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CD40 ligation mediates plaque-associated tau phosphorylation in β-amyloid overproducing mice
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
CD40 ligation mediates plaque-associated tau phosphorylation in β-amyloid overproducing mice Vincent Laporte⁎, Ghania Ait-Ghezala, Claude-Henry Volmar, Christopher Ganey, Nowell Ganey, Marcie Wood, Michael Mullan The Roskamp Institute, 2040, Whitfield Avenue, Sarasota FL, 34243, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological
Accepted 1 June 2008
hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid
Available online 19 June 2008
burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in
Keywords:
transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576).
Alzheimer's disease
Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1
Amyloid
antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-
CD40
positive dystrophic neurites were exclusively associated with Congo red-positive plaques as
CD40 ligand
previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio
Mice
of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and
Transgenic
p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with
Tg2576
CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40–CD40L
Microtubule-associated protein tau
interaction has an effect on tau phosphorylation independent of beta-amyloid pathology,
Hyperphosphorylated tau
and that this effect may occur through a decrease of cdk5 and p35/p25.
cdk5
© 2008 Elsevier B.V. All rights reserved.
Dystrophic neurite
1.
Introduction
Alzheimer's disease (AD) is characterized by the extracellular deposition of amyloid β-peptide (Aβ) (which is derived from the processing of the β-amyloid precursor protein [APP]) in senile plaques and intracellular accumulation of neurofibrillary tangles composed principally of phosphorylated tau protein (Selkoe, 2001). Abnormal phosphorylation of tau proteins is the most established cause of dysfunctional tau in AD and occurs at 19 specific amino acids throughout tau sequence (Quadros et al., 2007). Abnormal neuronal processes known as dystrophic neurites (DN) are found closely associated with the amyloid deposits. Once DN are embedded
in the edge of amyloid plaques, the latter are referred to as neuritic plaques. In severe AD cases, DN are immunolabeled for phosphorylated tau (Dickson et al., 2005; Su et al., 1998). Transgenic mice Tg2576 expressing human APP with the Swedish mutation (APPsw, K670N/M671L) have been a useful tool for studying AD-like brain amyloidosis in the past (Hsiao, 1998; Mullan et al., 1992). Unfortunately, Tg2576 mice do not develop neurofibrillary tangles or neuronal loss like human AD patients. However, DN have been located in the vicinity of amyloid deposits in the Tg2576, suggesting that these mice are a good model of neuritic dystrophy caused by amyloid deposition (Noda-Saita et al., 2004; Otth et al., 2002; Tomidokoro et al., 2001). Phosphorylated tau-positive DN were
⁎ Corresponding author. Fax: +1 941 752 2948. E-mail address: [email protected] (V. Laporte). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.032
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Fig. 1 – Congo red staining. Representative photographs of Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice brain stained with Congo red.
reported to be associated exclusively with Congo red-positive plaques (Noda-Saita et al., 2004). We have previously shown that the expression of a non-functional CD40 or CD40 ligand (CD40L) in human APPsw-bearing mice reduces AD-related pathologies such as microgliosis, astrocytosis and Ab load (Laporte et al., 2006; Tan et al., 2002a). In addition, the expression of genes related to tau phosphorylation, such as cdk5, calpain (the protein involved in the activation of cdk5), ERK1/2 or p38MAPK is disturbed in cultured human microglia after treatment with CD40L (Ait-Ghezala et al., 2005). The CD40–CD40L pathway mediates glial and perhaps neuro-inflammatory activity (Abdel-Haq et al., 1999; Calingasan et al., 2002), and in order to assess the impact of these inflammatory influences on tau pathology, we studied the phosphorylation of tau in a Tg2576 (Tg APPsw) model of AD in which CD40 or CD40L were disrupted. In addition, because a previous study from our laboratory strongly implicated cdk5 in CD40–CD40L signaling (a 15 fold change in the cdk5 expression level was obtained after CD40L treatment of microglia (Ait-Ghezala et al., 2005), we examined the expression level of cdk5 and its related protein in these animal models and in a neuronal cell line over-expressing CD40 when treated with CD40L.
2.
Results
We have previously shown that disruption of CD40 reduced amyloid deposits [as detected with an anti-Aβ antibody (4G8)] in transgenic mouse models of AD (Laporte et al., 2006). Similar results have been shown in AD mouse models deficient in CD40L (Tan et al., 2002a). In the current study, Congo red staining in Tg APPsw mice is reduced when CD40 or CD40L is genetically disrupted. At 22–24 months of age, the reduction in Congo red staining in the parietal cortex was 66% and 77% in Tg APPsw mice deficient for CD40L and CD40, respectively (Figs. 1 and 2a). The decrease obtained in CD40L deficient (def.) mice is in conformity with previously published data (Tan et al., 2002a). Further, aberrant neurites were labeled with the anti-phosphorylated tau antibodies AT8, CP13, PG5, and PHF-1 throughout the cerebral cortex and the hippocampus of Tg APPsw mice at the age of 22–24 months. Previous reports have shown that AT8 and CP13 antibodies did not stain anything in the parenchyma of Tg APPsw mice that was
not closely related to the amyloid deposits (Ishizawa et al., 2003; Noda-Saita et al., 2004). Similar staining patterns were obtained with PG5 and PHF1 antibodies. No neurofibrillary tangles were detected with MC1 (kindly provided by Dr. P. Davies), an antibody that recognizes an abnormal conformation of tau (data not shown). The phosphorylation of tau in the parietal cortex (method described below and illustrated as procedure A in Fig. 9) was decreased by 70 to 87% and by 88 to 94% in the case of a CD40L or CD40 deficiency, respectively, for the epitopes studied in the parietal cortex (Fig. 2b). As shown in Fig. 2c, the ratio of phosphorylated tau to Congo red burden in parietal cortex is highly decreased in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice. Then, we analyzed the levels of Congo red-positive material and phosphorylated tau within the plaques (as explained in Fig. 9 as procedure B). The size of the examined plaques and their distribution onto a size scale predefined by the plaques studied in Tg APPsw brains were compared. As shown in Table 1, there is a modest decrease in the average size of the plaques in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice when compared to controls. The decrease of the mean is accompanied by a slight shift in the distribution of the plaque in the predefined size scale. However, none of these changes were significantly different, though more than 600 plaques were analyzed in this study. As shown in Figs. 3 and 4, when CD40L or CD40 genes are disrupted in Tg APPsw mice, phosphorylated tau decreases in the vicinity of amyloid deposits. Surprisingly, CD40L deficiency is more potent than CD40 deficiency in this regard. Indeed, lack of CD40L reduces AT8, CP13, PG5, or PHF1-positive dystrophic neurites by 96%, 93%, 93%, and 84%, respectively, whereas absence of CD40 reduces those by 55%, 55%, 60%, and 69%, respectively. In a post hoc comparison of the means, all the differences between Tg APPsw mice and their counterparts lacking CD40 or CD40L were significant (Fig. 4). The differences between Tg APPsw/CD40L def. and Tg APPsw/CD40 def. were significant for all the phosphorylated tau antibodies used except PHF1. However, the trend obtained with this staining was the same as those obtained with the others. Noda-Saita et al. (2004) have shown that the mean ratio of areas of AT8positive dystrophic neurites to Congo red in Tg APPsw is unchanged with age (from 11 to 20.5 months old) and is approximately 10%. A similar ratio (8.7% ± 1.2) was found in these mice at 22 to 24 months of age (Noda-Saita et al., 2004).
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The examination of the link between the amount of Congo red-positive deposits and the level of phosphorylated tau in a given plaque in Tg APPsw mice shows that the Pearson product–moment correlation is between 0.53 and 0.73, depending on the anti-phosphorylated tau antibody used (AT8, CP13, PG5, or PHF1), reflecting a linear relationship between the two variables (Fig. 5). The phosphorylated tau-positive dystrophic neurites was not specifically co-localized with astrocytes, activated microglia or neurofilament H-stained dystrophic neurites in Tg APPsw mice (Fig. 6). Although in AD brain, dystrophic neurites that are positive for neurofilament have been shown to be also stained for phosphorylated tau (Su et al., 1998), our results are supported by a recent study that shows that AT8-positive speckles are not co-localized with α-internexin-labeled dystrophic neurites in mice model of AD (Woodhouse et al., in press). To elucidate the molecular basis underlying the CD40 or CD40L deficiency-induced selective phosphorylation of tau in the Tg APPsw mice, and because the level of cdk5 transcript is increased in primary human microglia treated with CD40L (Ait-Ghezala et al., 2005), we measured the level of expression of this kinase, which has been previously implicated in tau phosphorylation and maybe regulated by CD40 in cell cycling (Hirai et al., 2004). Cdk5 is known to phosphorylate tau in vitro and in vivo (Quadros et al., 2007). Its activities are regulated after binding with its activator protein, p25, which derives from p35. As expected, expression level of cdk5, p35, and p25 show an increasing trend in Tg APPsw mice aged 13 months when compared to age-match control wild-type mice although the difference is not significant (Fig. 7a). However, we found that CD40–CD40L pathway disruption affected the amount of cdk5, p35, and p25. Indeed, in Tg APPsw/CD40L def. mice the level of expression of cdk5 is drastically reduced. A decrease of cdk5 was also observed in Tg APPsw/CD40 def. mice. Furthermore, the level of p35 and p25 in the Tg APPsw/ CD40L def. and Tg APPsw/CD40 def. mice was also reduced (Fig. 7b). Consistently, the level of expression of cdk5, p35 and p25 in the human neuroblastoma cell line SHSY-5Y challenged with CD40L is increased noticeably (Fig. 8). The non-transfected cells respond very well to CD40L treatment, thus implying that neurons express functional CD40 as previously reported (Tan et al., 2002b). Interestingly, over-expression of CD40 in the SHSY-5Y cell line is sufficient for inducing a Fig. 2 – Analysis of Congo red and phosphorylated tau in the cortex. Quantification of the area occupied by (a) Congo red and (b) phosphorylated tau in 4 areas of the parietal cortex of Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. animals. (c) Ratio of the phosphorylated-tau to the Congo red staining in 4 areas of the parietal cortex of the three mice strains studied. ANOVA revealed between-group differences, and post hoc comparison showed differences between brains from Tg APPsw and Tg APPsw/CD40L def. or Tg APPsw/CD40 def. but not between Tg APPsw/CD40L def. and Tg APPsw/CD40 def. (*p < 0.05; **p < 0.01; ***p < 0.001).
The mean ratio measured for the areas of CP13-, PG5- or PHF1positive dystrophic neurites to Congo red in these mice were 10.1% ± 0.8, 5.5% ± 0.5, and 7.5% ± 0.9, respectively.
Table 1 – Evaluation of the size of the examined plaques and their distribution on a size scale in the three genotypes studied Genotype
APPsw APPsw/CD40L deficient APPsw/CD40 deficient a
Percentage of plaques examined which size is
Plaque size
<231 µm2
231– 565 µm2
N565 µm2
Mean (µm2)
33.3 a 36.2
33.3 a 31.2
33.3 a 32.6
601 ± 41 564 ± 51
38.8
30.1
30.1
549 ± 40
The examined plaques were split in three equal third to define the size scale.
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Fig. 3 – Photographs of amyloid deposit surrounded with dystrophic neurites. Representative photographs of Congo redpositive amyloid deposit in Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice stained with anti-phosphorylated tau antibody AT8, CP13, PG5 or PHF1. Note the loss of phosphorylated tau staining (arrows) in Tg APPsw/CD40L def. and Tg APPsw/ CD40 def. without reduction of the plaques size.
change in the expression level of the 3 proteins of interest (Fig. 8). It is important to note that the response recorded was very time-sensitive (5 min). Indeed, an overnight treatment did not induce a change in cdk5, p35 or p25 expression level (data not shown).
3.
Discussion
In AD cases, the phosphorylated tau staining that is observed in the vicinity of amyloid deposits is associated with dystrophic neurites (Dickson et al., 2005; Su et al., 1998). In this and other studies, phosphorylated tau-positive dystrophic neurites have been observed in the vicinity of amyloid deposits in TgAPPsw mice, but the exact cellular localization of the speckles of phosphorylated tau in this transgenic mouse model has not been widely studied. It has been recently described that AT8-positive dystrophic neurites do not colocalize with α-internexin-labeled dystrophic neurites (Wood-
house et al., in press). Here, we show that the localization of phosphorylated tau speckles that decorate amyloid deposits is different than that of neurofilament H, GFAP, or CD45. Together, these data suggest that the phosphorylated taupositive structures are oligodendritic, represent a distinct subset of dystrophic neurites or are not intracellular. Previous studies have shown that reduction of functional CD40L or CD40 mitigates amyloid deposition, astrocytosis, and microgliosis in transgenic mouse models of AD (Laporte et al., 2006; Tan et al., 2002a). We report here that these membrane molecules are also involved in the phosphorylation of tau, and that the cdk5 and p35/p25 pathway may be involved in this mechanism. However, in order to be certain that only cdk5 and its activator p35/p25 are involved in this biological effect, the level of expression of other kinases involved in the phosphorylation of tau or phosphatases engaged in its dephosphorylation should be examined. In recent years, growing evidence has linked tau hyperphosphorylation to neuroinflammation. Indeed, CXCR2 ligand
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Fig. 4 – Analysis of phosphorylated tau associated with amyloid deposit. Phosphorylated tau-positive dystrophic neurites area to Congo red area in the amyloid plaques found in Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice when (a) AT8, (b) CP13, (c) PG5 or (d) PHF1 antibody was used. Data are presented as mean ± SEM. Significant difference of the means assessed by post hoc comparison is indicated by the marks (*p < 0.05; **p < 0.01; ***p < 0.001).
(namely, GROα/KC or IL6) induced tau phosphorylation in a primary culture of mouse neurons (Quintanilla et al., 2004; Xia and Hyman, 2002). Tau hyperphosphorylation (at several sites also phosphorylated in AD) has been found in the brainstem of rats with experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (Schneider et al., 2004). Transgenic mice over-expressing a human mutant tau present tauopathy-like pathology at 5 months of age, and these changes co-localize with inflammatory markers (Bellucci et al., 2004). Finally, LPS-treated 3×-Tg-AD mice, an animal model for AD that features both amyloid deposits and neurofibrillary tangles, develop a more intense tau phosphorylation (Kitazawa et al., 2005). It is important to note that in the vast majority of these reports, the hyperphosphorylation of tau was concomitant with a dysregulation of the cdk5/p25 pathway (Bellucci et al., 2004; Kitazawa et al., 2005; Quintanilla et al., 2004; Schneider et al., 2004). CD40–CD40L interaction promotes proliferation of B-cells. It induces cell-cycle progression by increasing the level of cyclin-dependent kinases and decreasing the level of cdk inhibitor (Hirai et al., 2004). Here, we report that the absence of CD40 or CD40L reduces the level of cdk5 in mice. Among the hypotheses that have been proposed to explain the pathogen-
esis of AD is the proposal that cell-cycle abnormality is a causative factor that initiates all other pathological events (Webber et al., 2005). It is tempting for us to suggest that the abnormalities of the cell-cycle in AD are triggered by an excessive CD40 ligation that activates mitotic signaling pathways such as the p38-mitogen-activated protein kinase (MAPK) or the p44/42 MAPK pathways (Craxton et al., 1998; Tan et al., 2002b). Nolan et al. (2004) have reported that the inflammatory response to microbial sepsis was reduced in CD40 def. but not CD40L def. mice, suggesting that a CD40L-independent CD40 activation was possible through bacterial HSP70. Human HSP70 is recognized and internalized by CD40 (Becker et al., 2002). Our data demonstrate that the reduction of tau phosphorylation and the decrease of cdk5 expression in Tg APPsw mice are more pronounced when CD40L, rather than CD40, is disrupted. It is possible that a similar mechanism is responsible for these differences, and that CD40 and CD40L contribute to AD pathology individually. These contributions have yet to be defined. In this report, we quantified the phosphorylation of tau associated with congophilic plaques. Several lines of evidence suggest that in the Alzheimer brain, tau pathology occurs
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Fig. 5 – Correlation analysis between phosphorylated tau staining and Congo red staining in individual plaques in Tg APPsw mice. Scatter charts representing the linear relationship between phosphorylated tau staining and Congo red-positive amyloid deposit when (a) AT8, (b) CP13, (c) PG5, or (d) PHF1 antibody was used. The Pearson product–moment correlation is 0.57 for AT8, 0.73 for CP13, 0.53 for PG5, and 0.62 for PHF1-stained phosphorylated tau.
downstream of Aβ/amyloid accumulation. We have previously shown that reduction of amyloid burden is contingent upon reduced availability of functional CD40 or CD40L (Laporte et al., 2006; Tan et al., 2002a). Thus, we would expect a reduction of tau pathology if amyloid pathology is its precedent. This we observe. However, for a given amount of amyloid pathology (i.e. for equivalent plaque size), we would expect to observe the same amount of tau pathology if the latter is a direct consequence of the former. However, this we do not observe: interruption of CD40–CD40L functionality results in a relative loss of tau pathology for a given amount of amyloid deposition. This implies that CD40–CD40L activity impacts tau phosphorylation independently (from the determination of amyloid deposition). Conversely, we may argue that the amount of soluble Aβ oligomers (particular subsets of which are known to be especially neurotoxic) is reduced in CD40L or CD40 def. animals, and that these subsets (not the deposited amyloid) control tau phosphorylation. However, it is widely assumed that the local deposition of amyloid depends upon the local concentration of soluble Aβ oligomers, and that local conditions around plaques of equal size are similar regardless of animal genotype. There-
fore, we would expect equivalent tau pathology in each genotype which we do not observe. Much evidence supports the downstream effects of Aβ/ amyloid on tau pathology. However, the effect of LPS on tau phosphorylation in 3×-Tg-AD mice was not mediated by amyloid pathology since an equivalent treatment in non transgenic mice induced an elevation of cerebral AT8-immunoreactivity (Kitazawa et al., 2005). In conclusion, our data and other recent reports support the idea that inflammatory pathways such as those controlled by CD40–CD40L have a direct effect on the phosphorylation of tau.
4.
Experimental procedures
4.1.
Animals
CD40 or CD40L disrupted mice were purchased from the Jackson Laboratories. These genetic variations occur on a C57BL/6 background, and were constructed as described (Kawabe et al., 1994; Renshaw et al., 1994). Tg2576 mice (Tg APPsw) also with a
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Fig. 6 – Cellular localization of the phosphorylated tau speckles. Representative images were acquired after immunofluorescence labeling for phosphorylated tau (a, d, g, arrows, green), GFAP-stained astrocytes (b, arrow heads, red), neurofilament H-positive dystrophic neurites (e, arrow heads, red), and CD45-labeled microglia (h, arrow heads, red). When images were overlayed, no co-localization between phosphorylated tau speckles and the astrocytes, the microglia and the NFH-labeled dystrophic neurites was observed (c, f, i).
C57B6/SJL background were constructed as described (Hsiao et al., 1996). CD40 or CD40L disrupted mice were crossed with Tg APPsw mice and the following offspring were produced: Tg APPsw/CD40 def. and Tg APPsw/CD40L def. Offspring were characterized by polymerase chain reaction-based genotyping for mutant APP (to determine Tg APPsw status) and for the neomycin selection vector (to type for CD40 or CD40L deficiency). Littermates were used as controls throughout the experiments. Animals were given food and water ad libitum. They were housed and maintained in the Roskamp Institute Animal Facility, and all experiments were in compliance with protocols approved by the Roskamp Institutional Animal Care and Use Committee. The mice were then euthanized at 22 to 24 months old for pathologic analysis. The numbers of animal used in this study were as follow: Tg APPsw (n = 4), Tg APPsw/CD40L def. (n = 5), and Tg APPsw/CD40 def. (n = 5).
4.2.
Antibodies
Monoclonal antibody AT8 (Pierce Biotechnology, IL), which recognizes human phosphorylated tau at Ser202 and Thr205, was diluted 1:400. Monoclonal antibodies PHF-1, CP13, and PG5, which recognize human phosphorylated tau at S396/S404, S202, and S409, respectively, were kindly provided by Dr. Peter Davies
(Albert Einstein College of Medicine, NY) and diluted at 1:100. cdk5 and p35 antibodies were purchased from Santa Cruz Biotechnology, CA. Anti-GFAP (Dako, CA), anti-CD45 (Serotec, NC) and anti-neurofilament H (Millipore, MA) were used at 1:1000, 1:50 and 1:300 respectively.
4.3.
Cell culture and immunoprecipitation
The SHSY-5Y cell line transfected with vector containing human CD40 gene or an empty vector (for construct see AitGhezala et al., 2007) was plated for 24h before being treated with 2 µg/ml CD40L for 5 min. Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, IL) containing Complete Protease Inhibitor Cocktail (Roche Applied Science, IN) plus sodium orthovanadate, and the plates were placed directly at − 80°C. Then, lysed cells were collected, flash frozen in liquid nitrogen, and centrifuged at 21,000g to clear the lysate. An equal amount of protein (100 µg) from each lysate was incubated overnight with 1µg of antibody at 4°C on a rotating shaker. Then, 100 µl of Protein A Sepharose beads prepared as recommended by the manufacturer (GE Healthcare, NJ) was added and incubated with the mix for 2 h at 4°C. After centrifugation at 800g, the beads were washed three times with cold PBS, and the protein was collected in
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Fig. 7 – Level of expression of cdk5, p25 and p35 in CD40 or CD40L deficient Tg APPsw mice. Immunoblotting analysis of cdk5, p35, and p25 in wild–type (a, lanes 1–4), Tg APPsw (a, lanes 5–8; b, lanes 1 and 2), Tg APPsw/CD40L def. (b, lanes 3 and 4) and Tg APPsw/CD40 def. mice (b, lanes 5 and 6), with β-actin used as a protein loading control. The mice used in these experiments were 13 months of age (a) and 22–24 months of age (b). The histograms represent the ratio of cdk5, p35 or p25 to β-actin signal ± SEM. Differences showed by post hoc comparison between groups are indicated by the marks (*p < 0.05; **p < 0.01; ***p < 0.001).
30 µl of Laemmli buffer and electrophoretically separated on a SDS-PAGE gel.
4.4.
Immunohistochemistry and immunofluorescence
One hemisphere of each brain was immersed in 4% paraformaldehyde at 4°C overnight and processed in paraffin. Briefly, the hemispheres were embedded into paraffin using TissueTek (Sakura, USA) and cut into 6-μm-thick sagittal sections with a microtome (2030 Biocut, Reichert/Leica, Germany). Prior to staining, sections were deparaffinized in xylene, and rehydrated in an ethanol to water gradient. Endogenous peroxidase activity was quenched with a 20min-H2O2 treatment (0.3% in water), and sections were rinsed and incubated with blocking buffer (Protein Block Serum-free, DakoCytomation) for 20 min. Then, the Vector MOM (MouseOn-Mouse) immunodetection kit was used as recommended by the manufacturer (Vector Laboratories, CA). Antibodies were detected using the avidin–peroxidase complex from a Vectastain ABC Elite kit (Vector Laboratories), and labeling was revealed with the VECTOR SG Substrate kit. Congo red staining was performed using the Amyloid Stain, Congo Red Kit from Sigma according to the manufacturer's instructions.
For each animal, 3 sections of the whole brain were stained. From these sections, the parietal cortex was identified using anatomical landmarks as previously described (Paxinos and Franklin, 2001). Next, four 40×-magnified photographs encompassing approximately 80–90% of the parietal cortex were randomly captured. From these photographs, the area occupied by Congo red staining and phosphorylated tau staining
Fig. 8 – Effect of CD40L on the level of expression of cdk5, p25 and p35 in CD40L-treated human neuroblastoma cell line. Immunoblotting analysis of cdk5, p35, and p25 in SHSY-5Y cell line over-expressing CD40 (lanes 3 and 4) or not (lanes 1 and 2), after being treated with CD40L (5 min; 2 µg/ml). Prior being separated on a gel, the proteins of interest were immunoprecipitated from the cell lysates.
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Fig. 9 – Process of quantitative image analysis. Double staining of phosphorylated tau-positive DN (black speckles) and Congo red-positive plaques (red staining) were performed using sagittal sections of Tg2576 mice. Color images of the parietal cortex at 40× or of each plaque at 100× were obtained by a microscope equipped with a digital camera. DN or plaque structures were quantified subsequently after selecting the appropriate color (see Experimental procedures for details).
was evaluated. The ratio of phosphorylated tau to Congo red was calculated for each section, and an average was obtained for each individual mouse. These averages were used to estimate the overall ratio of phosphorylated tau to Congo red for each genotype (illustrated in Fig. 9 as procedure A). Additionally, a plaque by plaque analysis of the phosphorylated tau-positive DN area to the Congo red-positive area was performed as previously described (Noda-Saita et al., 2004) with some modifications (illustrated in Fig. 9 as procedure B). Congophilic plaques were randomly selected in the hippocampus and the whole cortex, and were photographed at a high magnification (100×) so that DN were clearly visible. Approximately 15 plaques were selected per brain section. As each plaque was processed individually, the analysis was independent of the number of plaques present in each section. Phosphorylated tau-positive DN and Congo red-positive areas were successively quantified in each plaque analyzed. Briefly, areas to be quantified (as defined by color) were selected using Image-Pro Plus software (Media Cybernetics, MD) and areas of identical color were summed to obtain the total area of phosphorylated tau or Congo red staining in one plaque. The same parameters were applied to each plaque. Then, the ratio of the phosphorylated tau-positive DN to the Congo redpositive area of each plaque was calculated. For each
genotype, ratios were averaged to obtain an estimate of the overall staining. The relationships between staining and genotype were examined by one-way analysis of variance and post hoc testing. To compare the size of the Congo red-positive plaques in each genotype, the following procedure was performed: the plaques examined in Tg APPsw mice were separated into thirds in order to define three plaque size ranges (< 231 µm2; between 231 and 565 µm2; N 565 µm2); then, the plaques observed in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice were sorted according to their size and the proportion of plaques that fall within each size range was calculated. For double immunofluorescence staining, rabbit anti-GFAP, rat anti-CD45, or rabbit anti-neurofilament H was applied to the sections for 1 h at RT and revealed with the appropriate rhodamine-conjugated secondary antibody (anti-rat or antirabbit IgG from Pierce, 1:200). Then, AT8 staining was performed as described above, except that the labeling was detected with fluorescein–avidin at 1:50 (Vector Laboratories).
4.5.
Immunoblotting
One hemisphere of each brain was homogenized by sonication in 600 µl of M-PER (Pierce, IL) in the presence of a protease
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inhibitor cocktail (Calbiochem, CA) and phosphatase inhibitors (sodium orthovanadate and PMSF) and centrifuged at 10,000g for 10 min at 4 °C. Supernatants were subjected to SDSPAGE electrophoresis and immunoblotted with antibodies against cdk5 or p35 (diluted 1:500 and 1:200, respectively). A biotinylated-secondary antibody and a streptavidin-conjugated horseradish peroxidase were used to detect p25 with p35 antibody. Membranes were stripped and re-probed with anti-β-actin antibody to control for protein load.
Acknowledgments This work has been supported by a VA Merit Award to M.M. and the generosity of Diane and Robert Roskamp. We thank Dr. P. Davies for his generous gift of phosphorylated tau antibodies used in this study.
REFERENCES
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a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
CD40 ligation mediates plaque-associated tau phosphorylation in β-amyloid overproducing mice Vincent Laporte⁎, Ghania Ait-Ghezala, Claude-Henry Volmar, Christopher Ganey, Nowell Ganey, Marcie Wood, Michael Mullan The Roskamp Institute, 2040, Whitfield Avenue, Sarasota FL, 34243, USA
A R T I C LE I N FO
AB S T R A C T
Article history:
Neuritic dystrophy with amyloid burden and neurofibrillary tangles are pathological
Accepted 1 June 2008
hallmarks of Alzheimer's disease. Genetic disruption of CD40 or CD40L alleviates amyloid
Available online 19 June 2008
burden, astrocytosis, and microgliosis in transgenic animal models of Alzheimer's disease. It has been reported that phosphorylated tau-positive dystrophic neurites are observed in
Keywords:
transgenic mice over-expressing human mutant beta-amyloid precursor protein (Tg2576).
Alzheimer's disease
Here, we studied the pattern of phosphorylated tau (labeled with AT8, CP13, PG5, and PHF1
Amyloid
antibodies) and plaques using immunohistochemical techniques. Phosphorylated tau-
CD40
positive dystrophic neurites were exclusively associated with Congo red-positive plaques as
CD40 ligand
previously reported. Further, we show that CD40L or CD40 deficiency reduces the mean ratio
Mice
of dystrophic neurite area to congophilic plaque area and the level of expression of cdk5 and
Transgenic
p35/p25 in mice. In addition, we show that in a human neuroblastoma cell line treated with
Tg2576
CD40L, cdk5 and p35/p25 are increased. Together, our data suggest that CD40–CD40L
Microtubule-associated protein tau
interaction has an effect on tau phosphorylation independent of beta-amyloid pathology,
Hyperphosphorylated tau
and that this effect may occur through a decrease of cdk5 and p35/p25.
cdk5
© 2008 Elsevier B.V. All rights reserved.
Dystrophic neurite
1.
Introduction
Alzheimer's disease (AD) is characterized by the extracellular deposition of amyloid β-peptide (Aβ) (which is derived from the processing of the β-amyloid precursor protein [APP]) in senile plaques and intracellular accumulation of neurofibrillary tangles composed principally of phosphorylated tau protein (Selkoe, 2001). Abnormal phosphorylation of tau proteins is the most established cause of dysfunctional tau in AD and occurs at 19 specific amino acids throughout tau sequence (Quadros et al., 2007). Abnormal neuronal processes known as dystrophic neurites (DN) are found closely associated with the amyloid deposits. Once DN are embedded
in the edge of amyloid plaques, the latter are referred to as neuritic plaques. In severe AD cases, DN are immunolabeled for phosphorylated tau (Dickson et al., 2005; Su et al., 1998). Transgenic mice Tg2576 expressing human APP with the Swedish mutation (APPsw, K670N/M671L) have been a useful tool for studying AD-like brain amyloidosis in the past (Hsiao, 1998; Mullan et al., 1992). Unfortunately, Tg2576 mice do not develop neurofibrillary tangles or neuronal loss like human AD patients. However, DN have been located in the vicinity of amyloid deposits in the Tg2576, suggesting that these mice are a good model of neuritic dystrophy caused by amyloid deposition (Noda-Saita et al., 2004; Otth et al., 2002; Tomidokoro et al., 2001). Phosphorylated tau-positive DN were
⁎ Corresponding author. Fax: +1 941 752 2948. E-mail address: [email protected] (V. Laporte). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.06.032
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Fig. 1 – Congo red staining. Representative photographs of Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice brain stained with Congo red.
reported to be associated exclusively with Congo red-positive plaques (Noda-Saita et al., 2004). We have previously shown that the expression of a non-functional CD40 or CD40 ligand (CD40L) in human APPsw-bearing mice reduces AD-related pathologies such as microgliosis, astrocytosis and Ab load (Laporte et al., 2006; Tan et al., 2002a). In addition, the expression of genes related to tau phosphorylation, such as cdk5, calpain (the protein involved in the activation of cdk5), ERK1/2 or p38MAPK is disturbed in cultured human microglia after treatment with CD40L (Ait-Ghezala et al., 2005). The CD40–CD40L pathway mediates glial and perhaps neuro-inflammatory activity (Abdel-Haq et al., 1999; Calingasan et al., 2002), and in order to assess the impact of these inflammatory influences on tau pathology, we studied the phosphorylation of tau in a Tg2576 (Tg APPsw) model of AD in which CD40 or CD40L were disrupted. In addition, because a previous study from our laboratory strongly implicated cdk5 in CD40–CD40L signaling (a 15 fold change in the cdk5 expression level was obtained after CD40L treatment of microglia (Ait-Ghezala et al., 2005), we examined the expression level of cdk5 and its related protein in these animal models and in a neuronal cell line over-expressing CD40 when treated with CD40L.
2.
Results
We have previously shown that disruption of CD40 reduced amyloid deposits [as detected with an anti-Aβ antibody (4G8)] in transgenic mouse models of AD (Laporte et al., 2006). Similar results have been shown in AD mouse models deficient in CD40L (Tan et al., 2002a). In the current study, Congo red staining in Tg APPsw mice is reduced when CD40 or CD40L is genetically disrupted. At 22–24 months of age, the reduction in Congo red staining in the parietal cortex was 66% and 77% in Tg APPsw mice deficient for CD40L and CD40, respectively (Figs. 1 and 2a). The decrease obtained in CD40L deficient (def.) mice is in conformity with previously published data (Tan et al., 2002a). Further, aberrant neurites were labeled with the anti-phosphorylated tau antibodies AT8, CP13, PG5, and PHF-1 throughout the cerebral cortex and the hippocampus of Tg APPsw mice at the age of 22–24 months. Previous reports have shown that AT8 and CP13 antibodies did not stain anything in the parenchyma of Tg APPsw mice that was
not closely related to the amyloid deposits (Ishizawa et al., 2003; Noda-Saita et al., 2004). Similar staining patterns were obtained with PG5 and PHF1 antibodies. No neurofibrillary tangles were detected with MC1 (kindly provided by Dr. P. Davies), an antibody that recognizes an abnormal conformation of tau (data not shown). The phosphorylation of tau in the parietal cortex (method described below and illustrated as procedure A in Fig. 9) was decreased by 70 to 87% and by 88 to 94% in the case of a CD40L or CD40 deficiency, respectively, for the epitopes studied in the parietal cortex (Fig. 2b). As shown in Fig. 2c, the ratio of phosphorylated tau to Congo red burden in parietal cortex is highly decreased in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice. Then, we analyzed the levels of Congo red-positive material and phosphorylated tau within the plaques (as explained in Fig. 9 as procedure B). The size of the examined plaques and their distribution onto a size scale predefined by the plaques studied in Tg APPsw brains were compared. As shown in Table 1, there is a modest decrease in the average size of the plaques in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice when compared to controls. The decrease of the mean is accompanied by a slight shift in the distribution of the plaque in the predefined size scale. However, none of these changes were significantly different, though more than 600 plaques were analyzed in this study. As shown in Figs. 3 and 4, when CD40L or CD40 genes are disrupted in Tg APPsw mice, phosphorylated tau decreases in the vicinity of amyloid deposits. Surprisingly, CD40L deficiency is more potent than CD40 deficiency in this regard. Indeed, lack of CD40L reduces AT8, CP13, PG5, or PHF1-positive dystrophic neurites by 96%, 93%, 93%, and 84%, respectively, whereas absence of CD40 reduces those by 55%, 55%, 60%, and 69%, respectively. In a post hoc comparison of the means, all the differences between Tg APPsw mice and their counterparts lacking CD40 or CD40L were significant (Fig. 4). The differences between Tg APPsw/CD40L def. and Tg APPsw/CD40 def. were significant for all the phosphorylated tau antibodies used except PHF1. However, the trend obtained with this staining was the same as those obtained with the others. Noda-Saita et al. (2004) have shown that the mean ratio of areas of AT8positive dystrophic neurites to Congo red in Tg APPsw is unchanged with age (from 11 to 20.5 months old) and is approximately 10%. A similar ratio (8.7% ± 1.2) was found in these mice at 22 to 24 months of age (Noda-Saita et al., 2004).
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The examination of the link between the amount of Congo red-positive deposits and the level of phosphorylated tau in a given plaque in Tg APPsw mice shows that the Pearson product–moment correlation is between 0.53 and 0.73, depending on the anti-phosphorylated tau antibody used (AT8, CP13, PG5, or PHF1), reflecting a linear relationship between the two variables (Fig. 5). The phosphorylated tau-positive dystrophic neurites was not specifically co-localized with astrocytes, activated microglia or neurofilament H-stained dystrophic neurites in Tg APPsw mice (Fig. 6). Although in AD brain, dystrophic neurites that are positive for neurofilament have been shown to be also stained for phosphorylated tau (Su et al., 1998), our results are supported by a recent study that shows that AT8-positive speckles are not co-localized with α-internexin-labeled dystrophic neurites in mice model of AD (Woodhouse et al., in press). To elucidate the molecular basis underlying the CD40 or CD40L deficiency-induced selective phosphorylation of tau in the Tg APPsw mice, and because the level of cdk5 transcript is increased in primary human microglia treated with CD40L (Ait-Ghezala et al., 2005), we measured the level of expression of this kinase, which has been previously implicated in tau phosphorylation and maybe regulated by CD40 in cell cycling (Hirai et al., 2004). Cdk5 is known to phosphorylate tau in vitro and in vivo (Quadros et al., 2007). Its activities are regulated after binding with its activator protein, p25, which derives from p35. As expected, expression level of cdk5, p35, and p25 show an increasing trend in Tg APPsw mice aged 13 months when compared to age-match control wild-type mice although the difference is not significant (Fig. 7a). However, we found that CD40–CD40L pathway disruption affected the amount of cdk5, p35, and p25. Indeed, in Tg APPsw/CD40L def. mice the level of expression of cdk5 is drastically reduced. A decrease of cdk5 was also observed in Tg APPsw/CD40 def. mice. Furthermore, the level of p35 and p25 in the Tg APPsw/ CD40L def. and Tg APPsw/CD40 def. mice was also reduced (Fig. 7b). Consistently, the level of expression of cdk5, p35 and p25 in the human neuroblastoma cell line SHSY-5Y challenged with CD40L is increased noticeably (Fig. 8). The non-transfected cells respond very well to CD40L treatment, thus implying that neurons express functional CD40 as previously reported (Tan et al., 2002b). Interestingly, over-expression of CD40 in the SHSY-5Y cell line is sufficient for inducing a Fig. 2 – Analysis of Congo red and phosphorylated tau in the cortex. Quantification of the area occupied by (a) Congo red and (b) phosphorylated tau in 4 areas of the parietal cortex of Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. animals. (c) Ratio of the phosphorylated-tau to the Congo red staining in 4 areas of the parietal cortex of the three mice strains studied. ANOVA revealed between-group differences, and post hoc comparison showed differences between brains from Tg APPsw and Tg APPsw/CD40L def. or Tg APPsw/CD40 def. but not between Tg APPsw/CD40L def. and Tg APPsw/CD40 def. (*p < 0.05; **p < 0.01; ***p < 0.001).
The mean ratio measured for the areas of CP13-, PG5- or PHF1positive dystrophic neurites to Congo red in these mice were 10.1% ± 0.8, 5.5% ± 0.5, and 7.5% ± 0.9, respectively.
Table 1 – Evaluation of the size of the examined plaques and their distribution on a size scale in the three genotypes studied Genotype
APPsw APPsw/CD40L deficient APPsw/CD40 deficient a
Percentage of plaques examined which size is
Plaque size
<231 µm2
231– 565 µm2
N565 µm2
Mean (µm2)
33.3 a 36.2
33.3 a 31.2
33.3 a 32.6
601 ± 41 564 ± 51
38.8
30.1
30.1
549 ± 40
The examined plaques were split in three equal third to define the size scale.
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Fig. 3 – Photographs of amyloid deposit surrounded with dystrophic neurites. Representative photographs of Congo redpositive amyloid deposit in Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice stained with anti-phosphorylated tau antibody AT8, CP13, PG5 or PHF1. Note the loss of phosphorylated tau staining (arrows) in Tg APPsw/CD40L def. and Tg APPsw/ CD40 def. without reduction of the plaques size.
change in the expression level of the 3 proteins of interest (Fig. 8). It is important to note that the response recorded was very time-sensitive (5 min). Indeed, an overnight treatment did not induce a change in cdk5, p35 or p25 expression level (data not shown).
3.
Discussion
In AD cases, the phosphorylated tau staining that is observed in the vicinity of amyloid deposits is associated with dystrophic neurites (Dickson et al., 2005; Su et al., 1998). In this and other studies, phosphorylated tau-positive dystrophic neurites have been observed in the vicinity of amyloid deposits in TgAPPsw mice, but the exact cellular localization of the speckles of phosphorylated tau in this transgenic mouse model has not been widely studied. It has been recently described that AT8-positive dystrophic neurites do not colocalize with α-internexin-labeled dystrophic neurites (Wood-
house et al., in press). Here, we show that the localization of phosphorylated tau speckles that decorate amyloid deposits is different than that of neurofilament H, GFAP, or CD45. Together, these data suggest that the phosphorylated taupositive structures are oligodendritic, represent a distinct subset of dystrophic neurites or are not intracellular. Previous studies have shown that reduction of functional CD40L or CD40 mitigates amyloid deposition, astrocytosis, and microgliosis in transgenic mouse models of AD (Laporte et al., 2006; Tan et al., 2002a). We report here that these membrane molecules are also involved in the phosphorylation of tau, and that the cdk5 and p35/p25 pathway may be involved in this mechanism. However, in order to be certain that only cdk5 and its activator p35/p25 are involved in this biological effect, the level of expression of other kinases involved in the phosphorylation of tau or phosphatases engaged in its dephosphorylation should be examined. In recent years, growing evidence has linked tau hyperphosphorylation to neuroinflammation. Indeed, CXCR2 ligand
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Fig. 4 – Analysis of phosphorylated tau associated with amyloid deposit. Phosphorylated tau-positive dystrophic neurites area to Congo red area in the amyloid plaques found in Tg APPsw, Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice when (a) AT8, (b) CP13, (c) PG5 or (d) PHF1 antibody was used. Data are presented as mean ± SEM. Significant difference of the means assessed by post hoc comparison is indicated by the marks (*p < 0.05; **p < 0.01; ***p < 0.001).
(namely, GROα/KC or IL6) induced tau phosphorylation in a primary culture of mouse neurons (Quintanilla et al., 2004; Xia and Hyman, 2002). Tau hyperphosphorylation (at several sites also phosphorylated in AD) has been found in the brainstem of rats with experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis (Schneider et al., 2004). Transgenic mice over-expressing a human mutant tau present tauopathy-like pathology at 5 months of age, and these changes co-localize with inflammatory markers (Bellucci et al., 2004). Finally, LPS-treated 3×-Tg-AD mice, an animal model for AD that features both amyloid deposits and neurofibrillary tangles, develop a more intense tau phosphorylation (Kitazawa et al., 2005). It is important to note that in the vast majority of these reports, the hyperphosphorylation of tau was concomitant with a dysregulation of the cdk5/p25 pathway (Bellucci et al., 2004; Kitazawa et al., 2005; Quintanilla et al., 2004; Schneider et al., 2004). CD40–CD40L interaction promotes proliferation of B-cells. It induces cell-cycle progression by increasing the level of cyclin-dependent kinases and decreasing the level of cdk inhibitor (Hirai et al., 2004). Here, we report that the absence of CD40 or CD40L reduces the level of cdk5 in mice. Among the hypotheses that have been proposed to explain the pathogen-
esis of AD is the proposal that cell-cycle abnormality is a causative factor that initiates all other pathological events (Webber et al., 2005). It is tempting for us to suggest that the abnormalities of the cell-cycle in AD are triggered by an excessive CD40 ligation that activates mitotic signaling pathways such as the p38-mitogen-activated protein kinase (MAPK) or the p44/42 MAPK pathways (Craxton et al., 1998; Tan et al., 2002b). Nolan et al. (2004) have reported that the inflammatory response to microbial sepsis was reduced in CD40 def. but not CD40L def. mice, suggesting that a CD40L-independent CD40 activation was possible through bacterial HSP70. Human HSP70 is recognized and internalized by CD40 (Becker et al., 2002). Our data demonstrate that the reduction of tau phosphorylation and the decrease of cdk5 expression in Tg APPsw mice are more pronounced when CD40L, rather than CD40, is disrupted. It is possible that a similar mechanism is responsible for these differences, and that CD40 and CD40L contribute to AD pathology individually. These contributions have yet to be defined. In this report, we quantified the phosphorylation of tau associated with congophilic plaques. Several lines of evidence suggest that in the Alzheimer brain, tau pathology occurs
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Fig. 5 – Correlation analysis between phosphorylated tau staining and Congo red staining in individual plaques in Tg APPsw mice. Scatter charts representing the linear relationship between phosphorylated tau staining and Congo red-positive amyloid deposit when (a) AT8, (b) CP13, (c) PG5, or (d) PHF1 antibody was used. The Pearson product–moment correlation is 0.57 for AT8, 0.73 for CP13, 0.53 for PG5, and 0.62 for PHF1-stained phosphorylated tau.
downstream of Aβ/amyloid accumulation. We have previously shown that reduction of amyloid burden is contingent upon reduced availability of functional CD40 or CD40L (Laporte et al., 2006; Tan et al., 2002a). Thus, we would expect a reduction of tau pathology if amyloid pathology is its precedent. This we observe. However, for a given amount of amyloid pathology (i.e. for equivalent plaque size), we would expect to observe the same amount of tau pathology if the latter is a direct consequence of the former. However, this we do not observe: interruption of CD40–CD40L functionality results in a relative loss of tau pathology for a given amount of amyloid deposition. This implies that CD40–CD40L activity impacts tau phosphorylation independently (from the determination of amyloid deposition). Conversely, we may argue that the amount of soluble Aβ oligomers (particular subsets of which are known to be especially neurotoxic) is reduced in CD40L or CD40 def. animals, and that these subsets (not the deposited amyloid) control tau phosphorylation. However, it is widely assumed that the local deposition of amyloid depends upon the local concentration of soluble Aβ oligomers, and that local conditions around plaques of equal size are similar regardless of animal genotype. There-
fore, we would expect equivalent tau pathology in each genotype which we do not observe. Much evidence supports the downstream effects of Aβ/ amyloid on tau pathology. However, the effect of LPS on tau phosphorylation in 3×-Tg-AD mice was not mediated by amyloid pathology since an equivalent treatment in non transgenic mice induced an elevation of cerebral AT8-immunoreactivity (Kitazawa et al., 2005). In conclusion, our data and other recent reports support the idea that inflammatory pathways such as those controlled by CD40–CD40L have a direct effect on the phosphorylation of tau.
4.
Experimental procedures
4.1.
Animals
CD40 or CD40L disrupted mice were purchased from the Jackson Laboratories. These genetic variations occur on a C57BL/6 background, and were constructed as described (Kawabe et al., 1994; Renshaw et al., 1994). Tg2576 mice (Tg APPsw) also with a
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Fig. 6 – Cellular localization of the phosphorylated tau speckles. Representative images were acquired after immunofluorescence labeling for phosphorylated tau (a, d, g, arrows, green), GFAP-stained astrocytes (b, arrow heads, red), neurofilament H-positive dystrophic neurites (e, arrow heads, red), and CD45-labeled microglia (h, arrow heads, red). When images were overlayed, no co-localization between phosphorylated tau speckles and the astrocytes, the microglia and the NFH-labeled dystrophic neurites was observed (c, f, i).
C57B6/SJL background were constructed as described (Hsiao et al., 1996). CD40 or CD40L disrupted mice were crossed with Tg APPsw mice and the following offspring were produced: Tg APPsw/CD40 def. and Tg APPsw/CD40L def. Offspring were characterized by polymerase chain reaction-based genotyping for mutant APP (to determine Tg APPsw status) and for the neomycin selection vector (to type for CD40 or CD40L deficiency). Littermates were used as controls throughout the experiments. Animals were given food and water ad libitum. They were housed and maintained in the Roskamp Institute Animal Facility, and all experiments were in compliance with protocols approved by the Roskamp Institutional Animal Care and Use Committee. The mice were then euthanized at 22 to 24 months old for pathologic analysis. The numbers of animal used in this study were as follow: Tg APPsw (n = 4), Tg APPsw/CD40L def. (n = 5), and Tg APPsw/CD40 def. (n = 5).
4.2.
Antibodies
Monoclonal antibody AT8 (Pierce Biotechnology, IL), which recognizes human phosphorylated tau at Ser202 and Thr205, was diluted 1:400. Monoclonal antibodies PHF-1, CP13, and PG5, which recognize human phosphorylated tau at S396/S404, S202, and S409, respectively, were kindly provided by Dr. Peter Davies
(Albert Einstein College of Medicine, NY) and diluted at 1:100. cdk5 and p35 antibodies were purchased from Santa Cruz Biotechnology, CA. Anti-GFAP (Dako, CA), anti-CD45 (Serotec, NC) and anti-neurofilament H (Millipore, MA) were used at 1:1000, 1:50 and 1:300 respectively.
4.3.
Cell culture and immunoprecipitation
The SHSY-5Y cell line transfected with vector containing human CD40 gene or an empty vector (for construct see AitGhezala et al., 2007) was plated for 24h before being treated with 2 µg/ml CD40L for 5 min. Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, IL) containing Complete Protease Inhibitor Cocktail (Roche Applied Science, IN) plus sodium orthovanadate, and the plates were placed directly at − 80°C. Then, lysed cells were collected, flash frozen in liquid nitrogen, and centrifuged at 21,000g to clear the lysate. An equal amount of protein (100 µg) from each lysate was incubated overnight with 1µg of antibody at 4°C on a rotating shaker. Then, 100 µl of Protein A Sepharose beads prepared as recommended by the manufacturer (GE Healthcare, NJ) was added and incubated with the mix for 2 h at 4°C. After centrifugation at 800g, the beads were washed three times with cold PBS, and the protein was collected in
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Fig. 7 – Level of expression of cdk5, p25 and p35 in CD40 or CD40L deficient Tg APPsw mice. Immunoblotting analysis of cdk5, p35, and p25 in wild–type (a, lanes 1–4), Tg APPsw (a, lanes 5–8; b, lanes 1 and 2), Tg APPsw/CD40L def. (b, lanes 3 and 4) and Tg APPsw/CD40 def. mice (b, lanes 5 and 6), with β-actin used as a protein loading control. The mice used in these experiments were 13 months of age (a) and 22–24 months of age (b). The histograms represent the ratio of cdk5, p35 or p25 to β-actin signal ± SEM. Differences showed by post hoc comparison between groups are indicated by the marks (*p < 0.05; **p < 0.01; ***p < 0.001).
30 µl of Laemmli buffer and electrophoretically separated on a SDS-PAGE gel.
4.4.
Immunohistochemistry and immunofluorescence
One hemisphere of each brain was immersed in 4% paraformaldehyde at 4°C overnight and processed in paraffin. Briefly, the hemispheres were embedded into paraffin using TissueTek (Sakura, USA) and cut into 6-μm-thick sagittal sections with a microtome (2030 Biocut, Reichert/Leica, Germany). Prior to staining, sections were deparaffinized in xylene, and rehydrated in an ethanol to water gradient. Endogenous peroxidase activity was quenched with a 20min-H2O2 treatment (0.3% in water), and sections were rinsed and incubated with blocking buffer (Protein Block Serum-free, DakoCytomation) for 20 min. Then, the Vector MOM (MouseOn-Mouse) immunodetection kit was used as recommended by the manufacturer (Vector Laboratories, CA). Antibodies were detected using the avidin–peroxidase complex from a Vectastain ABC Elite kit (Vector Laboratories), and labeling was revealed with the VECTOR SG Substrate kit. Congo red staining was performed using the Amyloid Stain, Congo Red Kit from Sigma according to the manufacturer's instructions.
For each animal, 3 sections of the whole brain were stained. From these sections, the parietal cortex was identified using anatomical landmarks as previously described (Paxinos and Franklin, 2001). Next, four 40×-magnified photographs encompassing approximately 80–90% of the parietal cortex were randomly captured. From these photographs, the area occupied by Congo red staining and phosphorylated tau staining
Fig. 8 – Effect of CD40L on the level of expression of cdk5, p25 and p35 in CD40L-treated human neuroblastoma cell line. Immunoblotting analysis of cdk5, p35, and p25 in SHSY-5Y cell line over-expressing CD40 (lanes 3 and 4) or not (lanes 1 and 2), after being treated with CD40L (5 min; 2 µg/ml). Prior being separated on a gel, the proteins of interest were immunoprecipitated from the cell lysates.
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Fig. 9 – Process of quantitative image analysis. Double staining of phosphorylated tau-positive DN (black speckles) and Congo red-positive plaques (red staining) were performed using sagittal sections of Tg2576 mice. Color images of the parietal cortex at 40× or of each plaque at 100× were obtained by a microscope equipped with a digital camera. DN or plaque structures were quantified subsequently after selecting the appropriate color (see Experimental procedures for details).
was evaluated. The ratio of phosphorylated tau to Congo red was calculated for each section, and an average was obtained for each individual mouse. These averages were used to estimate the overall ratio of phosphorylated tau to Congo red for each genotype (illustrated in Fig. 9 as procedure A). Additionally, a plaque by plaque analysis of the phosphorylated tau-positive DN area to the Congo red-positive area was performed as previously described (Noda-Saita et al., 2004) with some modifications (illustrated in Fig. 9 as procedure B). Congophilic plaques were randomly selected in the hippocampus and the whole cortex, and were photographed at a high magnification (100×) so that DN were clearly visible. Approximately 15 plaques were selected per brain section. As each plaque was processed individually, the analysis was independent of the number of plaques present in each section. Phosphorylated tau-positive DN and Congo red-positive areas were successively quantified in each plaque analyzed. Briefly, areas to be quantified (as defined by color) were selected using Image-Pro Plus software (Media Cybernetics, MD) and areas of identical color were summed to obtain the total area of phosphorylated tau or Congo red staining in one plaque. The same parameters were applied to each plaque. Then, the ratio of the phosphorylated tau-positive DN to the Congo redpositive area of each plaque was calculated. For each
genotype, ratios were averaged to obtain an estimate of the overall staining. The relationships between staining and genotype were examined by one-way analysis of variance and post hoc testing. To compare the size of the Congo red-positive plaques in each genotype, the following procedure was performed: the plaques examined in Tg APPsw mice were separated into thirds in order to define three plaque size ranges (< 231 µm2; between 231 and 565 µm2; N 565 µm2); then, the plaques observed in Tg APPsw/CD40L def. and Tg APPsw/CD40 def. mice were sorted according to their size and the proportion of plaques that fall within each size range was calculated. For double immunofluorescence staining, rabbit anti-GFAP, rat anti-CD45, or rabbit anti-neurofilament H was applied to the sections for 1 h at RT and revealed with the appropriate rhodamine-conjugated secondary antibody (anti-rat or antirabbit IgG from Pierce, 1:200). Then, AT8 staining was performed as described above, except that the labeling was detected with fluorescein–avidin at 1:50 (Vector Laboratories).
4.5.
Immunoblotting
One hemisphere of each brain was homogenized by sonication in 600 µl of M-PER (Pierce, IL) in the presence of a protease
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inhibitor cocktail (Calbiochem, CA) and phosphatase inhibitors (sodium orthovanadate and PMSF) and centrifuged at 10,000g for 10 min at 4 °C. Supernatants were subjected to SDSPAGE electrophoresis and immunoblotted with antibodies against cdk5 or p35 (diluted 1:500 and 1:200, respectively). A biotinylated-secondary antibody and a streptavidin-conjugated horseradish peroxidase were used to detect p25 with p35 antibody. Membranes were stripped and re-probed with anti-β-actin antibody to control for protein load.
Acknowledgments This work has been supported by a VA Merit Award to M.M. and the generosity of Diane and Robert Roskamp. We thank Dr. P. Davies for his generous gift of phosphorylated tau antibodies used in this study.
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