aDepartment of Physiology and Biophysics, Howard University, Washington, DC, 20059, USAbDepartment of Biology, College of Science, George Mason University, Fairfax, VA, 22030, USA
A R T I C L E I N F O
CA2/3 region of the hippocampus. Furthermore, the study suggests a decreased CB1 receptor
neuropathology associated with Alzheimer's disease.
B R A I N R E S E A R C H 1 3 7 6 ( 2 0 1 1 ) 9 4 1 0 0
ava i l ab l e a t www.sc i enced i r ec t . com
www.e l sev i e r . com/ loca te /b ra i n resAlzheimer's disease (AD) is a devastating neurodegenerativedisease characterized by progressive memory loss, cognitivedecline and widespread loss of neurons and their synapses inthe cerebral cortex, entorhinal area, hippocampus, ventralstriatum and basal forebrain (Selkoe, 2001 and Wisniewskiand Terry, 1973). At the molecular level, the known abnormal-
cell death (Troncoso et al., 1996). Based on studies of transgenicmice that express mutant APP and or presenilin-1 (PS1), it ishypothesized that amyloid beta 42 (A42) accumulation anddiffuse plaque formation is associated with local microglialactivation, cytokine release, reactive astrocytosis and a multi-protein inflammatory response (Eikelenboom et al, 1994;McGeer and McGeer, 1995 and Rogers et al., 1996). Generation 2011 Elsevier B.V. All rights reserved.
1. Introduction (APP), hyperphosphorylation of tau protein, and apoptotic-likeities include abnormal processing of amylo
Corresponding author. Fax: +1 202 806 4479.E-mail address: Kmanaye@howard.edu (KAbbreviations: AD, Alzheimer's Disease; A
arachidonylglycerol; CB1, cannabinoid receptglial fibrillary acidic protein; PS1, presenilin 1
0006-8993/$ see front matter 2011 Elsevdoi:10.1016/j.brainres.2010.12.061expression may result in diminished anti-inflammatory processes, exacerbating theiNOSTNFA B S T R A C T
Cannabinoids have neuroprotective effects that are exerted primarily through cannabinoidCB1 receptors in the brain. This study characterized CB1 receptor distribution in the doubletransgenic (dtg) APPswe/PS1E9mousemodel for Alzheimer's disease. Immunohistochemicallabeling of CB1 protein in non-transgenic mice revealed that CB1 was highly expressed inthe hippocampus, with the greatest density of CB1 protein observed in the combinedhippocampal subregions CA2 and CA3 (CA2/3). CB1 immunoreactivity in the CA1 and CA2/3hippocampal regions was significantly decreased in the dtg APPswe/PS1E9 mice compared tonon-transgenic littermates. Reduced CB1 expression in dtg APPswe/PS1E9 mice wasassociated with astroglial proliferation and elevated expression of the cytokines induciblenitric oxide synthase and tumor necrosis factor alpha. This finding suggests an anti-inflammatory effect of cannabinoids that is mediated by CB1 receptor, particularly in theAccepted 19 December 2010Available online 28 December 2010
Keywords:Alzheimer's diseaseInflammationAstrocytesSara Kalifaa,b, Eva K. Polstona, Joanne S. Allarda, Kebreten F. Manayea,Research Report
Distribution patterns of cannabhippocampus of APPswe/PS1E9id precursor protein
.F. Manaye)., amyloid beta; APP, amor 1; CB2, cannabinoid rec; iNOS, inducible nitric o
ier B.V. All rights reservenoid CB1 receptors in theouble transgenic miceof cytokines by glia can potentiate excitotoxicitywhich can lead
yloid precursor protein; AEA, Arachidonylethanolamide; 2-AG, 2-eptor 2; CNS, central nervous system; dtg, double transgenic; GFAP,xide synthase; TNF, tumor necrotic factor alpha; tg, transgenic
1 3to neuronal dysfunction and death. Therefore down-regulationof glial activation may have a favorable effect on the inductionand progression of neurodegenerative diseases. One type ofagent that may have potential in this regard is cannabinoids(Ramrez et al., 2005). Cannabinoids are a group of terpeno-phenolic compounds that are present in cannabis sativaplant (marijuana). In 1997, two receptors, CB1 and CB2, thatare responsible for mediating the effects of cannabinoids,were characterized, localized and cloned (Matsuda, 1997).Whereas CB1 receptor is most abundant in the centralnervous system (CNS), CB2 cannabinoid receptor is foundboth in peripheral immune tissues (Galiegue et al., 1995) andin CNS (Onaivi et al, 2006). The discovery of the cannabinoidreceptor and the availability of highly selective and potentcannabinoid mimetics led to the identification of a familyof lipid neurotransmitters that serve as natural ligands forthe CB1 receptor. Arachidonylethanolamide (AEA), namedanandamide 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 twoextensively characterized endocannabinoids. Both ananda-mide and 2-AG, act as true endogenous cannabinoids' bybinding and functionally activating one or both cannabinoidreceptor subtypes.
Cannabinoids act as neuroprotective agents against exci-totoxicity in vitro and acute brain damage in vivo (Ramrezet al., 2005). Cannabinoids are also being investigated aspotential therapeutic agents for neurological and neurode-generative disorders (Baker et al., 2000 and Mechoulam etal., 2002). Neuroprotection by cannabinoids is thought tooccur through CB1-mediated inhibition of voltage-sensitiveCa2+ channels to reduce Ca2+ influx, glutamate release andexcitotoxicity (Shen and Thayer, 1998 and Piomelli et al.,2000). In addition, cannabinoids have been shown to haveantioxidative and anti-inflammatory effects (Hampson et al.,1998 and Marsicano et al., 2002). The endocannabinoidsystem appears to be a relevant modulator of physiologicalfunctions in the central nervous system (Di Marzo andDeutsch, 1998). However, little is known about the role of theendocannabinoid system in neurodegenerative diseasesinvolving neuroinflammation such as Alzheimer's disease.
Transgenic mouse models that express human genesassociated with familial AD, including APP and PS1, haveprovided important tools for understanding neural reactionsto the deposition of mutant A proteins, and for developingnovel approaches for the therapeutic management of AD inhumans (Games et al., 1995; Hsiao et al., 1995; Malherbe et al.,1996; Hardy, 1997; Johnson-Wood et al., 1997; Sturchler-Pierratet al., 1997; Morgan et al., 2000; Wang et al., 2003). In thepresent study, we employed the double transgenic (dtg)APPswe/PS1E9 mouse to investigate the relationship betweenCB1 receptor expression, neuroinflammation, and AD-likeneuropathology. Stereological approaches were used to quan-tify numbers of CB1-immunoreactive (CB1-IR) neurons andglial fibrillary acidic protein-immunoreactive (GFAP-IR) cellsin the hippocampus of middle aged dtg APPswe/PS1E9 malemice and age-matched non-transgenic (non-tg) littermates.Western blot was then used to compare expression levels of
B R A I N R E S E A R C Htwo pro-inflammatory cytokines, iNOS and TNF, between thedtg APPswe/PS1E9 and non-tg littermate mice.2.1. CA2/3 contains higher numbers of CB1-immunoreactive(CB1-IR) cells than CA1 region of the hippocampus. In addition,dtg APPswe/PS1E9 mice showed a significant reduction in thenumber of CB1-IR hippocampal neurons compared to non-tglittermates
Low and high magnification photomicrographs showingpatterns of CB1 labeling in the mouse hippocampus arepresented in Figs. 1(A and B). CB1 receptor was abundantlyexpressed in CA1 andCA2/3 fields of the hippocampal neuronsand, specifically, in large pyramidal cells. Stereological estima-tions of the numbers of CB1-IR cells in the CA1 and CA2/3regions of the hippocampus in middle-aged (1012 month old)mice revealed that the numbers of CB1-IR cells were signifi-cantly higher in the CA2/3 region of the hippocampusthan 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 dtgAPPswe/PS1E9 mice were significantly lower in both the CA1(27% fewer cells) and CA2/3 (23% fewer cells) hippocampalregions compared to non-tg littermates (p=0.013).
2.2. There was a significant increase in the numbers ofGFAP-immunoreactive cells, particularly in the CA1 region, inthe dtg APPswe/PS1E9 mice compared to the non-dtg group
Photomicrographs presented in Figs. 2(A and B) show GFAPimmunoreactivity in the hippocampus of dtg APPswe/PS1E9mice and non-tg litter mates. GFAP-IR astrocytes were distrib-uted throughout the hippocampus of both the dtg APPswe/PS1E9mice and the non-tg littermates. As shown, hippocampalGFAP-IR cells were noticeably denser in the dtg APPswe/PS1E9mice than in the non-tg litter mates. Statistical analysis of thestereological results revealed a significant 90% increase inGFAPpositive cells in the CA1 hippocampal region of dtg APPswe/PS1E9 animals compared to non-tg littermates (p=0.002;Fig. 2C). Regional analyses depicted in Fig. 2C showed that innon-tg control animals the distribution of GFAP positive cellswas uniform throughout the hippocampus. In contrast, numb-ers of GFAP-IR cells in the hippocampus of dtg APPswe/PS1E9mice were significantly higher in the CA1 region than in CA2/3(p=0.006), with the number of GFAP-IR cell in the CA2/3region of dtg APPswe/PS1E9 mice being similar to that of thenon-tg littermates.
2.3. Genotype-related increase in iNOS and TNF- proteinexpression in dtg APPswe/PS1E9 mice compared to non-dtg mice
Results from Western blot analyses of iNOS and TNF-protein expression levels are presented in Fig. 3. Hippocampalexpression levels of both iNOS (Fig. 3A, p0.001) and TNF-(Fig. 3B, p0.001) were significantly elevated in the dtgAPPswe/PS1E9mice compared to non-tg age-matched controls.
3. Discussion2. Results
957 6 ( 2 0 1 1 ) 9 4 1 0 0Cannabinoid receptors are abundant in discrete areas of thebrain. In the hippocampus, a region that plays a crucial role in
1 396 B R A I N R E S E A R C Hlearning and memory, particularly high levels of endocanna-binoids (Di Marzo et al., 2000) and CB1 receptors (Tsou et al.,1998) are present. In this study, intense labeling of CB1-
Fig. 1 Photomicrographs of CB1-IR neurons in thehippocampus at low (A; scale bar=250 m) and high (B; scalebar=25 m)magnification. Panel C shows numbers of CB1-IRneurons in the CA1 and CA2/3 hippocampal regions of dtgAPPswe/PS1E9 mice and non-tg littermates. Numbers ofCB1-IR cells were significantly lower in the dtg APPswe/PS1E9mice in both the CA1 and CA2/3 (p=0.001) regions. CB1-IRcells were lower in CA1 compared to CA2/3 in both groups.immunoreactive cells was observed in the hippocampus ofthe middle aged mice, confirming previous immunohisto-chemical (Pettit et al., 1998 and Tsou et al., 1998), autoradio-graphical (Herkenham et al., 1991) and in situ hybridizationfindings (Mailleux and Vanderhaeghen, 1992 and Matsudaet al., 1990). Previous studies have shown that CB1 receptorexpression is not observed in microglia or astrocytes inhippocampus, suggesting that the labeled cells in the presentstudy are neurons (Marchalant et al., 2007).
Immunoreactivity for CB1 receptor protein significantlydiffered between the sub-regions of the hippocampus. TheCB1-immunoreactive cell count revealed that CA2/3 expresseshigher levels of CB1-IR cells than CA1 subfield of the hippo-campus (p=0.001). Previous studies corroborate this finding(Herkenham et al., 1991 and Tsou et al., 1998). However, thereare also reports of no regional differences in CB1 expressionthroughout the hippocampus (Pettit et al., 1998; Egertova andElphick, 2000; Lu et al., 1999). Alternatively, one study hasreported an opposite finding of higher CB1 receptor levelswithin the CA1 region (Liu et al., 2003). These discrepanciesmay be due to the different methodologies and animalmodelsused 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 electronmicroscopy on monkey brain.
Cannabinoid receptor binding decreases in several brainregions in a variety of neurodegenerative diseases, includ-ing Alzheimer's disease (Glass et al., 1993; Richfield andHerkenham, 1994 and Westlake et al., 1994). The presentstudy established that the number of CB1-immunoreactiveneurons in CA1 and CA2/3 regions of the hippocampus of themiddle aged (1012 months) dtg APPswe/PS1E9 mice weresignificantly reduced (p=0.013) compared to non-dtg litter-mate control mice. This supports the findings of other worksshowing reduced CB1 receptor expression in Alzheimer'sdisease brains (Ramrez et al., 2005; Westlake et al., 1994).
Astrocytes have the ability to respond to pathological sit-uations by engaging in a series of structural and functionalchanges collectively referred to as astrogliosis (Eng andGhirnikar, 1994; Itagaki et al., 1989 and Schubert et al., 2001).Prominent reactive astrogliosis is seen in several diseasesincluding Alzheimer's disease (Eng and Ghirnikar, 1994). Thepresent study shows a significant increase (p=0.002) in thenumber of astrocytes in the CA1 regions of the hippocampus ofthedtgAPPswe/PS1E9mice compared to thenon-tg littermates,supporting previous results from our laboratory (Manaye et al.,2007). In dtg APPswe/PS1E9 mice, the number of GFAP-immunoreactive cells was significantly elevated in CA1 butnot CA2/3. This is consistentwith the observation that the CA1subfield is the primary hippocampal target in AD, with the CA3subfield showing less pathology (Van Hoesen et al., 1991).Further study will be required to determine the specific ef-fects of CB1 receptor activation on astrocyte proliferation.
Expression levels of the proinflammatory cytokines, TNF-and iNOS, were elevated in the 1012 month old dtg APPswe/PS1E9 mice, in contrast to age matched non-dtg mice. Thesefindings implicate TNF- and iNOS as mediators in theneuroinflammatory process associated with AD pathology in
7 6 ( 2 0 1 1 ) 9 4 1 0 0this animal model. Previous work demonstrated that bothaberrant iNOS expression and peroxynitrite damage take
1 3B R A I N R E S E A R C Hplace in the AD brain (Lth et al., 2002). It has also been shownthat genetic deletion of iNOS substantially protected micefromA toxicity (Nathan et al., 2005). TNF- on the other handhas been implicated recently as a critical mediator of long-term potentiation (LTP) reduction by A (Wang et al., 2005).
The present study demonstrated region-specific changes inlevels of CB1-immunoreactive neurons and astrogliosis in thedtg APPswe/PS1E9 mouse model of AD pathology. Neuronaldamage can increase the production of endocannabinoids(Marsicano et al., 2002). Our finding of lower CB1 expression inthe CA1 region, which is particularly susceptible to neurode-generation in AD, suggests a possible role for endocannabi-noids as anti-inflammatory treatment for Alzheimer's disease.Hence, our results may be important for future clinical inves-tigations into neuroprotective cannabinoid treatment.
4. Experimental procedures
Male dtg APPswe/PS1E9 [(APPswe, PS1dE9)85Dbo/J; PrP promoter]and non-tg mice were raised from birth in the vivarium at theLaboratory of Experimental Gerontology...