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Introduction
Alzheimer's disease (AD) is the most common cause of
dementia in the aged population. It is characterized by the
presence of extracellular senile plaques composed of
β-amyloid and intracellular neurofibrillary tangles (NFT)
constituting primarily of the abnormally hyperphosphorylated
microtubule-associated protein tau[1_4]. A profound loss of basal
forebrain cholinergic neurons accompanied by dysfunction
of central cholinergic neurotransmission was also obvious
in AD patients[5].
As the mechanism leading to the hallmark pathological
alterations is still not fully understood, there is still no
specific cure for the therapy of the disease. So far, the most
effective pharmacological strategies to attenuate the
impaired cognitive function of AD patients have been targeted
at the supplementation of acetylcholine with
acetylcholinesterase inhibitors[6]. However, it is just a palliative strategy
aimed at the temporary improvement of the cognitive
function[7]. With the progress in understanding the molecular
and cellular pathophysiology of AD, pharmacological
interventions aimed at modifying the development and progress
of the disease have been developed. These approaches
include β-amyloid immunization, treatment with secretase
inhibitors, and the inhibition of tau-related neurofibrillary
degeneration[6,8].
Dehydroevodiamine (DHED), one of the quinazoline
alkaloids isolated from Evodia rutaecarpa
Bentham, was found to be a non-competitive cholinesterase
inhibitor[9]. Studies have shown that it has anti-amnesic effects in a
scopolamine-induced amnesia model and can prevent impairment of
learning and memory and neuronal loss in rat models of
cognitive disturbance with minimal side-effects and effective
dosing[9,10]. It is also demonstrated that DHED can attenuate
Aβ-induced amnesia in mice, which suggests that DHED
may be an ideal drug candidate for the AD-type dementia
treatment[11]. The formation of neurofibrillary tangle is the
recognized pathology positively correlated with the extent
of neuronal loss and the degree of clinical dementia in
AD[12,13]. Neurofibrillary tangles are primarily composed of the
abnormally hyperphosphorylated tau. It has been found that tau
hyperphosphorylation is an early event in the evolution of
the disease[1_3]. Thus, we are curious as to whether DHED
has an effect on the AD-like tau hyperphosphorylation and
the possible underlying mechanism.
The phosphorylation of tau is strictly regulated by a panel
of protein kinases and protein phosphatases (PP). Among
the known phosphatases, PP-2A is the most active enzyme
in dephosphorylating the abnormally hyperphosphorylated
tau isolated from AD patients[14_16] or induced in the rat
brain[17], and the activity of PP-2A is significantly reduced in the AD
brains[18]. Therefore, in the present study, we treated the
metabolically competent rat brain slice with calyculin A (CA),
a potent and specific inhibitor of PP-2A and PP-1, to induce
AD-like tau hyperphosphorylation, and investigated the
effect of DHED on tau phosphorylation and the
activity-dependent modification of PP-2A. We found that DHED could
prevent the CA-induced tau hyperphosphorylation in rat
brain slices and efficiently decreased the inhibitory
phosphorylation of PP-2A at Tyr307.
Materials and methods
Animals Male Sprague-Dawley rats, weighing 200_240
g, were obtained from the Center of Laboratory Animals of
Tongji Medical College, Huazhong University of Science and
Technology (Wuhan, China). All the animal experiments were
performed according to the Policies on the Use of Animals
and Humans in Neuroscience Research revised and approved
by the Society for Neuroscience in 1995.
Chemicals and antibodies DHED was purified by HPLC
from the unripe fruit of Evodia rutaecarpa
Bentham with 98% purity (Kunming Biovalley Institute of Materia Medica,
Kunming, China). The drug was dissolved in 5% DMSO
solution and was prepared immediately prior to
use[19]. CA (Discodermia calyx) and monoclonal antibody (mAb) Tau-1
against unphosphorylated tau were purchased from
Chemi-con International (Temecula, CA, USA). Polyclonal
antibody (pAb) R134d against total tau and pAb R123d against
PP-2A catalytic subunit were gifts from Dr IQBAL and Dr
GRUNDKE-IQBAL (New York State Institute for Basic Research, Staten Island, NY, USA). pAb pS262 against
phosphorylated tau at Ser262 and pAb pS396 against
phosphorylated tau at Ser396 were purchased from Biosource
International (Camarillo, CA, USA). pAb against
phosphorylated Tyr307 of PP-2A catalytic subunit (anti-p-PP2A) was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA,
USA). pAb anti-β-actin and mAb anti-α-tubulin were
purchased from Sigma Chemical Co (St Louis, MO, USA).
Preparation and treatment of rat brain slices
The rats were deeply anaesthetized and the brains were rapidly
removed and cooled in oxygenated (95%
O2, 5% CO2) artificial cerebrospinal fluid (aCSF) containing 126 mmol/L NaCl,
3.5 mmol/L KCl, 1.2 mmol/L
NaH2PO4, 1.3 mmol/L
MgCl2, 2.0 mmol/L CaCl2, 11 mmol/L D-(+)-glucose, and 25 mmol/L
NaHCO3 (pH 7.4) for 7_8 min at 4 °C. The forebrain was
isolated and sagittally divided. Then, 400 µm thick coronal
slices were sectioned with a Mcllwain Tissue Chopper (Mickle
Laboratory Engineering Co Ltd, Gomshall, Surrey,
England). The slices were equilibrated at room temperature for 1 h and
immediately incubated at 33 °C in oxygenated aCSF either
alone (control) or in the presence of DHED with different
concentrations (10, 100, and 200 µmol/L, respectively). After
incubation for 1 h, CA was added into the aCSF to induce tau
hyperphosphorylation. After incubation for another 2 h, the
brain slices were removed, washed twice and divided into 2
parts. One part was homogenized at 4 °C in the buffer
containing 50 mmol/L Tris-HCl (pH 7.0) 10 mmol/L
β-mercaptoe-thanol, 1.0 mmol/L EDTA, 0.1 mmol/L phenylmethylsulfonyl
fluoride, 2.0 mmol/L benzamidine, 1.0 mmol/L
Na3VO4, 100 mmol/L NaF, and 2.0 mg/mL each of aprotinin, leupeptin, and
pepstatin A at a ratio of 1:9 (g/mL). The other part was fixed
in 4% paraformaldehyde for immunohistochemistry.
Lactate dehydrogenase (LDH) activity assay
The LDH released into aCSF from rat brain slices during incubation
was determined colorimetrically with a commercial kit from
Jiancheng Bioengineering Institute (Nanjing, China)
according to the manufacturer's protocol. The assay was carried
out on 96-well microplates, and the results were read by
DG3022-microplate reader (TECAN, Grodig, Salzburg, Australia) at a wavelength of 440 nm.
Western blotting The protein concentration of
homo-genates from the brain slices was quantitated by the
bicinchoninic acid method using protein assay reagent
(Pierce, Rockford, IL, USA). Homogenates were separated
by electrophoresis through 10% SDS-PAGE. After being
transferred to polyvinylidene difluoride membranes
(Amer-sham Pharmacia Biotech, Uppsala, Sweden) and probed with
anti-tau and PP-2A antibodies as described above, the
density of the bands was quantitatively analyzed by Kodak
Digital Science 1D software (Eastman Kodak, New Haven, CT,
USA).
Immunohistochemistry At the end of incubation, the
slices were collected, washed with phosphate-buffered
saline, and fixed in 4% paraformaldehyde in 0.1 mol/L
phosphate buffer (pH 7.4) at room temperature for 5 h. Then the
slices were dehydrated with 70% ethanol, 80% ethanol, 95%
ethanol, 100% ethanol, and xylene sequentially. The fixed
slices were then embedded in paraffin and cut into 6
µm-thick serial sections. All the slides with tissue sections were
dried at 37 °C for 2 d before they were immunostained with
the antibodies as described above. The immunoactivities
were detected using an avidin-biotin-peroxidase system and
visualized with diaminobenzidine (DAB; Sigma, St Louis, MO,
USA).
Statistical analysis All data were expressed as mean±SD
and analyzed by using SPSS 13.0 statistical software (SPSS,
Chicago, IL, USA). One-way ANOVA followed by least
significant difference post-hoc tests was used to determine the
differences among the groups.
Results
Effect of DHED on CA-induced tau
hyperphosphoryla-tion Data received by Western blotting (Figure 1A, upper
panel) and quantitative analysis (Figure 1A, lower panel)
showed a remarkably increased immunoreaction of tau to
pS262 and a reduced binding of tau to Tau-1 after incubating
the brain slices with CA for 2 h, suggesting
hyperphos-phorylation of tau at Ser262 (pS262) and
Ser198/199/202
(Tau-1, the antibody reacts with the unphosphorylated
epitope of tau; Figure 1A). No obvious change in total tau
(R134d) and the phosphorylated tau at Ser396 (pS396) was
observed when treated with CA (Figure 1A). The
administration of DHED dramatically arrested the CA-induced tau
hyperphosphoryla-tion at Tau-1 and pS262 epitopes (Figure
1A). It also decreased the basal phosphorylation level of
tau at Ser396, although CA failed to induce
hyperphos-phorylation at this site (Figure 1A). These results suggested
that DHED could efficiently attenuate CA-induced tau
hyperphosphorylation at multiple AD-related sites in the rat
brain slices.
To determine the viability of the brain slices during the
treatment with CA and DHED, we measured the LDH level
released from the slices into the culture medium. No
obvious differences were observed among the different groups
(Figure 1B). These data suggested that CA at the
concentration used for the study did not cause a markedly increased
cell death in the cultured rat brain slices, and DHED also did
not affect the cell viability.
Effect of DHED and CA on the distribution of
phosphorylated tau According to the results observed by Western
blotting, we chose DHED at a concentration of 200 µmol/L to
study the distribution of the phosphorylated tau. We
further cut the fixed brain slices into 6 μm sections and
measured the distribution of the phosphorylated tau and the
effects of DHED by immunohistochemistry. Compared with
the control group, dramatically increased pS262 staining was
observed in the perinuclear of the pyramidal neurons in CA4
region of the cornu ammonis, and decreased Tau-1 staining
was apparent in the cytoplasm and neural fibers of the CA3
and CA4 regions in sections treated with CA. DHED
markedly reversed the hyperphosphorylation of tau at these
regions (Figure 2).
Effect of DHED and CA on Tyr307-phosphorylated
PP-2A To explore the possible mechanism involved in the
attenuation of DHED in the CA-induced tau
hyperphos-phoryla-tion, Western blotting was used to measure the
phosphorylated level of Tyr307 in the PP-2A catalytic subunit,
probably contributing to the inactivation of
PP-2A[20]. The phosphorylated level of
Tyr307 in the PP-2A catalytic subunit was elevated in the CA-treated slices, while DHED at
the concentration of 200 µmol/L efficiently decreased the
inhibitory phosphorylation of PP-2A at Tyr307. No obvious
difference in total PP-2A was detected among the various
groups (Figure 3).
Discussion
The amount of NFT in affected neurons is directly
associated with dementia symptoms in AD patients. The major
component of NFT is the hyperphosphorylated tau, an early
event in the evolution of AD[1_3]. Therefore, tau
hyperphos-phorylation has been proposed to play a pivotal role in AD
patients[21]. However, there has been no specific and
effective drug to arrest tau hyperphosphorylation. In the present
study, we demonstrated by Western blotting that DHED
could protect against CA-induced tau hyperphosphorylation
at Ser262 and Ser198/199/202 sites in rat brain slices in a
dose-dependent manner. By immunohistochemistry, we
further confirmed that CA could induce tau
hyperphos-phorylation in the hippocampus, the most critical brain
region responsible for spatial learning and memory, and also
the region heavily burdened with NFT in AD
patients[22]. It is known that tau is hyperphosphorylated at multiple sites in
the AD brains[23]. The phosphorylation of tau at Ser262 alone,
a critical site located within the microtubule binding domain,
dramatically decreases the binding of tau to the
microtubules and thus inhibits the assembly of
microtubules[24]. It may eventually lead to the collapse of cytoskeleton and
neurodegeneration. Our results demonstrated that DHED
could prevent tau from hyperphosphorylation in some key
sites, thus, we speculate that DHED may be able to maintain
the stability of microtubules and thus hold out a normal
architecture of the neurons. We reported in a previous study
that CA could induce hyperphosphorylation of tau at
Ser396/404 by using the paired helical filament (PHF)-1 antibody in
N2a cells[25]. However, we did not see increased
phosphorylation of tau at Ser396 (using the pS396 antibody) when
treated with CA in the rat brain slice in the present study.
This discrepancy can be induced by different sources of the
materials used for the studies. It also suggests that CA may
predominantly affect Ser404, but not Ser396 because PHF-1
reacts with both sites, but pS396 only binds to p-Ser396.
Although CA failed to induce tau hyperphosphorylation at
Ser396, DHED could still decrease the basal
phosphorylation level of tau at this site.
The phosphorylation of tau is strictly regulated by a panel
of protein kinases and protein phosphatases. Among the
latter, PP-2A is believed to be the most crucial phosphatase
in regulating tau
dephosphorylation[14_17]. In the AD brains,
the levels of mRNA and the major heterotrimer holoenzyme,
as well as the activity of PP-2A, were reduced significantly[18,26,27]. In vitro studies also demonstrated that PP-2A was
the most active phosphatase in dephosphorylating the
abnormally hyperphosphorylated and aggregated tau
isolated from the AD brain and restoring the biological activity
of tau in promoting microtubule
assembly[14,15]. Therefore, PP-2A might be a promising target to recover normal tau
function in the AD brains. However, until now, no effective
agent has been reported to selectively activate PP-2A in
tangle-bearing neurons. In the present study, we found that
DHED treatment could ameliorate tau hyperphosphorylation,
and in the meantime, it decreased the phosphorylation level
of PP-2A at Tyr307, probably contributing to the
inactivation of the catalytic subunit of the phosphatase. Our results
strongly indicate that DHED may exert its protective effect
on CA-induced tau hyperphosphorylation at least partially
through antagonizing the CA-induced PP-2A inhibition. As
an acetylcholinesterase inhibitor, DHED inhibits the activity
of cholinesterase. In a previous study, we found that the
inhibition of PP-2A by okadaic acid decreased the level of
acetylcholine in the rat brain[28]. It has also been reported
recently that therapeutic acetylcholinesterase inhibitors,
such as donepezil and galanthamine, can prevent glutamate
neurotoxicity via nicotinic acetylcholine receptors and
inhibitors for a non-receptor type tyrosine kinase Fyn, and
janus-activated kinase 2 suppressed the neuroprotective
effect of donepezil and
galanthamine[29]. These studies suggest the possible connections of cholinergic metabolism
function, PP-2A activity/the tyrosine phosphorylation, and
the tyrosine kinase regulation. The exact mechanism for the
ameliorative effect of DHED in the CA-induced inhibitory
tyrosine phosphorylation of PP-2A needs further
investiga-tion. It should be noted that DHED treatments at lower
concentrations (10 and 100 µmol/L) prevented CA-induced tau
hyperphosphorylation at Ser198/199/202 sites (Tau-1
epitopes), but exerted no effect on the inhibitory
modification of PP-2A, indicating the possible involvement of other
tau kinases and phosphatases.
As DHED is easy to pass through the blood-brain barrier
and has been reported to have the least side-effects and the
lowest dosing among the cholinesterase
inhibitors[9,10,30], in addition to the present findings, it may be a promising
candidate for the drug development of AD.
Acknowledgements
We thank Dr Khalid IQBAL, Dr Inge GRUNDKE-IQBAL,
Dr Cheng-xin GONG, and Dr Fei LIU at New York State
Institute for Basic Research for technical support.
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