Canna~Fangled Abstracts

Loss of CB1 receptors leads to decreased cathepsin D levels and accelerated lipofuscin accumulation in the hippocampus.

By August 22, 2013No Comments

elsevier_logoLoss of CB1 receptors leads to decreased cathepsin D levels and accelerated lipofuscin accumulation in the hippocampus

  • a Institute of Molecular Psychiatry, University of Bonn, Sigmund-Freud-Straße 25, 53127 Bonn, Germany
  • b Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany
  • c Life and Medical Sciences (LIMES), Membrane Biology and Lipid Biochemistry Unit, University of Bonn, c/o Kekulé-Institut für Organische Chemie und Biochemie der Universität, Gerhard-Domagk-Str. 1, 53121 Bonn, Germany

Highlights

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Increased lipofuscin accumulation in the hippocampus of Cnr1−/− mice.

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Lack of CB1 receptor leads to decreased lysosomal capacity.

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Enhanced autophagy in the hippocampus of old Cnr1−/− mice.

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Autophagic flux is enhanced in the lack of CB1 receptors.

 

Abstract

Early onset of age-related changes in the brain of cannabinoid 1 receptor knockout (Cnr1−/−) mice suggests that cannabinoid 1 (CB1) receptor activity significantly influences the progression of brain aging. In the present study we show that lack of CB1 receptors leads to a significant increase in lipofuscin accumulation and a reduced expression and activity of cathepsin D, lysosomal protease implicated in the degradation of damaged macromolecules, in the hippocampus of 12-month-old mice. The impaired clearance of damaged macromolecules due to the low cathepsin D levels and not enhanced oxidative stress may be responsible for the lipofuscin accumulation because macromolecule oxidation levels were comparable between the genotypes within the same age group. The altered levels of autophagy markers p62 and LC3-II suggest that autophagy is upregulated in CB1 knockout mice. Increased autophagic flux in the absence of CB1 receptors is probably a compensatory mechanism to partially counteract decreased lysosomal degradation capacity. Together, these results suggest that CB1 receptor activity affects lysosomal activity, degradation of damaged macromolecules and thus it may influence the course and onset of brain aging.

Keywords

  • Endocannabinoid system;
  • Hippocampus;
  • Oxidative stress;
  • LC3;
  • p62;
  • Autophagy

Figures and tables from this article:

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Fig. 1. Increased lipofuscin accumulation in the CA3 region of the hippocampus in aged CB1 knockout mice. (A) Representative confocal microscopy images of the CA3 region of the hippocampus in young and old wild type (Cnr1+/+) and CB1 receptor knockout (Cnr1−/−) mice. Brain slices were stained with 4′,6-diamidino-2-phenylindole (DAPI) to visualize the nuclei of the cells (blue fluorescence). Lipofuscin accumulation is indicated by the presence of yellowish autofluorescence (number of slices evaluated per group = 18). Scale bar: 100 μm. (B) Quantification of lipofuscin autofluorescence in the CA3 region of the hippocampus in young and old Cnr1+/+ and Cnr1−/− mice. Columns represent group mean values; error bars represent standard errors of mean (SEM). +++p < 0.001 difference between the genotypes within the same age group; ***p < 0.001 difference between the age groups, both Bonferroni’s post hoc test. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Age-dependent changes in the amount of oxidized macromolecules in the hippocampus of Cnr1+/+ and Cnr1−/−mice (A) No difference in lipid peroxidation measured as the concentration of thiobarbituric acid-reactive substances between age groups or genotypes (n = 4–8 per group). (B) Similar age-related increase in protein oxidation assessed as the amount of protein carbonyls in Cnr1+/+ and Cnr1−/− mice (***p < 0.001 Bonferroni’s post hoc test; n = 6–8 per group). (C) Age-related increase in the amount of oxidized DNA measured as 8-hydroxyguanosine staining intensity did not differ between the genotypes (***p < 0.001 difference between the age groups within the same genotype, Bonferroni’s post hoc test, n = 4).
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Fig. 3. Expression of lysosomal markers (cathepsin D and LAMP1) in the hippocampus of Cnr1+/+ and Cnr1−/− mice (n = 4–5). (A) Representative Western blots showing the expression of different isoforms of cathepsin D in young and old Cnr1+/+ and Cnr1−/− mice. (B) Densitometric analysis of Western blot data revealed decreased expression of the intermediate, 48 kDa, form and mature isoforms–heavy chain, 34 kDa, and light chain, 14 kDa, in young and old Cnr1−/−mice. *, **, *** indicate p < 0.05, p < 0.01, p < 0.001 significant difference between the genotypes using Bonferroni’s post hoc test. (C) Cathepsin D activity is significantly reduced in the hippocampus of Cnr1−/− mice. ** indicate p < 0.01 in Student’s t-test. (D) No significant difference in LAMP1 protein levels between age groups or genotypes, however, a tendency towards higher LAMP1 density in the old CB1 knockout mice could be noted (n = 4).
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Fig. 4. Autophagic markers (LC3, p62) in the hippocampus of Cnr1+/+ and Cnr1−/− mice (A) and measurements of autophagic flux in hippocampal neurons (B and C). (A) Lipidation of LC3 (LC3-II level) was increased in 12-month-old Cnr1−/− mice (*p < 0.05 in Bonferroni’s post hoc test; n = 3–5). Cnr1−/− animals had lower p62 levels in the hippocampus compared to Cnr1+/+ mice (**p < 0.01 significant difference between the genotypes within the same age-group using Bonferroni’s post hoc test; n = 4–5). (B) Western blot analysis of autophagic markers in hippocampal neurons from Cnr1+/+ and Cnr1−/− mice (n = 3). Basal level of LC3-II was lower in Cnr1−/− hippocampal neurons. There was no effect of rapamycin treatment on LC3-II levels, but the level of p62 was slightly decreased. Treatment with bafilomycin (20 nM) resulted in a significant increase in LC3-II and p62 levels in both Cnr1+/+ and Cnr1−/− neurons, indicating the block of autophagic flux. **p < 0.01 treatment effect (two-way ANOVA). (C) Autophagic flux is significantly upregulated in the absence of CB1 receptors. LC3-II accumulation after bafilomycin treatment in comparison to basal levels was calculated for Cnr1+/+ and Cnr1−/− neurons. Fold increase in LC3-II levels after the treatment was significantly higher in the absence of CB1 receptors, indicating higher autophagy rate (*p < 0.05, unpaired t-test).
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Supplementary Fig. 1. Age-related increase in lipofuscin autofluorescence in cortex (A) and striatum (B) is similar between Cnr1+/+ and Cnr1−/− mice. (***p < 0.001 significant difference between the age groups within the same genotype using Bonferroni’s t-test; n = 4–6).
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Supplementary Fig. 2. Lipofuscin accumulation in the cortex is significantly higher in old female mice both in (A) Cnr1+/+and (B) Cnr1−/− mice (+++p < 0.001 difference between the sexes; ***p < 0.001 difference between the age groups, both Bonferroni’s post hoc test; n = 2–3).
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Supplementary Fig. 3. No age or genotype related changes in lipid (A and B) and protein (C and D) oxidation in cortex and striatum of Cnr1+/+ and Cnr1−/− mice.
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Supplementary Fig. 4. Similar age-dependent increase in DNA oxidative damage in cortex (A) and striatum (B) in Cnr1+/+ and Cnr1−/− mice. (C and D) Age-related increase in DNA oxidative damage in the cortex was significanly higher in females in both genotypes. (+p < 0.05; +++p < 0.001 difference between the sexes; ***p < 0.001 difference between the age groups, each Bonferroni’s post hoc test; n = 2–3).
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Supplementary Fig. 5. Expression of cathepsin D (CD) mRNA in the hippocampus of old Cnr1−/− mice was lower as in age-matched Cnr1+/+ animals (**p < 0.01 in Bonferroni’s post hoc test).
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Supplementary Fig. 6. Total ceramide content and profile of individual ceramide species did not vary between Cnr1+/+and Cnr1−/− mice. (A) Total ceramide content (in μg per mg protein, n = 4–8 (B) Ceramide profiles were similar between the two genotypes and age groups tested. C16:0 ceramides were present at detectable levels only in aged mice (indicated by arrows).
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Supplementary Fig. 7. Significantly increased expression of p62 mRNA in the hippocampus in ageing in Cnr1+/+ but not in Cnr1−/− mice. p62 levels were significantly lower in old Cnr1−/− mice as in the age-matched Cnr1+/+ mice (***p < 0.001 significant difference between the genotypes within the same age group. ++p < 0.01 significant difference between young and old Cnr1+/+ mice using Bonferroni’s post hoc test; n = 6–7).
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Supplementary Fig. 8. No significant difference in phospho-mTOR/total mTOR and phospho-Akt/total Akt ratios between the age-groups or genotypes in the hippocampus (n = 4). (A) mTOR phosphorylation (B) Akt phosphorylation.
Corresponding author contact information
Corresponding author at: Institute of Molecular Psychiatry, c/o Life & Brain Center, University of Bonn, Sigmund-Freud-Straße 25, 53127 Bonn, Germany. Tel.: +49 2286885317; fax: +49 6885301.
1
Present address: Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Ludwig-Erhard-Allee 2, 53175 Bonn, Germany.

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