[Frontiers in Bioscience E3, 1375-1384, June 1, 2011]

Long-term Phenylbutyrate administration prevents memory deficits in Tg2576 mice by decreasing A Beta

Ana Ricobaraza1, Mar Cuadrado-Tejedor1,2, Ana Garcia-Osta1

1Division of Neurosciences, CIMA, University of Navarra, Pamplona, Spain1, 2Department of Anatomy, Faculty of Medicine, University of Navarra, Pamplona, Spain2


1. Abstract
2. Introduction
3. Material and methods
3.1. Mouse model and treatment
3.2. Morris water maze test (mwm)
3.3. Determination of A Beta levels
3.4. Immunohistochemistry
3.5. Production of protein extracts
3.6. Immunoblotting
3.7. Data analysis and statistical procedures
4. Results
4.1. Morris water maze test (MWM)
4.2. A Beta pathology
4.3. Tau pathology
5. Discussion
6. Acknowledgments
7. References


Aberrations in protein folding, processing, and/or degradation are common features of neurodegenerative diseases, such as Alzheimer's disease (AD). Sodium 4-phenylbutyrate (PBA) is a well-known histone deacetylase inhibitor, which increases gene transcription of a number of genes, and also exerts neuroprotective effects. PBA acts as a chemical chaperone reducing the load of mutant or unfolded proteins during cellular stress. Previously, we reported that 5-week administration of PBA reinstated memory loss and dendritic spine densities in the Tg2576 mouse model of AD. In this study we reported that chronic administration of PBA, starting before the onset of disease symptoms (6 month-old) prevents age-related memory deficits in Tg2576 mice. The amelioration of the memory impairment is associated to a decrease in amyloid beta pathology and the glial fibrillary acidic protein (GFAP), suggesting that inflammation was reduced in PBA-treated animals. Together, the beneficial effects of PBA make it a promising agent for the prevention of AD.


Alzheimer�s disease (AD) is a neurodegenerative disorder clinically characterized by a progressive cognitive decline. The hallmark pathological features of AD involve misfolding and aggregation of proteins including the accumulation of amyloid Beta (A Beta) protein in plaques (neuritic plaques), and the aggregation of hyperphosphorylated Tau protein in intraneuronal filaments that originate the neurofibrillary tangles (1). A Beta is processed from the amyloid precursor protein (APP), a transmembrane protein folded and modified in the endoplasmic reticulum (ER) and transported through the Golgi complex to the outer membrane (2, 3). The accumulation of proteins in the ER may both, disturb its function and induce ER stress, a cellular response that restores cellular homeostasis. However, if the provoking insult is prolonged, ER stress may trigger apoptotic cell death (4). According to the amyloid hypothesis, the accumulation of A Beta in the brain is the fundamental cause of the disease (5), and could be, at least in part, the responsible of the ER stress that different authors have demonstrated in AD brains (6, 7).

ER stress might activate the expression of glycogen synthase kinase 3 Beta (GSK-3 Beta), a well-known Tau kinase, and in that way, immunohistochemical studies have revealed a close connection between the ER stress and Tau pathology in neurons (8). Moreover, recent studies have indicated that ER stress may also trigger inflammatory responses to defend brain tissue against necrotic injuries (9). Importantly, a chronic inflammatory response is associated to the A Beta pathology and it is known that activated microglia and astrocytes closely associate to amyloid plaques in AD brains (10, 11). Overall, ER stress is clearly involved in the pathogenesis of AD. On one hand the pathological characteristics of AD (oxidative stress, impaired Ca2+ homeostasis, intracellular deposition of Tau proteins and amyloid- Beta peptides) may be caused by ER stress in neurons. On the other hand, these pathological features may also trigger ER stress and thus aggravate AD pathology (2, 9, 12).

Recently, we have demonstrated that the administration of the histone deacetylase (HDAC) inhibitor, 4-phenylbutyrate (PBA) for 5 weeks reversed the memory impairments in the AD mouse model Tg2576 mice at the age of 15 month (13). PBA is a short chain acid fat (SCAF), acts as a chemical chaperone and, furthermore, exerts anti inflammatory properties (14, 15), thus, may be a potential pharmacological therapy to revert AD-like symptoms. We have shown that the effect of PBA in Tg2576 mice was associated with a rescue of the synaptic dendritic markers and dendritic spine loss related to the memory failure (16). Tg2576 mice overexpress the human Swedish mutation of the APP (APPK670N, M671L) (17) and by 15 months of age they have considerable amyloid pathology, quantifiable inflammatory response (18) and serious learning impairment in the traditional spatial reference memory water maze (19, 20).

Although the 5-week treatment was significantly effective in the amelioration of memory deficits in aged mice, no changes were found in total A Beta cortical levels measured by ELISA (13). However, in further studies focused in the mechanism involved in PBA-induced memory restoration, a decrease in the intracellular accumulation of A Beta in CA1 pyramidal cells was revealed in transgenic mice receiving PBA treatment (16). These data suggested that PBA may facilitate the clearance of A Beta and this effect could be related to its chaperone-like activity and ability to mitigate ER stress (21). It has been postulated that PBA could have an anti inflammatory effect through inhibition of ER stress (14, 15, 22). The present study was aimed to determine whether a chronic treatment with PBA, starting at the onset of AD symptoms, would prevent the progressive amyloid pathology (and histopathological changes linked to the disease) as well as the associated immunological response. For this purpose, 6-month old Tg2576 mice were treated until 12 months of age, when behavioural and biochemical assays were carried out to asses the potential preventive effect of PBA in AD.


3.1. Mouse model and treatment

In this study, Tg2576 AD transgenic mice, which express the human 695-aa isoform of APP containing the Swedish double mutation (APPswe) ((APP695)Lys670→Asn, Met671→Leu) driven by a hamster prion promoter, were used. In the Tg2576 AD mouse model, A Beta peptide content in the brain accumulates exponentially between 7 and 12 months of age and mice show impaired memory in the water maze test at the age of 12 - 15 months (17, 23).

The aim of this study was to investigate whether PBA could prevent Alzheimer phenotype, thus, 6 month old Tg2576 mice (before the onset of symptoms) were administered daily with PBA (200 mg/kg, i.p.) or vehicle for 6 months. PBA solution was prepared by titrating equimolecular amounts of 4-phenylbutyric acid (Sigma, Madrid, Spain) and sodium hydroxide to pH 7.4. As a control, a group of strain- and age-matched non-transgenic mice was used. All procedures were carried out in accordance with European and Spanish regulations (86/609/CEE; RD1201/2005). This study was approved by the Ethical Committee of the University of Navarra (no. 018/05).

3.2. Morris Water Maze Test (MWM)

The MWM test was used to evaluate spatial memory function in response to the treatment as previously described (13, 17, 23). Briefly, mice were trained for three consecutive days (8 trials/day) swimming to a platform above the water (visible-platform). The same platform location was used for all visible-platform sessions and was changed for the invisible-platform training, conducted over the next 8 consecutive days (4 trials/day) and during which, the time required to reach the hidden platform was recorded. To test the retention, three probe trials were performed at the beginning of 4th, 7th, and 9th. In the probe trials the platform was removed from the pool and the percent of time spent in the quadrant where previously was the platform was recorded. All trials were monitored by a camera above the center of the pool connected to a SMART-LD program (Panlab S.L., Barcelona, Spain). All experimental procedures were performed blind to groups.

On day 10th, the platform was moved diagonally across its initial position in the invisible platform, without changing any distal visual cues, and relearning was assessed. Each animal performed the test as described above with the new platform location. Two probe trials were performed on days 12th and 14th. The time spent into the quadrant of the previous platform location and time spent in the new location quadrant, indicated learning of the new condition, was measured. In all cases, animals were killed 24 h after the last trial. The brains were removed and immediately frozen on dry ice before dissection. Some animals were perfused transcardially with paraformaldehyde for immunohistochemistry.

3.3. Determination of A Beta levels

For the analysis of total A Beta42 burden, mice frontal cortex were homogenized in a buffer containing 5 M guanidine HCl and 50 mM Tris HCl, pH 8, protease inhibitors (CompleteTM Protease Inhibitor Cocktail, Roche, Barcelona, Spain) and phosphatase inhibitors (0.1 mM Na3VO4, 1 mM NaF). A Beta42 levels were measured using a sensitive sandwich ELISA kit from Biosource (Camarillo, Ca, USA) following the manufacturer's instructions.

3.4. Immunohistochemistry

Floating tissue sections comprising hippocampal and cortex formation were processed for immunohistochemistry. Brain sections were rinsed (3 x 10 min) with 0.125 M phosphate buffer (pH 7.4). The endogenous peroxidase activity was eliminated by pre-treatment with 0.3% hydrogen peroxide in methanol for 45 min and washed in 0.125 M phosphate buffer solutions (PBS). The sections were then incubated in a blocking solution (PBS containing 0.5% Triton X-100, 0.1% BSA and 2% normal goat serum) for 2 h at room temperature and incubated overnight with primary antibodies in blocking solution. Subsequently, the sections were washed in PBS and incubated in a biotinilated secondary antibodies that matched the primary antibodies (1:200, Vector Laboratories, Burlingame, CA, USA) diluted in blocking solution for 2 h and then avidin-biotin complex for 1 h at room temperature. The reaction was developed using the diaminobenzidine (DAB) kit (Vector Laboratories). For 3D6 immunostaining, sections were incubated in 70% formic acid for 10 min to expose the epitope. The primary antibodies used were as follows: mouse monoclonal 3D6 (amino acids 1-5 of A Beta peptide, 1:4000, Chemicon, Temecula, CA), mouse monoclonal anti phospho-Tau (Ser202/Thr205) AT8 (1:50, Pierce Biotechnology Inc., Rockford), rabbit polyclonal anti-GFAP (1:1000, Sigma-Aldrich, St Luis, MO). For the double immunostaining, the secondary antibodies used were Alexa Fluor 488 goat anti-mouse, highly cross-absorbed, and Alexa Fluor 546 goat anti-rabbit, highly cross-absorbed (both 1:200, Molecular Probes, Eugene, Oregon, USA). Sections were mounted on super frost plus slides, air dried for 24 h, rinsed in toluene (2 x 5 min), and cover slip with DPX mounting medium. To ensure comparable immunostaining, sections were processed together under identical conditions. For the assessment of nonspecific primary immunostaining, some sections from each experimental group were incubated without the primary antibodies; in this case no immunostaining was observed. Nonspecific secondary immunostaining was also evaluated by incubating sections with primary and its nonspecific secondary antibodies; again, no immunostaining was observed.

3.5. Production of protein extracts

Mice were sacrificed by cervical dislocation and hippocampi quickly dissected from the brains. Total tissue homogenates were obtained by homogenizing the hippocampus in a cold lysis buffer with protease inhibitors (0.2 M NaCl, 0.1 M HEPES, 10% glycerol, 200 mM NaF, 2 mM Na4P2O7, 5 mM EDTA, 1 mM EGTA, 2 mM DTT, 0.5 mM PMSF, 1 mM Na3VO4, 1 mM benzamidine, 10 mg/ml leupeptin, 400 U/ml aprotinin), centrifuged at 14,000 x g 4�C for 20 min and the supernatant was aliquoted and stored at -80�C. Total protein concentrations were determined using the BioRad Bradford protein assay (BioRad Laboratories).

For APP carboxy-terminal fragments determination, the hippocampus was homogenized in a buffer containing SDS 2%, Tris-HCl (10 mM, pH 7.4), protease inhibitors (Complete™ Protease Inhibitor Cocktail, Roche) and phosphatase inhibitors (0.1 mM Na3VO4, 1 mM NaF). The homogenates were sonicated for 2 min and centrifuged at 100,000 g for 1 h. Aliquots of the supernatant were frozen at −80 �C and protein concentration was determined by the Bradford method using the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA).

3.6. Immunoblotting

Protein samples were mixed with an equal volume of 2 x Laemmli sample buffer, resolved onto SDS-polyacrylamide gels and transferred to nitrocellulose membrane. The membranes were blocked with 5% milk, 0.05% Tween-20 in PBS or TBS followed by overnight incubation with the following primary antibodies: mouse monoclonal anti-p-Tau AT8 (1:1000, Pierce Biotechnology), mouse monoclonal anti Tau (clone Tau46, 1:1000, Sigma-Aldrich), rabbit polyclonal anti-pGSK3-Ser9 (1:1000, Cell Signalling Technology, Beverly, MA), rabbit polyclonal anti-pAkt-Ser473 (1:1000, Cell Signalling Technology), rabbit polyclonal anti-GSK3 (1:1000), rabbit polyclonal anti-GFAP (1:100, Sigma-Aldrich) and mouse monoclonal anti-actin (1:4000, Santa Cruz Biotechnology, Santa Cruz, CA) in the corresponding buffer. Following two washes in PBS/Tween-20 or TBS/Tween20 and one PBS or TBS alone, immunolabeled protein bands were detected by using HRP-conjugated anti-rabbit or anti-mouse antibody (Santa Cruz; dilution 1:5000) following an enhanced chemiluminescence system (ECL, GE Healthcare Bioscience, Buckinghamshire, UK), and autoradiographic exposure to HyperfilmTMECL (GE Healthcare Bioscience ). Signals quantification was performed using Scion Image software (Scion Corporation).

For western blot analysis of APP-derived fragments, aliquots of the protein extracts were mixed with XT sample buffer™plus XT reducing agent™ or Tricine sample buffer™ (Bio-Rad) and boiled for 5 min. Proteins were separated in a Criterion™ precast Bis-Tris 4-12% gradient precast gel (Bio-Rad) and transferred to nitrocellulose membranes. The membranes were blocked with 5% milk, 0.05% Tween-20 in TBS followed by overnight incubation with the following primary antibodies: mouse monoclonal 3D6 (amino acids 1-5 of A Beta peptide, 1:4000, Chemicon), rabbit polyclonal anti-APP C-terminal (amino acids 676-695) (1:2000, Sigma-Aldrich).

3.7. Data analysis and statistical procedures

The results were processed for statistical analysis using SPSS package for Windows, version 15.0 (SPSS, Chicago, IL, USA). Unless otherwise indicated, results are presented as mean�SEM. In the MWM and fear conditioning test, escape latencies during training were analyzed using one-way analysis of variance (ANOVA) followed by Scheffé post hoc test. In the MWM, Friedman�s test was performed to determine the intra group comparisons over trials. Biochemical data were analyzed using one-way ANOVA followed by Scheffé post hoc test.


4.1. Morris Water Maze test (MWM)

To test whether PBA prevents memory deficits in AD transgenic mice, we assessed learning/memory performance at 12 months of age after six months of treatment with PBA or vehicle. At the end of the treatment no significant differences were found between the groups in their performance in both the visible and the invisible platform training. Escape latencies from the hidden platform were analyzed using the Friedman�s repeated measure nonparametric test for the intra-group comparisons. The mean latencies spent to reach the platform decreased significantly over the training sessions for all the groups: non-transgenic (Non-Tg; χ2r=19.67, p<0.01), Tg2576-saline (χ2r=17.67, p<0.01) and Tg2576-PBA (χ2r=14.61, p<0.01) mice. Specifically, intra-group comparisons of escape latencies showed a significant effect of the training in all the groups, reflecting its ability to learn the platform location.

After the 12 th, 24th and 32 th trial, all mice were subjected to a probe trial in which they swam in the pool for 60 s with the platform removed. Memory retention was measured as the percentage of time (within 60 s) that mice spent in the target quadrant (held the platform during training). The probe on days 7th and 9th showed that transgenic mice treated with saline spent significantly less time in the target quadrant than non transgenic mice. Transgenic mice that underwent PBA treatment spent a percent of time in the target quadrant that not differ from that of age-matched controls (Figure 1B). These data suggest that PBA, administered chronically to Tg2576 mice, prevents the deficits in the water maze memory retention assay.

One day after the probe in day 9, the platform was moved to the opposite quadrant location and mice were trained for 4 days (four trials per day, days 10th to 13th) in this reversed setting (reversal learning). Tg2576 saline mice showed significantly longer escape times over the training blocks on days 11 and 12, whereas the non-transgenic and PBA-treated mice more rapidly reached the platform in the new location (Figure 1C). These results demonstrate that a persistent search in the previous target zone resulted in a significant delay for Tg2576 saline mice to find the new target location. The probe test, in which the platform was again removed, revealed significant differences in performance between groups (Figure 1D). In the quadrant that had previously contained the platform (old platform), a gradual reduction in percent time can be seen for all the groups. A shift to the 'new' goal position (new platform) was observed on day 12 for PBA-treated mice, whereas non-transgenic mice reached similar memory retention in the reversed setting (new location) two days later (14th probe day). Transgenic-saline treated mice spent less than 25% of time (random learning) in the new location at the end of the task, indicative of memory retention impairment (Figure 1D).

Thus, at the age of 12 month, Tg2576 mice had clear deficits in spatial memory consolidation and in the reversal task. Reversion of these deficits by chronic treatment with PBA supports a role of PBA in plasticity remodelling and memory enhancement in the AD transgenic mice.

4.2. A Beta pathology

The effect of PBA treatment on the A Beta levels in Tg2576 mice brain was determined in the cerebral cortex by sandwich ELISA. As shown in Figure 2A, a significant difference (50% reduction, p < 0.05, Student΄s t test) was seen in A Beta42 levels in Tg2576 mice treated with PBA compared to saline-treated transgenic mice. No A Beta was detected in wild type littermates (data not shown). The levels of the carboxyl terminal fragments of APP, C83 and C99, were analyzed using Tris/tricine PAGE 16.5% gels for a better resolution. No differences in intensity of any of the fragments were found between the two groups of transgenic mice, suggesting that PBA does not affect APP processing (Figure 2B).

A Beta immunoreactivity was assayed using 3D6 antibody to stain intraneuronal A Beta and extracellular deposits. Fewer senile plaques were detected in the frontal cortex and hippocampus of mice receiving chronic PBA treatment compared to Tg2576-saline treated mice (Figure 2C). Interestingly, 3D6 immnuoreactivity displayed largely reduced levels of intraneuronal A Beta in the pyramidal cells of CA3 and CA1 and in the granular layer and the hilus of dentate gyrus (GD) (Figure 2D) of mice treated with PBA compared to saline-treated animals.

Inflammatory processes are intimately involved in several crucial events in the pathological cascade, in particular with dense-core A Beta plaques. Previous studies had shown that PBA have anti inflammatory effect (14). Analysis of astroglial activation by double staining for A Beta42 (using 3D6 antibody) and the astrocytic marker molecule, glial fibrillary acidic protein (GFAP), were carried out in mice hippocampus. Immunostaining revealed that GFAP-positive cells were mostly located around amyloid plaques in the brain of Tg2576 mice (Figure 3A). Remarkably, amyloid plaques and activated astrocytic cells were decreased in the brain of Tg2576 mice receiving PBA for 6 months. In the brain of non-transgenic mice, neither 3D6 nor GFAP immunostaining was detectable (data not shown).

In order to confirm these immunohistochemical findings, western blotting of hippocampal homogenates was carried out. Statistical comparison of GFAP band optic density normalized with Beta-actin showed significant differences among the different groups (Figure 3B). Expression levels of GFAP in Tg2576-PBA treated mice (82 %) were significantly decreased (p< 0.05) when compared to that of untreated Tg2576 mice (121 %), suggesting that PBA prevents the astrocytosis observed in the untreated Tg2576 animals.

4.3. Tau pathology

Hiperphosphorylation of Tau is another pathological mark that contributes to the development of AD. An enhancement in Tau phosphorylation in transgenic animals was observed by immunocytochemistry using the AT8 antibody, which recognizes aberrantly hyperphosphorylated epitopes. In fact, positive neurons for AT8 staining were easily found in the CA3 region and the DG of the hippocampus in these animals but were not presented in the samples from their respective non-transgenic age-matched controls or in the transgenic mice receiving PBA (Fig 4A). The levels of Tau phosphorylation in the mice hippocampus were measured by western blot analysis using the same antibody (AT8) and normalized to total Tau (T46). As depicted in Figure 4B, a one-way ANOVA showed a significant group effect (p< 0.05). Scheffe's multiple-comparison test revealed that phosphorylation of Tau was significantly increased in the hippocampus of 12-month-old Tg2576 (124 %, p < 0.05) compared to non-transgenic mice (100 %) and was only partially reversed in transgenic mice treated with PBA (114 %) (Figure 4B). Western blot analysis showed that PBA moderately reduced Tau phosphorylation in transgenic mice, whereas the effect observed using immunohistochemistry, seems more consistent. This could be due to the differences between the two techniques, and, although the immnuhistochemistry is not quantitative, allows it to detect changes in specific areas (i.e.: CA3 and DG) which can be undetectable if the whole hippocampus is analyzed by western blot.

Tau hyperphosphorylation may be the result of dysregulation of different pathways, including the pGSK3/pAkt pathway. The levels of inactive GSK3 Beta, phosphorylated at Ser 9 (pGSK3 Beta-Ser9), normalized to GSK3 Beta were measured. A one-way ANOVA followed by Scheffe's multiple-comparison test revealed that pGSK3 Beta-Ser9 levels were markedly decreased in the hippocampus of transgenic mice treated with vehicle (36 %) compared to non-transgenic mice (100 %). No significant differences in pGSK3ί-Ser9 levels were found in transgenic mice treated with PBA (125 %) compared to non-transgenic mice (Figure 4C). In the same extracts the level of pAkt, which phosphorylates GSK3 Beta at Ser 9, were analyzed. The expression of pAkt in transgenic mice that received PBA was no significantly different from that of non-transgenic mice (80 % vs non-Tg: 100 %); in contrast, the saline-treated Tg2576 mice showed a marked reduction in pAkt (20 %; p< 0.01 vs non-Tg and p< 0.05 vs Tg2576-PBA) (Figure 4D). These data suggest that PBA induces a reduction in pTau levels through the modulation of the pAkt/GSK3 pathway.


Few studies have tested whether or not a treatment at the pre-plaque stage, that would parallel the MCI stage of AD, would prevent/delay the progressive cognitive decline in AD models. Our previous studies demonstrated that 5-week treatment with PBA restored the memory impairments in aged Tg2576 mice without decreasing total brain A Beta burden (13, 16). Here, we investigated whether PBA would be effective in preventing A Beta pathology and the associated memory deterioration in an AD mouse model when the treatment starts before the onset of the disease's symptoms. For this aim, 6 month-old Tg2576 mice were administered daily with PBA (200 mg/kg, i.p.) or vehicle for 6 months. At the end of the treatment, memory function in mice was tested using the Morris water maze test with a reversal phase, in which animals have to extinguish their initial learning of the platform's position and acquire a direct path to a new goal position (24). A platform reversal appears to be a suitable test to detect subtle changes in synaptic plasticity leading to a cognitive deficit that may be eventually prevented by PBA treatment.

In this study, and corroborated by others (23, 25), 12 month Tg2576 mice were able to acquire and use a spatial map in the standard version of the Morris water maze task, whereas they were impaired in probe trials (memory consolidation). PBA treatment prevented their impairment in memory retention, showing a similar performance to non transgenic mice. Analysis of reversal trial performance showed that, whereas untreated transgenic mice did not learn the new platform location, both, the non-transgenic and transgenic PBA-treated mice showed decreasing latencies escape in the new platform position with repeated reversal trials. Pompl et al., (26) also demonstrated that, at the age of 9 month, Tg2576 performed similar to non-transgenic mice in the spatial learning phase (invisible platform) of MWM but performed worse in the reversal learning phase. Our data demonstrated that transgenic mice receiving chronic PBA treatment performed in the reversal learning phase similar to non transgenic mice, suggestive of an improvement of synaptic plasticity.

Eventhough 5 week-treatment with PBA did not modified A Beta burden in aged mice with extensive amyloid pathology (13), in the present study we show that when the PBA treatment starts before the onset of amyloid pathology, a decrease in total A Beta levels detected in mice brain might contribute to the memory restoration. Other therapeutic approaches that decrease the level of soluble A Beta in animal models, even in the absence of detectable changes in plaque load, ameliorate cognitive deficits (27, 28). These results demonstrate that PBA, administered before the onset of the disease symptoms, may prevent spatial learning and memory decline of Tg2576 mice by decreasing A Beta42 accumulation in the brain. This could be due to the PBA cytoprotective chaperone effect that facilitates the clearance of A Beta42 without affecting its production, since APP processing was not affected.

When cells are subjected to protein misfolding in the ER, a stress cascade with pathological consequences, including inflammation and cell death, is triggered. In a recent study we demonstrated that PBA clear intraneuronal A Beta staining in CA1 and affects the levels of ER stress markers (16). On analyzing inflammation-associated transcripts in 12-month Tg2576 mice, an up-regulation of GFAP, which reflects astro- and micro- gliosis occurs (18, 29) (30). GFAP-positive astrocytes are located in the vicinity of the A Beta plaques (31) and may promote A Beta plaque formation and maturation (19). Noteworthy, here we report a reduction in astrocyte activation (GFAP expression) in the hippocampus in PBA-treated mice compared to untreated transgenic mice, which correlates with a reduction in A Beta burden. Thus, it may be hypothesized that a reduction in intracellular A Beta, regulates ER stress markers and mediate events that reduce GFAP immunoreactivity.

ER stress activates GSK3b through dephosphorylation of Ser9 (32). Previously, we showed that PBA inhibited GSK3b in Tg2576 mice hippocampus after a 5 week-treatment (13). These results suggest that PBA protects neurons from ER stress and leads to GSK3b inhibition, which in turns prevents tau phosphorylation and contributes to restore neuronal plasticity. Here, using immunoshistochemitry and western blot analysis, we corroborated that PBA is able to decreased pTau in the hippocampus of Tg2576 mice, which could be mediated by the GSK3 Beta inactivation, as deduced by the increase in GSK3 Beta-Ser 9 levels detected in the hippocampal extracts.

Overall, there are two possible mechanisms in the preventive and beneficial effect of PBA in Tg2576 after 6 month-treatment: (1) PBA decreases the inflammatory reaction engendered by amyloid deposits and/or (2) PBA modulates the clearance of A Beta and amyloid deposition, and thereby reduces ER stress and inflammatory reaction.


This study has been supported by the Ministry of Education and Science (SAF2008-02342), and UTE project FIMA, Spain. We thank Dr. R. Franco for critical reading of the manuscript. We thank María Espelosín, Susana Ursúa and Esther Gimeno for technical help, and the animal facility of CIMA for technical support.


1. K. Blennow, M. J. de Leon and H. Zetterberg: Alzheimer's disease. Lancet, 368, 387-403 (2006)

2. S. C. Domingues, A. G. Henriques, W. Wu, E. F. Da Cruz e Silva and O. A. Da Cruz e Silva: Altered subcellular distribution of the Alzheimer's amyloid precursor protein under stress conditions. Ann N Y Acad Sci, 1096, 184-95 (2007)

3. A. Weidemann, G. Konig, D. Bunke, P. Fischer, J. M. Salbaum, C. L. Masters and K. Beyreuther: Identification, biogenesis, and localization of precursors of Alzheimer's disease A4 amyloid protein. Cell, 57, 115-26 (1989)

4. M. Schroder: Endoplasmic reticulum stress responses. Cell Mol Life Sci, 65, 862-94 (2008)

5. J. Hardy and D. J. Selkoe: The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science, 297, 353-6 (2002)

6. J. J. Hoozemans, E. S. van Haastert, D. A. Nijholt, A. J. Rozemuller, P. Eikelenboom and W. Scheper: The unfolded protein response is activated in pretangle neurons in Alzheimer's disease hippocampus. Am J Pathol, 174, 1241-51 (2009)

7. F. M. LaFerla, K. N. Green and S. Oddo: Intracellular amyloid-beta in Alzheimer's disease. Nat Rev Neurosci, 8, 499-509 (2007)

8. U. Unterberger, R. Hoftberger, E. Gelpi, H. Flicker, H. Budka and T. Voigtlander: Endoplasmic reticulum stress features are prominent in Alzheimer disease but not in prion diseases in vivo. J Neuropathol Exp Neurol, 65, 348-57 (2006)
PMid:20035627 PMCid:2806266

9. A. Salminen, A. Kauppinen, T. Suuronen, K. Kaarniranta and J. Ojala: ER stress in Alzheimer's disease: a novel neuronal trigger for inflammation and Alzheimer's pathology. J Neuroinflammation, 6, 41 (2009)

10. P. Cras, M. Kawai, S. Siedlak, P. Mulvihill, P. Gambetti, D. Lowery, P. Gonzalez-DeWhitt, B. Greenberg and G. Perry: Neuronal and microglial involvement in beta-amyloid protein deposition in Alzheimer's disease. Am J Pathol, 137, 241-6 (1990)

11. S. Haga, K. Akai and T. Ishii: Demonstration of microglial cells in and around senile (neuritic) plaques in the Alzheimer brain. An immunohistochemical study using a novel monoclonal antibody. Acta Neuropathol, 77, 569-75 (1989)

12. C. Pereira, E. Ferreiro, S. M. Cardoso and C. R. de Oliveira: Cell degeneration induced by amyloid-beta peptides: implications for Alzheimer's disease. J Mol Neurosci, 23, 97-104 (2004)

13. A. Ricobaraza, M. Cuadrado-Tejedor, A. Perez-Mediavilla, D. Frechilla, J. Del Rio and A. Garcia-Osta: Phenylbutyrate ameliorates cognitive deficit and reduces tau pathology in an Alzheimer's disease mouse model. Neuropsychopharmacology, 34, 1721-32 (2009)

14. X. Qi, T. Hosoi, Y. Okuma, M. Kaneko and Y. Nomura: Sodium 4-phenylbutyrate protects against cerebral ischemic injury. Mol Pharmacol, 66, 899-908 (2004)

15. W. Qi, J. Mu, Z. F. Luo, W. Zeng, Y. H. Guo, Q. Pang, Z. L. Ye, L. Liu, F. H. Yuan and B. Feng: Attenuation of diabetic nephropathy in diabetes rats induced by streptozotocin by regulating the endoplasmic reticulum stress inflammatory response. Metabolism (2010)

16. A. Ricobaraza, M. Cuadrado-Tejedor, S. Marco, I. Perez-Otano and A. Garcia-Osta: Phenylbutyrate rescues dendritic spine loss associated with memory deficits in a mouse model of Alzheimer disease. Hippocampus (2010)

17. K. Hsiao, P. Chapman, S. Nilsen, C. Eckman, Y. Harigaya, S. Younkin, F. Yang and G. Cole: Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science, 274, 99-102 (1996)

18. S. A. Frautschy, F. Yang, M. Irrizarry, B. Hyman, T. C. Saido, K. Hsiao and G. M. Cole: Microglial response to amyloid plaques in APPsw transgenic mice. Am J Pathol, 152, 307-17 (1998)

19. K. Terai, A. Iwai, S. Kawabata, Y. Tasaki, T. Watanabe, K. Miyata and T. Yamaguchi: beta-amyloid deposits in transgenic mice expressing human beta-amyloid precursor protein have the same characteristics as those in Alzheimer's disease. Neuroscience, 104, 299-310 (2001)

20. T. Kawarabayashi, L. H. Younkin, T. C. Saido, M. Shoji, K. H. Ashe and S. G. Younkin: Age-dependent changes in brain, CSF, and plasma amyloid (beta) protein in the Tg2576 transgenic mouse model of Alzheimer's disease. J Neurosci, 21, 372-81 (2001)

21. R. C. Rubenstein and P. L. Zeitlin: Sodium 4-phenylbutyrate downregulates Hsc70: implications for intracellular trafficking of DeltaF508-CFTR. Am J Physiol Cell Physiol, 278, C259-67 (2000)

22. S. M. Rahman, I. Qadri, R. C. Janssen and J. E. Friedman: Fenofibrate and PBA prevent fatty acid-induced loss of adiponectin receptor and pAMPK in human hepatoma cells and in hepatitis C virus-induced steatosis. J Lipid Res, 50, 2193-202 (2009)

23. M. N. Reed, P. Liu, L. A. Kotilinek and K. H. Ashe: Effect size of reference memory deficits in the Morris water maze in Tg2576 mice. Behav Brain Res, 212, 115-20

24. C. V. Vorhees and M. T. Williams: Morris water maze: procedures for assessing spatial and related forms of learning and memory. Nat Protoc, 1, 848-58 (2006)

25. M. A. Westerman, D. Cooper-Blacketer, A. Mariash, L. Kotilinek, T. Kawarabayashi, L. H. Younkin, G. A. Carlson, S. G. Younkin and K. H. Ashe: The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer's disease. J Neurosci, 22, 1858-67 (2002)

26. P. N. Pompl, M. J. Mullan, K. Bjugstad and G. W. Arendash: Adaptation of the circular platform spatial memory task for mice: use in detecting cognitive impairment in the APP(SW) transgenic mouse model for Alzheimer's disease. J Neurosci Methods, 87, 87-95 (1999)

27. T. A. Comery, R. L. Martone, S. Aschmies, K. P. Atchison, G. Diamantidis, X. Gong, H. Zhou, A. F. Kreft, M. N. Pangalos, J. Sonnenberg-Reines, J. S. Jacobsen and K. L. Marquis: Acute gamma-secretase inhibition improves contextual fear conditioning in the Tg2576 mouse model of Alzheimer's disease. J Neurosci, 25, 8898-902 (2005)

28. J. C. Dodart, K. R. Bales, K. S. Gannon, S. J. Greene, R. B. DeMattos, C. Mathis, C. A. DeLong, S. Wu, X. Wu, D. M. Holtzman and S. M. Paul: Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci, 5, 452-7 (2002)

29. W. C. Benzing, J. R. Wujek, E. K. Ward, D. Shaffer, K. H. Ashe, S. G. Younkin and K. R. Brunden: Evidence for glial-mediated inflammation in aged APP(SW) transgenic mice. Neurobiol Aging, 20, 581-9 (1999)

30. G. P. Lim, T. Chu, F. Yang, W. Beech, S. A. Frautschy and G. M. Cole: The curry spice curcumin reduces oxidative damage and amyloid pathology in an Alzheimer transgenic mouse. J Neurosci, 21, 8370-7 (2001)

31. W. F. Yu, Z. Z. Guan, N. Bogdanovic and A. Nordberg: High selective expression of alpha7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer's disease and patients carrying Swedish APP 670/671 mutation: a possible association with neuritic plaques. Exp Neurol, 192, 215-25 (2005)

32. L. Song, P. De Sarno and R. S. Jope: Central role of glycogen synthase kinase-3beta in endoplasmic reticulum stress-induced caspase-3 activation. J Biol Chem, 277, 44701-8 (2002)

Abbreviations: AD: Alzheimer�s disease, APP: amyloid precursor protein, A Beta: amyloid Beta protein, ER: endoplasmic reticulum, PBA: 4-Phenylbutyrate, GFAP: glial fibrillary acid protein

Key Words: Alzheimer, Phenylbutyrate, Beta-amyloid, Inflammation, Synaptic Plasticity

Send correspondence to: Ana Garcia-Osta, Division of Neurosciences, CIMA, University of Navarra, Avda. de Pío XII 55. 31080 Pamplona, Spain, Tel: 34-948-194700, Fax: 34-948-194715, E-mail:agosta@unav.es