PS1ΔE9 double transgenic mice

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available at www.sciencedirect.com

www.elsevier.com/locate/brainres

Research Report

Distribution patterns of cannabinoid CB1 receptors in the hippocampus of APPswe/PS1ΔE9 double transgenic mice Sara Kalifaa,b , Eva K. Polstona , Joanne S. Allarda , Kebreten F. Manayea,⁎ a

Department of Physiology and Biophysics, Howard University, Washington, DC, 20059, USA Department of Biology, College of Science, George Mason University, Fairfax, VA, 22030, USA

b

A R T I C LE I N FO

AB S T R A C T

Article history:

Cannabinoids have neuroprotective effects that are exerted primarily through cannabinoid

Accepted 19 December 2010

CB1 receptors in the brain. This study characterized CB1 receptor distribution in the double

Available online 28 December 2010

transgenic (dtg) APPswe/PS1ΔE9 mouse model for Alzheimer's disease. Immunohistochemical labeling of CB1 protein in non-transgenic mice revealed that CB1 was highly expressed in

Keywords:

the hippocampus, with the greatest density of CB1 protein observed in the combined

Alzheimer's disease

hippocampal subregions CA2 and CA3 (CA2/3). CB1 immunoreactivity in the CA1 and CA2/3

Inflammation

hippocampal regions was significantly decreased in the dtg APPswe/PS1ΔE9 mice compared to

Astrocytes

non-transgenic littermates. Reduced CB1 expression in dtg APPswe/PS1ΔE9 mice was

iNOS

associated with astroglial proliferation and elevated expression of the cytokines inducible

TNFα

nitric oxide synthase and tumor necrosis factor alpha. This finding suggests an antiinflammatory effect of cannabinoids that is mediated by CB1 receptor, particularly in the CA2/3 region of the hippocampus. Furthermore, the study suggests a decreased CB1 receptor expression may result in diminished anti-inflammatory processes, exacerbating the neuropathology associated with Alzheimer's disease. © 2011 Elsevier B.V. All rights reserved.

1.

Introduction

Alzheimer's disease (AD) is a devastating neurodegenerative disease characterized by progressive memory loss, cognitive decline and widespread loss of neurons and their synapses in the cerebral cortex, entorhinal area, hippocampus, ventral striatum and basal forebrain (Selkoe, 2001 and Wisniewski and Terry, 1973). At the molecular level, the known abnormalities include abnormal processing of amyloid precursor protein

(APP), hyperphosphorylation of tau protein, and apoptotic-like cell death (Troncoso et al., 1996). Based on studies of transgenic mice that express mutant APP and or presenilin-1 (PS1), it is hypothesized that amyloid beta 42 (Aβ42) accumulation and diffuse plaque formation is associated with local microglial activation, cytokine release, reactive astrocytosis and a multiprotein inflammatory response (Eikelenboom et al, 1994; McGeer and McGeer, 1995 and Rogers et al., 1996). Generation of cytokines by glia can potentiate excitotoxicity which can lead

⁎ Corresponding author. Fax: +1 202 806 4479. E-mail address: [email protected] (K.F. Manaye). Abbreviations: AD, Alzheimer's Disease; Aβ, amyloid beta; APP, amyloid precursor protein; AEA, Arachidonylethanolamide; 2-AG, 2arachidonylglycerol; CB1, cannabinoid receptor 1; CB2, cannabinoid receptor 2; CNS, central nervous system; dtg, double transgenic; GFAP, glial fibrillary acidic protein; PS1, presenilin 1; iNOS, inducible nitric oxide synthase; TNFα, tumor necrotic factor alpha; tg, transgenic 0006-8993/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2010.12.061

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to neuronal dysfunction and death. Therefore down-regulation of glial activation may have a favorable effect on the induction and progression of neurodegenerative diseases. One type of agent that may have potential in this regard is cannabinoids (Ramírez et al., 2005). Cannabinoids are a group of terpenophenolic compounds that are present in cannabis sativa plant (marijuana). In 1997, two receptors, CB1 and CB2, that are responsible for mediating the effects of cannabinoids, were characterized, localized and cloned (Matsuda, 1997). Whereas CB1 receptor is most abundant in the central nervous system (CNS), CB2 cannabinoid receptor is found both in peripheral immune tissues (Galiegue et al., 1995) and in CNS (Onaivi et al, 2006). The discovery of the cannabinoid receptor and the availability of highly selective and potent cannabinoid mimetics led to the identification of a family of lipid neurotransmitters that serve as natural ligands for the CB1 receptor. Arachidonylethanolamide (AEA), named anandamide from the Sanskrit word meaning ‘internal bliss’ (Devane et al., 1992) and 2-arachidonylglycerol (2-AG) (Mechoulam et al., 1995; Sugiura et al., 1995) are the two extensively characterized endocannabinoids. Both anandamide and 2-AG, act as true ‘endogenous cannabinoids' by binding and functionally activating one or both cannabinoid receptor subtypes. Cannabinoids act as neuroprotective agents against excitotoxicity in vitro and acute brain damage in vivo (Ramírez et al., 2005). Cannabinoids are also being investigated as potential therapeutic agents for neurological and neurodegenerative disorders (Baker et al., 2000 and Mechoulam et al., 2002). Neuroprotection by cannabinoids is thought to occur through CB1-mediated inhibition of voltage-sensitive Ca2+ channels to reduce Ca2+ influx, glutamate release and excitotoxicity (Shen and Thayer, 1998 and Piomelli et al., 2000). In addition, cannabinoids have been shown to have antioxidative and anti-inflammatory effects (Hampson et al., 1998 and Marsicano et al., 2002). The endocannabinoid system appears to be a relevant modulator of physiological functions in the central nervous system (Di Marzo and Deutsch, 1998). However, little is known about the role of the endocannabinoid system in neurodegenerative diseases involving neuroinflammation such as Alzheimer's disease. Transgenic mouse models that express human genes associated with familial AD, including APP and PS1, have provided important tools for understanding neural reactions to the deposition of mutant Aβ proteins, and for developing novel approaches for the therapeutic management of AD in humans (Games et al., 1995; Hsiao et al., 1995; Malherbe et al., 1996; Hardy, 1997; Johnson-Wood et al., 1997; Sturchler-Pierrat et al., 1997; Morgan et al., 2000; Wang et al., 2003). In the present study, we employed the double transgenic (dtg) APPswe/PS1ΔE9 mouse to investigate the relationship between CB1 receptor expression, neuroinflammation, and AD-like neuropathology. Stereological approaches were used to quantify numbers of CB1-immunoreactive (CB1-IR) neurons and glial fibrillary acidic protein-immunoreactive (GFAP-IR) cells in the hippocampus of middle aged dtg APPswe/PS1ΔE9 male mice and age-matched non-transgenic (non-tg) littermates. Western blot was then used to compare expression levels of two pro-inflammatory cytokines, iNOS and TNFα, between the dtg APPswe/PS1ΔE9 and non-tg littermate mice.

2.

95

Results

2.1. CA2/3 contains higher numbers of CB1-immunoreactive (CB1-IR) cells than CA1 region of the hippocampus. In addition, dtg APPswe/PS1ΔE9 mice showed a significant reduction in the number of CB1-IR hippocampal neurons compared to non-tg littermates Low and high magnification photomicrographs showing patterns of CB1 labeling in the mouse hippocampus are presented in Figs. 1(A and B). CB1 receptor was abundantly expressed in CA1 and CA2/3 fields of the hippocampal neurons and, specifically, in large pyramidal cells. Stereological estimations of the numbers of CB1-IR cells in the CA1 and CA2/3 regions of the hippocampus in middle-aged (10–12 month old) mice revealed that the numbers of CB1-IR cells were significantly higher in the CA2/3 region of the hippocampus than in the CA1 region in both dtg and non-tg mice (p = 0.001). As shown in Fig. 1C, numbers of CB1-IR neurons in dtg APPswe/PS1ΔE9 mice were significantly lower in both the CA1 (27% fewer cells) and CA2/3 (23% fewer cells) hippocampal regions compared to non-tg littermates (p = 0.013).

2.2. There was a significant increase in the numbers of GFAP-immunoreactive cells, particularly in the CA1 region, in the dtg APPswe/PS1ΔE9 mice compared to the non-dtg group Photomicrographs presented in Figs. 2(A and B) show GFAP immunoreactivity in the hippocampus of dtg APPswe/PS1ΔE9 mice and non-tg litter mates. GFAP-IR astrocytes were distributed throughout the hippocampus of both the dtg APPswe/ PS1ΔE9 mice and the non-tg littermates. As shown, hippocampal GFAP-IR cells were noticeably denser in the dtg APPswe/PS1ΔE9 mice than in the non-tg litter mates. Statistical analysis of the stereological results revealed a significant 90% increase in GFAP positive cells in the CA1 hippocampal region of dtg APPswe/ PS1ΔE9 animals compared to non-tg littermates (p = 0.002; Fig. 2C). Regional analyses depicted in Fig. 2C showed that in non-tg control animals the distribution of GFAP positive cells was uniform throughout the hippocampus. In contrast, numbers of GFAP-IR cells in the hippocampus of dtg APPswe/PS1ΔE9 mice were significantly higher in the CA1 region than in CA2/3 (p = 0.006), with the number of GFAP-IR cell in the CA2/3 region of dtg APPswe/PS1ΔE9 mice being similar to that of the non-tg littermates.

2.3. Genotype-related increase in iNOS and TNF-α protein expression in dtg APPswe/PS1ΔE9 mice compared to non-dtg mice Results from Western blot analyses of iNOS and TNF-α protein expression levels are presented in Fig. 3. Hippocampal expression levels of both iNOS (Fig. 3A, p ≤ 0.001) and TNF-α (Fig. 3B, p ≤ 0.001) were significantly elevated in the dtg APPswe/PS1ΔE9 mice compared to non-tg age-matched controls.

3.

Discussion

Cannabinoid receptors are abundant in discrete areas of the brain. In the hippocampus, a region that plays a crucial role in

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Fig. 1 – Photomicrographs of CB1-IR neurons in the hippocampus at low (A; scale bar = 250 μm) and high (B; scale bar = 25 μm) magnification. Panel C shows numbers of CB1-IR neurons in the CA1 and CA2/3 hippocampal regions of dtg APPswe/PS1ΔE9 mice and non-tg littermates. Numbers of CB1-IR cells were significantly lower in the dtg APPswe/PS1ΔE9 mice in both the CA1 and CA2/3 (p = 0.001) regions. CB1-IR cells were lower in CA1 compared to CA2/3 in both groups.

learning and memory, particularly high levels of endocannabinoids (Di Marzo et al., 2000) and CB1 receptors (Tsou et al., 1998) are present. In this study, intense labeling of CB1-

immunoreactive cells was observed in the hippocampus of the middle aged mice, confirming previous immunohistochemical (Pettit et al., 1998 and Tsou et al., 1998), autoradiographical (Herkenham et al., 1991) and in situ hybridization findings (Mailleux and Vanderhaeghen, 1992 and Matsuda et al., 1990). Previous studies have shown that CB1 receptor expression is not observed in microglia or astrocytes in hippocampus, suggesting that the labeled cells in the present study are neurons (Marchalant et al., 2007). Immunoreactivity for CB1 receptor protein significantly differed between the sub-regions of the hippocampus. The CB1-immunoreactive cell count revealed that CA2/3 expresses higher levels of CB1-IR cells than CA1 subfield of the hippocampus (p = 0.001). Previous studies corroborate this finding (Herkenham et al., 1991 and Tsou et al., 1998). However, there are also reports of no regional differences in CB1 expression throughout the hippocampus (Pettit et al., 1998; Egertova and Elphick, 2000; Lu et al., 1999). Alternatively, one study has reported an opposite finding of higher CB1 receptor levels within the CA1 region (Liu et al., 2003). These discrepancies may be due to the different methodologies and animal models used in each study. Liu et al. (2003) and Egertova and Elphick (2000) used Western blot on rat brain tissue while Pettit et al. (1998) used rats and Lu et al. (1999) used light and electron microscopy on monkey brain. Cannabinoid receptor binding decreases in several brain regions in a variety of neurodegenerative diseases, including Alzheimer's disease (Glass et al., 1993; Richfield and Herkenham, 1994 and Westlake et al., 1994). The present study established that the number of CB1-immunoreactive neurons in CA1 and CA2/3 regions of the hippocampus of the middle aged (10–12 months) dtg APPswe/PS1ΔE9 mice were significantly reduced (p = 0.013) compared to non-dtg littermate control mice. This supports the findings of other works showing reduced CB1 receptor expression in Alzheimer's disease brains (Ramírez et al., 2005; Westlake et al., 1994). Astrocytes have the ability to respond to pathological situations by engaging in a series of structural and functional changes collectively referred to as astrogliosis (Eng and Ghirnikar, 1994; Itagaki et al., 1989 and Schubert et al., 2001). Prominent reactive astrogliosis is seen in several diseases including Alzheimer's disease (Eng and Ghirnikar, 1994). The present study shows a significant increase (p = 0.002) in the number of astrocytes in the CA1 regions of the hippocampus of the dtg APPswe/PS1ΔE9 mice compared to the non-tg littermates, supporting previous results from our laboratory (Manaye et al., 2007). In dtg APPswe/PS1ΔE9 mice, the number of GFAPimmunoreactive cells was significantly elevated in CA1 but not CA2/3. This is consistent with the observation that the CA1 subfield is the primary hippocampal target in AD, with the CA3 subfield showing less pathology (Van Hoesen et al., 1991). Further study will be required to determine the specific effects of CB1 receptor activation on astrocyte proliferation. Expression levels of the proinflammatory cytokines, TNF-α and iNOS, were elevated in the 10–12 month old dtg APPswe/ PS1ΔE9 mice, in contrast to age matched non-dtg mice. These findings implicate TNF-α and iNOS as mediators in the neuroinflammatory process associated with AD pathology in this animal model. Previous work demonstrated that both aberrant iNOS expression and peroxynitrite damage take

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Fig. 2 – Photomicrograph of GFAP-IR cells in the hippocampal formation of (A) non-tg littermates and (B) dtg APPswe/PS1ΔE9 mice (Scale bars represent 50 μm). Numbers of GFAP-IR astrocytes were significantly higher in the CA1 region of hippocampus of dtg APPswe/PS1ΔE9 mice than in the non-tg control animals (C; p = 0.002). GFAP-IR cell numbers were significantly increased in CA1 region compared to CA2/3 region of dtg APPswe/PS1ΔE9 (P = 0.006).

place in the AD brain (Lüth et al., 2002). It has also been shown that genetic deletion of iNOS substantially protected mice from Aβ toxicity (Nathan et al., 2005). TNF-α on the other hand has been implicated recently as a critical mediator of longterm potentiation (LTP) reduction by Aβ (Wang et al., 2005). The present study demonstrated region-specific changes in levels of CB1-immunoreactive neurons and astrogliosis in the dtg APPswe/PS1ΔE9 mouse model of AD pathology. Neuronal damage can increase the production of endocannabinoids (Marsicano et al., 2002). Our finding of lower CB1 expression in the CA1 region, which is particularly susceptible to neurodegeneration in AD, suggests a possible role for endocannabinoids as anti-inflammatory treatment for Alzheimer's disease. Hence, our results may be important for future clinical investigations into neuroprotective cannabinoid treatment.

4.

Experimental procedures

4.1.

Mice

Male dtg APPswe/PS1ΔE9 [(APPswe, PS1dE9)85Dbo/J; PrP promoter] and non-tg mice were raised from birth in the vivarium at the Laboratory of Experimental Gerontology at the Gerontology Research Center (GRC, NIA/NIH, Baltimore, MD). These mice were raised from founders donated by Drs. David Borchelt and

Michael Lee at the Johns Hopkins University School of Medicine that overexpress APPswe and PS1ΔE9 mutations. Mice were grouped housed (two to five) in plastic cages with corncob bedding with ad lib access to food (NIH formula 07) and filtered water. Conditions within the vivarium were maintained on a 12:12 h light:dark cycle and a temperature of 22 ± 2 °C. The GRC is accredited and animal care and treatment followed the guidelines of the American Association for the Accreditation of Laboratory Animal Care.

4.2.

Tissue preparation

Mice were sacrificed by perfusion with 0.9% saline followed by 4% paraformaldehyde. Brains were removed, and cryoprotected in 30% sucrose solution before being stored at −80 °C until sectioning. Each brain was serially sectioned in the coronal plane on a cryostat at an instrument setting of 40 μm for section thickness. For stereological studies the hippocampus was sampled in a systemic manner, as detailed elsewhere (Mouton, 2002).

4.3.

Immunohistochemistry

Brains from 23 male mice were obtained from the Laboratory of Experimental Gerontology at the Gerontology Research Center (GRC, NIA/NIH) in Baltimore, MD. The numbers per

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Sections were washed in PBS 0.1 M then transferred into 5% normal bovine serum in 0.1 M PBS for 30 min at RT to block non-specific binding. Sections were incubated overnight using goat polyclonal antibodies directed against CB1 receptor protein (Santa Cruz Biotechnology, CB1 (N-15)) and rabbit anti GFAP antibody (polyclonal, Abcam Cambridge, MA, USA) diluted to 1:500 (CB1) and 1:1000 (GFAP) with 2% bovine serum and 0.3% TritonX-100 in PBS at 48 C. After incubation, sections were washed in PBS 0.1 M and incubated in biotinylated secondary anti-goat (for CB1) and anti rabbit (for GFAP) antibodies (Vector Laboratories, Burlingame, CA, USA) with normal bovine serum in PBS 0.1 M for 90 min at RT. Sections were washed in PBS 0.1 M and re-incubated for another 90 min in ABC solution from the Vector stain Kit (Vector Laboratories, Burlingame, CA, USA) at RT. Sections were rinsed in PBS 0.1 M and colorized using DAB (10 mg DAB, 40 ml PBS 0.1 M) for 6–10 min. All CB1-immunostained sections were lightly counterstained in a 0.1% solution of cresyl violet, rinsed, dehydrated through an ascending graded series of alcohol and cleared in xylene and coverslipped with DPX. The specificity of CB1 receptor staining was confirmed by omitting the primary antibody; no label was observed in these control sections.

4.4.

Fig. 3 – Western blot analyses (relative optical densities normalized to β-actin) of iNOS (A) and TNF-α (B) expression levels in the hippocampus of APPswe/PS1ΔE9 mice and non-tg littermates (error bars = mean ± SEM). Expression levels of both iNOS and TNF-α were significantly elevated in the Dtg APPswe/PS1ΔE9 animals compared to control animals (p ≤ 0.001).

group and genotypes of the mice were as follows: 10– 12 months old dtg APPswe/PS1ΔE9 (n = 6) and age-matched non-tg littermate controls (n = 5) for CB1 immunostaining and 10–12 months old dtg (n = 7) and age matched nontransgenic controls (n = 5) for GFAP immunostaining were used. For visualization of CB1- and GFAP-immunoreactive cells, systematic-serial sections through the hippocampus, a total of 10–12 sections per brain, were collected in 12-well plates and washed in 0.1 M PBS, incubated in 1% hydrogen peroxide for 30 min at room temperature (RT), washed again in PBS 0.1 M, and placed in 0.3%Triton X-100 for 10 min RT.

Stereology

With assistance from the Stereo Investigator system (MicroBrightField Inc., Williston, VT), design-based stereology was used to estimate mean total number of CB1- immunoreactive cells and GFAP-immunoreactive cells in CA subregions. The mean total numbers of CB1-IR pyramidal neurons and GFAPIR cells in CA1 and CA2/3 regions of hippocampus were quantified using the optical fractionator method (West et al., 1991 and Gundersen, 1986), as previously reported (Liu et al., 2008 and Manaye et al., 2007). Briefly, the reference spaces on each sampled section were outlined under low power magnification (4×), and CB1-IR cells and GFAP-IR cells were quantified using a high resolution, oil immersion objective (60×, 1.4 numerical aperture). CB1-IR pyramidal cells appeared as large brown cells, while non CB1-IR cells were identified on the basis of a Nissl-stained neuronal phenotype, i.e., a dark violet blue nucleolus and a well-formed nuclear membrane. Nonneuronal CB1-IR cells were identified based on their apparent morphology, i.e., glial cells were identified as small, dark violet cells. To avoid artifacts at the sectioning surface, e.g., lost caps, a guard volume was observed 2–3 μm above and below the dissector where no cells were counted. Sampling of all parameters was continued to a mean coefficient of error (CE) of 0.05 to 0.10, according to Gundersen et al. (1999). All stereological parameters were quantified with assistance from a computerized stereology system by an operator blind to treatment and according to established principles, as detailed previously (Lei et al., 2003; Mouton et al., 2009 and Mouton, 2002).

4.5.

Western blotting

Brains from 6 male dtg APPswe/PS1ΔE9 and 6 male non-tg control mice were obtained from the Laboratory of Experimental Gerontology at the Gerontology Research Center (GRC,

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NIA/NIH) in Baltimore, MD. The hippocampi of the left hemisphere from all the mice were dissected out. The hippocampi of the dtg APPswe/PS1ΔE9 and that of the control mice were homogenized separately in an ice-chilled solution (50 mM Tris–HCl at pH 7.4) with protease inhibitor (cocktail tablet complete, from Roche). The homogenate from each mouse was centrifuged (Beckman Avanti J-25 I) at 32,000 ×g for 30 min. The supernatant was collected and protein concentration was measured using the BCA Protein assay with bovine serum albumin as a standard. Equal amounts of protein (20 μg) were loaded onto a 4–15% SDS-PAGE then transferred to a polyvinylidenedifluoride (PVDF) membrane. The membrane was probed with primary antibody (mouse anti TNF-α and anti-iNOS antibodies 1:1000) followed by horseradish peroxidase-conjugated secondary antibody (Goat anti-mouse IgG, 1:10,000). Antibody detection was performed using enhanced Chemoluminescence reagents Super Signal West Dura. The membrane was exposed to Hyperfilm MP (Amersham Biosciences, U.S.A.) and developed using Kodak GBX developer and fixer. Densitometric analysis was conducted using Quantity One imaging software (BioRad, CA) to quantify the intensity of bands from five independent Western blots.

4.6.

Statistical analysis

Two-way analysis of variance followed by Bonferroni post hoc test was used to compare the effects of transgene and hippocampal subregions for measures of GFAP and CB-1 immunoreactivity. Student's t test was used for statistical analysis of iNOS and TNFα levels determined by Western blot.

Acknowledgments The authors wish to acknowledge support from the NIH/ NINDS Grant number: U54 NS42867, Drs. Donald Ingram and Peter Mouton for their donation of the brains, and the NIA Intramural Program in Baltimore, Maryland. We would also like to acknowledge Dr. Jharna Das for her assistance with the Western blot analysis.

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