Shultzman et al., 2007). Taken together with the current data, these studies suggest that microtubule-targeting drugs may protect against tau pathology and cognitive decline. To date, two tubulin-interacting agents, NAP and paclitaxel have been shown to reduce tau pathology in vivo. Paclitaxel was shown to diminish the NAP-tubulin interaction in vitro (Divinski et al., 2006), suggesting that these two agents work, in part, through a related mechanism. Although paclitaxel is a non-selective tubulin-interacting agent and is associated with severe adverse effects in humans, NAP exhibits preferential interaction with neuronal tubulin (Divinski et al., 2006) as well as brain bioavailability (Gozes et al., 2000, 2005).
Finally, Phase I clinical testing has demonstrated the safety and tolerability of intranasally administered NAP (Gozes et al., 2005). NAP may thus represent a prototype disease-modifying treatment for AD, targeting the major pathological hallmarks of the disease. The intranasal formulation of NAP (Chemical Abstract Service no. 211439-12-2) is currently in phase II clinical trials in an AD program initially targeting elderly patients suffering from amnestic mild cognitive impairment (Allon Therapeutics Inc., Vancouver, BC, Canada) (Melnikova, 2007).
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Acknowledgements |
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
ABBREVIATIONS: AD, Alzheimer's disease; NAP, NAPVSIPQ; 3xTg-AD, triple transgenic mice; MWM, Morris water maze.
Address correspondence to: Dr. Yasuji Matsuoka, Department of Neurology, Georgetown University Medical Center, 4000 Reservoir Road N.W., Washington, DC 20057. E-mail: ym56{at}georgetown.edu
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, P = 0.067; E and F).