Elsevier

Neurobiology of Aging

Volume 34, Issue 11, November 2013, Pages 2574-2584
Neurobiology of Aging

Regular article
Cannabinoid receptor 1 deficiency in a mouse model of Alzheimer's disease leads to enhanced cognitive impairment despite of a reduction in amyloid deposition

https://doi.org/10.1016/j.neurobiolaging.2013.05.027Get rights and content

Abstract

Alzheimer's disease (AD) is characterized by amyloid-β deposition in amyloid plaques, neurofibrillary tangles, inflammation, neuronal loss, and cognitive deficits. Cannabinoids display neuromodulatory and neuroprotective effects and affect memory acquisition. Here, we studied the impact of cannabinoid receptor type 1 (CB1) deficiency on the development of AD pathology by breeding amyloid precursor protein (APP) Swedish mutant mice (APP23), an AD animal model, with CB1-deficient mice. In addition to the lower body weight of APP23/CB1−/− mice, most of these mice died at an age before typical AD-associated changes become apparent. The surviving mice showed a reduced amount of APP and its fragments suggesting a regulatory influence of CB1 on APP processing, which was confirmed by modulating CB1 expression in vitro. Reduced APP levels were accompanied by a reduced plaque load and less inflammation in APP23/CB1−/− mice. Nevertheless, compared to APP23 mice with an intact CB1, APP23/CB1−/− mice showed impaired learning and memory deficits. These data argue against a direct correlation of amyloid plaque load with cognitive abilities in this AD mouse model lacking CB1. Furthermore, the findings indicate that CB1 deficiency can worsen AD-related cognitive deficits and support a potential role of CB1 as a pharmacologic target.

Introduction

Alzheimer's disease (AD) is the major neurodegenerative disease in humans. Clinically, cognitive deficits of AD patients correlate with cerebral atrophy in vulnerable brain regions, mainly in the frontal cortex and the hippocampal region. Pathologically, AD involves neurofibrillary degeneration and the progressive deposition of self-aggregating amyloid-β (Aβ) peptides in extracellular amyloid plaques that are surrounded by activated astrocytes and microglia (Johnston et al., 2011, Schellenberg and Montine, 2012). A major focus of AD research lies on the development of therapeutic strategies that block processing of amyloid precursor protein (APP) to Aβ peptides. Aβ is generated by the subsequent cleavage of APP by β- and γ-secretase. Cleavage of APP by β-secretase can be inhibited by stimulation of α-secretase that cleaves APP in the Aβ domain, thus preventing Aβ release (Haass and Selkoe, 2007, Huang and Mucke, 2012). Stimulation of α-secretase activity has shown neuroprotective effects (Postina et al., 2004). Besides the decrease of Aβ production and aggregation, attenuation of the AD-associated neuroinflammation and reduction of neuronal damage caused by oxidative stress are further important therapeutical strategies.

The neuroprotective effects of cannabinoids have been studied in a variety of neurodegenerative paradigms, and our understanding concerning the influence of cannabinoids on AD pathology is growing (Aso et al., 2012, Bisogno and Di Marzo, 2008, Esposito et al., 2011, Iuvone et al., 2009, Karl et al., 2012). Brains of AD patients show a strong overexpression of CB2 in activated microglia and of fatty acid amide hydrolase in activated astrocytes (Benito et al., 2003). A previous report showed a decreased number of cannabinoid receptor type 1 (CB1)–positive neurons in the frontal cortex of brains of AD patients in areas with amyloid plaques and activated microglial cells (Ramirez et al., 2005). This reduction of CB1-positive neurons is accompanied by an alteration of the expression and cellular localization of endocannabinoid synthesizing and degrading enzymes (diacylglycerol lipase and monoacylglycerol lipase) in brains of AD patients (Mulder et al., 2011). In addition, in vivo and in vitro data suggested that cannabinoid receptor agonists can protect neurons against amyloid-induced toxicity (Martin-Moreno et al., 2011, Martin-Moreno et al., 2012, Ramirez et al., 2005). Moreover, it was also shown that AD-associated Aβ peptides influence endocannabinoid signaling, and there is an interplay between endocannabinoid signaling and Aβ toxicity (Chen et al., 2011, Jung et al., 2012, Mazzola et al., 2003, Mulder et al., 2011, van der Stelt et al., 2006). Specifically, intracerebroventricular coadministration of the mixed synthetic CB1 and CB1 agonist Win55,212-2 with a toxic Aβ peptide to rats prevented Aβ-induced microglial activation, cognitive impairment, and neuronal loss. This study was extended by in vitro experiments disclosing inhibition of Aβ-induced microglial activation by CB1 and CB2 agonists and neuroprotective effects on primary rat neurons. Prolonged oral cannabinoid administration to TgAPP2576 mice also showed beneficial effects, such as a reduced inflammation, a lower cortical accumulation of Aβ peptides, and an improved cortical performance (Martin-Moreno et al., 2012). Several mechanisms by which cannabinoids might exert their neuroprotective and anti-inflammatory effects, such as the involvement of mitogen-activated protein kinases and glycogen synthase kinase 3-beta signaling pathways (Martin-Moreno et al., 2012, Milton, 2002), the inhibition of acetylcholinesterase activity and acetylcholinesterase-induced Aβ aggregation (Eubanks et al., 2006), and antioxidant properties (Marsicano et al., 2002a), have been proposed. The neuroprotective and anti-inflammatory activities of Win55,212-2 against Aβ-induced damage in rats were recently shown to be mediated by CB1 and CB2 (Fakhfouri et al., 2012). Recently, CB1 was found to be expressed in mitochondria, and a novel role for CB1 receptors in the regulation of energy metabolism in the brain was proposed (Benard et al., 2012). Because the energy demand of the brain is rather high, a dysfunction of mitochondrial CB1 or a deficiency could have significant consequences for neuronal function and survival. Collectively, these data indicate that cannabinoid receptors may be promising therapeutic targets in AD.

The described changes in the endocannabinoid signaling system in brains of AD patients and the observed beneficial effects after cannabinoid administration in vitro and in vivo prompted us to generate a novel mouse model by knocking out CB1 expression in APP23 mice, a recognized AD mouse model, to study the impact of CB1 deficiency on the development of AD pathology.

Section snippets

Reagents

Unless stated otherwise, all reagents were from regular commercial sources. All used antibodies and their sources are described in Table 1.

Cell culture

Mouse N2A neuroblastoma cells (provided by Claus Pietrzik, University Medical Center Mainz) were cultured in Dulbecco's Modified Eagle's Medium, 1 mM Minimum Essential Medium Sodium Pyruvate (both Gibco) supplemented with antibiotics and antimycotics (Invitrogen, Karlsruhe, Germany), and 10% active fetal calf serum (PAA, Pasching, Austria). N2A cells were

Reduced survival and body weight of APP23/CB1−/− mice

APP23/CB1−/− mice showed a higher mortality rate than APP23, CB1−/−, and WT mice, whereby about 50% of the deaths occurred during the first 5 months (Fig. 1A). Mice died suddenly, and postmortem autoptical analysis of dead animals revealed no obvious signs of pathologic changes. Furthermore, APP23/CB1−/− mice had a significantly lower body weight than APP23, CB1−/−, and WT mice (Fig. 1B). APP23 and CB1−/− mice also showed a reduced body weight compared with WT mice that is not unexpected

Discussion

In the present study, we describe a novel mouse model, APP23/CB1−/− mice, which was generated to investigate a role of CB1 in pathways and processes that are linked to AD pathogenesis. The APP23/CB1−/− mice showed a reduced body weight and premature death, although no obvious abnormalities regarding health status and cage behavior were detectable. In addition, we observed a significant reduction in the protein level of mutant APP, secreted sAPPα, its C-terminal α and β fragments, and Aβ1–40

Acknowledgements

The authors would like to thank U. Schmitt (Department of Psychiatry, Mainz) for invaluable help with water maze experiments, J. Trotter for anti-PSD95 antibody, A. Bilkei-Gorzo for help with statistics, M. Staufenbiel (Novartis, Basel) for providing APP23 mice, and C. Ziegler for helpful comments on the manuscript. This work was performed in the framework of the DFG research group (DFG FOR 926; grants to CB and BL) and the DFG CRC 1080 (grant to CB).

References (53)

  • P.V. Arriagada et al.

    Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease

    Neurology

    (1992)
  • P.V. Arriagada et al.

    Distribution of Alzheimer-type pathologic changes in nondemented elderly individuals matches the pattern in Alzheimer's disease

    Neurology

    (1992)
  • E. Aso et al.

    CB1 agonist ACEA protects neurons and reduces the cognitive impairment of AbetaPP/PS1 mice

    J. Alzheimers Dis.

    (2012)
  • A.A. Asuni et al.

    Change in tau phosphorylation associated with neurodegeneration in the ME7 model of prion disease

    Biochem. Soc. Trans.

    (2010)
  • G. Benard et al.

    Mitochondrial CB(1) receptors regulate neuronal energy metabolism

    Nat. Neurosci.

    (2012)
  • C. Benito et al.

    Cannabinoid CB2 receptors and fatty acid amide hydrolase are selectively overexpressed in neuritic plaque-associated glia in Alzheimer's disease brains

    J. Neurosci.

    (2003)
  • T. Bisogno et al.

    The role of the endocannabinoid system in Alzheimer's disease: facts and hypotheses

    Curr. Pharm. Des.

    (2008)
  • S. Brahmachari et al.

    Induction of glial fibrillary acidic protein expression in astrocytes by nitric oxide

    J. Neurosci.

    (2006)
  • W.J. Buchser et al.

    96-Well electroporation method for transfection of mammalian central neurons

    Biotechniques

    (2006)
  • M.E. Calhoun et al.

    Neuron loss in APP transgenic mice

    Nature

    (1998)
  • D. Cota et al.

    The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis

    J. Clin. Invest.

    (2003)
  • A.A. Davis et al.

    Deletion of M1 muscarinic acetylcholine receptors increases amyloid pathology in vitro and in vivo

    J. Neurosci.

    (2010)
  • S.T. Dheen et al.

    Microglial activation and its implications in the brain diseases

    Curr. Med. Chem.

    (2007)
  • G.T. Dodd et al.

    The peptide hemopressin acts through CB1 cannabinoid receptors to reduce food intake in rats and mice

    J. Neurosci.

    (2010)
  • C. Duyckaerts et al.

    Progression of Alzheimer histopathological changes

    Acta Neurol. Belg.

    (1998)
  • G. Esposito et al.

    Cannabidiol reduces Abeta-induced neuroinflammation and promotes hippocampal neurogenesis through PPARgamma involvement

    PloS One

    (2011)
  • Cited by (41)

    • G protein-coupled receptors in neurodegenerative diseases

      2019, GPCRs: Structure, Function, and Drug Discovery
    • Cannabinoid pharmacology/therapeutics in chronic degenerative disorders affecting the central nervous system

      2018, Biochemical Pharmacology
      Citation Excerpt :

      Whereas some authors reported that specific agonists of this receptor did not alter the Aβ production, aggregation, or clearance in two different transgenic AD models [40,62], others provided evidence about a crosstalk between CB1 receptors and APP. On one hand, deletion of these receptors resulted in reduced APP protein levels and Aβ plaque deposition in APP23 transgenic mice [63] but not in APP/PS1 mice [64]. Furthermore, APP overexpression altered membrane localization and inhibitory signaling activity of CB1 receptors in the hippocampus of Tg2576 mice [65].

    • Genetic deletion of CB<inf>1</inf> cannabinoid receptors exacerbates the Alzheimer-like symptoms in a transgenic animal model

      2018, Biochemical Pharmacology
      Citation Excerpt :

      Neither the levels of soluble Aβ peptide or Aβ deposition were affected by the reduced CB1 receptor expression in APP/PS1/CB1 +/− mice, supporting our assumption about a major role for CB1 receptor in the defense against Aβ detrimental effects rather than in the production of this peptide. However, Stumm et al. [34] revealed that APP23 transgenic mice knockout for CB1 receptor exhibited reduced APP protein levels and Aβ plaque deposition and a recent report demonstrated that APP is able to alter membrane localization and inhibitory signalling activity of CB1 receptor in the hippocampus of Tg2576 mice [22]. Thus, a direct or indirect participation of CB1 receptor in Aβ processing cannot be completely ruled out.

    • [ <sup>18</sup> F]FMPEP-d <inf>2</inf> PET imaging shows age- and genotype-dependent impairments in the availability of cannabinoid receptor 1 in a mouse model of Alzheimer's disease

      2018, Neurobiology of Aging
      Citation Excerpt :

      Preclinical studies with AD animal models have also yielded contradictory results. Reductions in amyloid plaque load accompanied with impaired learning and memory deficits were demonstrated in CB1R-deficient APP23 mice (APP23/CB1-/-) when compared with APP23 mice, suggesting that CB1R deficiency worsens learning and memory deficits in AD (Stumm et al., 2013). Significantly decreased CB1R expression has been observed in the hippocampus (HIPPO) of 10- to 12-month-old APPSWE/PS1ΔE9 mice in association with astrogliosis (Kalifa et al., 2011), whereas increased CB1R levels have been reported in the cortex of 14-month-old—but not in 7-month-old—APPSWE/PS1ΔE9 mice (Mulder et al., 2011).

    View all citing articles on Scopus
    1

    These two authors contributed equally to this study.

    2

    Present address: Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

    View full text