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Inhibition of striatonigral autophagy as a link between cannabinoid intoxication and impairment of motor coordination

  1. Cristina Blázquez
  2. Andrea Ruiz-Calvo
  3. Raquel Bajo-Grañeras
  4. Jérôme M Baufreton
  5. Eva Resel
  6. Marjorie Varilh
  7. Antonio C Pagano Zottola
  8. Yamuna Mariani
  9. Astrid Cannich
  10. José A Rodríguez-Navarro
  11. Giovanni Marsicano
  12. Ismael Galve-Roperh
  13. Luigi Bellocchio  Is a corresponding author
  14. Manuel Guzmán  Is a corresponding author
  1. Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED), Instituto Universitario de Investigación Neuroquímica (IUIN) and Department of Biochemistry and Molecular Biology, Complutense University, Spain
  2. Instituto Ramón y Cajal de Investigación Sanitaria (IRYCIS), Spain
  3. Centre National de la Recherche Scientifique (CNRS) and University of Bordeaux, Institut des Maladies Neurodégénératives, France
  4. Institut National de la Santé et de la Recherche Médicale (INSERM) and University of Bordeaux, NeuroCentre Magendie, Physiopathologie de la Plasticité Neuronale, France
Research Article
Cite this article as: eLife 2020;9:e56811 doi: 10.7554/eLife.56811
7 figures, 2 tables and 1 additional file

Figures

THC impairs autophagy in the mouse striatum.

Wild-type C57BL/6N mice were treated with THC (10 mg/kg as a single i.p. injection) or its vehicle. Four hours later, the striatum (St), cortex (Cx), hippocampus (Hc) and cerebellum (Cb) were dissected for Western blot analysis. (A) Effect of THC on autophagy markers in the different brain regions. (B) Relative levels of LC3-I and p62 in the different brain regions from vehicle-treated animals. In both panels, representative blots of each condition, together with optical density values relative to those of the respective loading controls, are shown (n = 6 animals per group). Blots were cropped for clarity. Electrophoretic migration of molecular weight markers is depicted on the right-hand side of each blot. **p<0.01 from vehicle-treated group by unpaired Student t-test. Raw numerical data and further statistical details are shown in Figure 1—source data 1.

Figure 1—source data 1

Source data for THC impairs autophagy in the mouse striatum.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig1-data1-v1.xlsx
THC impairs autophagy in primary striatal neurons.

Primary striatal neurons from C57BL/6N mice were exposed for 24 hr to THC (0.75 µM) or HU-210 (10 nM), alone or in combination with hydroxychloroquine (0.1 mM), E64d (0.1 µM) and/or pepstatin A (10 ng/ml), or their vehicles. (A) LC3-II immunoreactivity (number of cells with three or more LC3-positive dots relative to total cells; upper panel) and LC3-II/LAMP1 immunoreactivity (number of LAMP1-positive cells with three or more LC3 dots relative to total cells; lower panel). Representative images with encircled examples of double-positive cells are shown (n = 3–6 independent cell preparations per condition). (B) p62 immunoreactivity (p62 fluorescence intensity relative to total cells). Representative images of selected experimental conditions with encircled examples of high-intensity cells are shown (n = 3–6 independent cell preparations per condition). *p<0.05, **p<0.01 from vehicle-treated group by two-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 2—source data 1.

Figure 2—source data 1

Source data for THC impairs autophagy in primary striatal neurons.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
Temsirolimus prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.

Wild- type C57BL/6N mice were treated with temsirolimus (1 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. (A) Motor coordination (RotaRod test, time to fall relative to pre-treatment; n = 8–9 animals per group). (B, C) Western blot analysis of autophagy markers (panel B) and mTORC1 signaling pathway markers (panel C) in the striatum. Representative blots of each condition, together with optical density values relative to those of loading controls, are shown (n = 4 animals per group). Blots were cropped for clarity. Electrophoretic migration of molecular weight markers is depicted on the right-hand side of each blot. (D, E) Immunofluorescence analysis of p62 (p62 fluorescence intensity per DARPP32-positive cell; panel D) and phosphorylated ribosomal protein S6 (phospho-S6/DARPP32 double-positive cells relative to total cells; panel E) in the dorsal striatum (n = 4 animals per group). Representative images with encircled examples of a high-intensity cell (panel D) or double-positive cells (panel E) are shown. *p<0.05, **p<0.01 from vehicle-treated group, or #p<0.05, ##p<0.01 from THC-treated group, by one-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 3—source data 1.

Figure 3—source data 1

Source data for Temsirolimus prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Temsirolimus does not rescue THC-induced hypolocomotion.

Wild-type C57BL/6N mice (n = 6–9 animals per group) were treated with temsirolimus (1 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Animals were then subjected to the open field test for 10 min. Total distance travelled (cm), overall activity (counts), resting time (s), fast movements (counts) and stereotypic movements (counts) were determined. *p<0.05, **p<0.01 from vehicle-treated group by one-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 3—source data 1.

Figure 4 with 1 supplement
Trehalose prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.

Wild-type C57BL/6N mice were given trehalose (10 g/L) or plain water ad libitum for 24 hr, and, subsequently, were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. (A) Motor coordination (RotaRod test, time to fall relative to pre-treatment; n = 11–14 animals per group). (B, C) Western blot analysis of autophagy markers (panel B) and mTORC1 signaling pathway markers (panel C) in the striatum. Representative blots of each condition, together with optical density values relative to those of loading controls, are shown (n = 4 animals per group). (D, E) Immunofluorescence analysis of p62 (p62 fluorescence intensity per DARPP32-positive cell; panel D) and phosphorylated ribosomal protein S6 (phospho-S6/DARPP32 double-positive cells relative to total cells; panel E) in the dorsal striatum (n = 4 animals per group). Representative images with encircled examples of a high-intensity cell (panel D) or double-positive cells (panel E) are shown. *p<0.05, **p<0.01 from vehicle-treated group, or #p<0.05, ##p<0.01 from THC-treated group, by one-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 4—source data 1.

Figure 4—source data 1

Source data for Trehalose prevents the THC-induced impairment of striatal autophagy and motor coordination in vivo.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig4-data1-v1.xlsx
Figure 4—figure supplement 1
Trehalose does not rescue THC-induced hypolocomotion.

Wild-type C57BL/6N mice (n = 6–10 animals per group) were given trehalose (10 g/L in drinking water) or plain water ad libitum for 24 hr, and, subsequently, with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Animals were then subjected to the open field test for 10 min. Total distance travelled (cm), overall activity (counts), resting time (s), fast movements (counts) and stereotypic movements (counts) were determined. *p<0.05, **p<0.01 from vehicle-treated group by one-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 4—source data 1.

Figure 5 with 1 supplement
Cannabinoid CB1 receptors located on D1R-MSNs, but not on glutamatergic neurons, are required for the THC-induced impairment of striatal autophagy and motor coordination in vivo.

(A–C) D1R-CB1R KO mice and CB1R-floxed control littermates were treated with THC (10 mg/kg as a single i.p.injection) or its vehicle for 4 hr. Panel A, Motor coordination (RotaRod test, time to fall relative to pre-treatment; n = 7 animals per group). Panel B, Western blot analysis of autophagy markers in the striatum. Representative blots of each condition, together with optical density values relative to those of loading controls, are shown (n = 5 animals per group). Blots were cropped for clarity. Electrophoretic migration of molecular weight markers is depicted on the right-hand side of each blot. Panel C, Immunofluorescence analysis of p62 (p62 fluorescence intensity per DARPP32-positive cell) in the dorsal striatum (n = 4 animals per group). Representative images with an encircled example of high-intensity cell are shown. (D–F) Glu-CB1R KO mice and CB1R-floxed control littermates were treated with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Panel D, Motor coordination (RotaRod test, time to fall relative to pre-treatment; n = 5 animals per group). Panel E, Western blot analysis of autophagy markers in the striatum. Representative blots of each condition, together with optical density values relative to those of loading controls, are shown (n = 5 animals per group). Blots were cropped for clarity. Electrophoretic migration of molecular weight markers is depicted on the right-hand side of each blot. Panel F, Immunofluorescence analysis of p62 (p62 fluorescence intensity per DARPP32-positive cell) in the dorsal striatum (n = 4 animals per group). Representative images with an encircled example of high-intensity cell are shown. *p<0.05, **p<0.01 from the corresponding vehicle-treated group, or #p<0.05, ##p<0.01 from the corresponding THC-treated CB1R-floxed group, by two-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 5—source data 1.

Figure 5—source data 1

Source data for Cannabinoid CB1 receptors located on D1R-MSNs, but not on glutamatergic neurons, are required for the THC-induced impairment of striatal autophagy and motor coordination in vivo.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Rimonabant rescues THC-induced motor dyscoordination.

Wild-type C57BL/6N mice (n = 5 animals per group) were treated with SR141716 (rimonabant; 3 mg/kg as a single i.p. injection) or its vehicle for 20 min, and, subsequently, with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Animals were then subjected to the RotaRod test (time to fall relative to pre-treatment). **p<0.01 from vehicle-treated group, or ##p<0.01 from THC-treated group, by one-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 5—source data 1.

Figure 6 with 1 supplement
mTORC1 and p62 in D1R-MSNs participate in the THC-induced impairment of motor coordination in vivo.

(A) D1R-Cre mice and wild-type control littermates were injected stereotactically into the dorsal striatum with a CAG-DIO-dnRaptor rAAV, and left untreated for 4 weeks. Animals were subsequently treated with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr, and motor coordination was evaluated (RotaRod test, time to fall in seconds; n = 5 animals per group). **p<0.01 from vehicle-treated WT/post-treatment group, or ##p<0.01 from THC-treated WT/post-treatment group, by two-way ANOVA with Tukey’s multiple comparisons test. Representative images of c-myc tag and phosphorylated ribosomal protein S6 staining in the dorsal striatum, together with their quantification (c-myc-positive cells relative to total cells, or phospho-S6-positive cells relative to total cells), are shown (n = 4 animals per group). **p<0.01 from WT group by unpaired Student t-test (c-myc immunofluorescence); *p<0.05, **p<0.01 from vehicle-treated/WT group, or ##p<0.01 from THC-treated/WT group, by two-way ANOVA with Tukey’s multiple comparisons test (phospho-S6 immunofluorescence). (B) D1R-Cre mice and wild-type control littermates were injected stereotactically into the dorsal striatum with a CAG-DIO-p62 rAAV, and left untreated for 4 weeks. Animals were subsequently treated with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr, and motor coordination was evaluated (RotaRod test, time to fall in seconds; n = 5 animals per group). *p<0.05 from vehicle-treated WT/post-treatment group, or #p<0.05 from the respective WT/pre-treatment group, by two-way ANOVA with Tukey’s multiple comparisons test. Representative images of p62 staining in the dorsal striatum, together with their quantification (p62 fluorescence intensity relative to total cells), are shown (n = 4 animals per group). **p<0.01 from WT group by unpaired Student t-test. Raw numerical data and further statistical details are shown in Figure 6—source data 1.

Figure 6—source data 1

Source data for mTORC1 and p62 in D1R-MSNs participate in the THC-induced impairment of motor coordination in vivo.

https://cdn.elifesciences.org/articles/56811/elife-56811-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
THC activates the mTORC1 pathway in D1R-MSNs but not D2R-MSNs in vivo.

Drd1a-tdTomato;Drd2-EGFP mice were treated with THC (10 mg/kg as a single i.p. injection) or its vehicle for 4 hr. Immunofluorescence analysis of phosphorylated ribosomal protein S6 in the dorsal striatum (phospho-S6/tdTomato-double-positive cells relative to total tdTomato-positive cells, or phospho-S6/EGFP-double-positive cells relative to total EGFP-positive cells; n = 6 animals per group). Representative images with encircled examples of double-positive cells are shown. **p<0.01 from vehicle-treated D1R group, or #p<0.05 from THC-treated D1R group, by two-way ANOVA with Tukey’s multiple comparisons test. Raw numerical data and further statistical details are shown in Figure 6—source data 1.

Author response image 1

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Mus musculus, C57BL/6N, male)Cnr1fl/fl;Drd1aCreMonory et al., 2007; doi:10.1371/journal.pbio.0050269N/AConditional mutant mice in which the CB1 receptor gene (Cnr1) is absent from D1R (Drd1a)-expressing cells
Strain, strain background (Mus musculus, C57BL/6N, male)Drd1aCreLemberger et al., 2007; doi:10.1186/1471-2202-8-4N/ATransgenic mice expressing Cre recombinase in D1R (Drd1a)-expressing cells
Strain, strain background (Mus musculus, C57BL/6N, male)Cnr1fl/fl;Neurod6CreMonory et al., 2006; doi:10.1016/j.neuron.2006.07.006N/AConditional mutant mice in which the CB1 receptor gene (Cnr1) is absent from dorsal telencephalic glutamatergic (Neurod6-expressing) neurons
Strain, strain background (Mus musculus, C57BL/6N, male)Drd1a-tdTomato;Drd2-EGFPSuárez et al., 2014; doi:10.1016/j.biopsych.2013.05.006N/ATransgenic mice expressing the tdTomato and EGFP reporter genes under the control of the Drd1a gene promoter and the Drd2 gene promoter, respectively
Strain, strain background (Mus musculus, C57BL/6N, male)C57BL/6NHarlan LaboratoriesRRID:MGI:5902763Wild-type mice
Transfected construct (Homo sapiens)Myc-Raptor (ΔCT) expression vectorAddgene
Hara et al., 2002; doi:10.1016/s0092-8674(02)00833–4.
Koketsu et al., 2008; doi:10.1152/ajpendo.00253.2007
Plasmid #1859;
RRID:Addgene_1859
Vector backbone: pRK-5; construct generated by PCR-mediated deletion of1293 base pairs at the Raptor C-terminus
Transfected construct (Homo sapiens)HA-p62
expression vector
AddgenePlasmid #28027;
RRID:Addgene_28027
Vector backbone: pcDNA4/TO
Genetic reagent (Homo sapiens)CAG-DIO rAAV expression vectorKlugmann et al., 2005; doi:10.1016/j.mcn.2004.10.002
Bellocchio et al., 2016; doi:10.1523/JNEUROSCI.1192–16.2016
CAG-DIO rAAV
Hybrid serotype 1/2
Recombinant adeno-associated virus (rAAV) for Cre-driven transgene expression with a CAG promoter
Biological sample (Mus musculus)Primary striatal neuronsHarlan Laboratories (C57BL/6N mice)C57BL/6N
RRID:MGI:5902763
In vitro cell cultures
AntibodyAnti-LC3B
(rabbit polyclonal)
Sigma-AldrichCat. #L7543;
RRID:AB_796155
IF (1:300);
WB (1:4000)
AntibodyAnti-p62
(rabbit polyclonal)
Enzo Life SciencesCat. #BML-PW9860-0025;
RRID:AB_2052149
IF (1:250);
WB (1:1000)
AntibodyAnti-p62
(rabbit polyclonal)
ProgenCat. #GP62-C;
RRID:AB_2687531
WB (1:1000)
AntibodyAnti-LAMP1
(rabbit polyclonal)
AbcamCat. #ab25245
RRID:AB_449893
IF (1:1000)
AntibodyAnti-DARPP32
(mouse monoclonal)
BD BiosciencesCat. #611520;
RRID:AB_398980
IF (1:700)
AntibodyAnti-phospho-S6-S235/S236
(rabbit polyclonal)
Cell SignalingCat. #2211;
RRID:AB_331679
IF (1:300)
AntibodyAnti-phospho-S6-S240/S244
(rabbit polyclonal)
Cell SignalingCat. #5364;
RRID:AB_10694233
IF (1:800)
 AntibodyAnti-c-Myc
(mouse monoclonal)
Sigma-AldrichCat. #11-667-149-001;
RRID:AB_390912
IF (1:500)
AntibodyAnti-phospho-S6K-T389
(mouse monoclonal)
Cell SignalingCat. #9206;
RRID:AB_2285392
WB (1:1000)
AntibodyAnti-total-S6K
(rabbit polyclonal)
Cell SignalingCat. #9202;
RRID:AB_331676
WB (1:1000)
AntibodyAnti-phospho-ULK1-S757
(rabbit polyclonal)
Cell SignalingCat. #6888;
RRID:AB_10829226
WB (1:1000)
AntibodyAnti-phospho-ULK1-S555
(rabbit polyclonal)
Cell SignalingCat. #5869;
RRID:AB_10707365
WB (1:1000)
AntibodyAnti-total-ULK1
(rabbit polyclonal)
Cell SignalingCat. #8054;
RRID:AB_11178668
WB (1:1000)
AntibodyAnti-β-actin
(mouse monoclonal)
Sigma-AldrichCat. #A5441;
RRID:AB_476744
WB (1:4000)
AntibodyAnti-mouse monoclonal IgG
(HRP-linked whole antibody)
GE-Healthcare LifescienceCat. #NA931;
RRID:AB_772210
WB (1:5000)
AntibodyAnti-rabbit monoclonal IgG
(HRP-linked whole antibody)
GE-Healthcare LifescienceCat. #NA934;
RRID:AB_2722659
WB (1:5000)
AntibodyGoat anti-guinea pig IgG (H+L)
(HRP-linked secondary antibody)
InvitrogenCat. #A18769;
RRID:AB_2535546
WB (1:5000)
AntibodyGoat anti-mouse IgG (H+L)
(cross-adsorbed, Alexa Fluor 488)
InvitrogenCat. #A-11001;
RRID:AB_2534069
IF (1:500)
AntibodyGoat anti-mouse IgG (H+L)
(cross-adsorbed, Alexa Fluor 594)
InvitrogenCat. #A-11005;
RRID:AB_2534073
IF (1:500)
AntibodyGoat anti-mouse IgG (H+L)
(cross-adsorbed, Alexa Fluor 647)
InvitrogenCat. #A-21235;
RRID:AB_2535804
IF (1:500)
AntibodyGoat anti-rabbit IgG (H+L)
(cross adsorbed, Alexa Fluor 488)
InvitrogenCat. #A-11008;
RRID:AB_143165
IF (1:500)
AntibodyGoat anti-rabbit IgG (H+L)
(cross adsorbed, Alexa Fluor 594)
InvitrogenCat. #A-11012;
RRID:AB_2534079
IF (1:500)
AntibodyGoat anti-rabbit IgG (H+L)
(cross adsorbed, Alexa Fluor 647)
InvitrogenCat. #A-21244;
RRID:AB_2535812
IF (1:500)
Commercial assay or kitPapain dissociation system (PDS)WorthingtonCat. #LK 003153In vitro cell cultures
Chemical compound, drugΔ9-tetrahydro-cannabinol (THC)THC Pharm GmbHDronabinolIn vivo experiments (10 mg/kg, i.p.);in vitro experiments (0.75 μM)
Chemical compound, drugRimonabant (SR141716)Cayman ChemicalCat. #9000484In vivo experiments: (3 mg/kg, i.p.)
Chemical compound, drugTemsirolimusLC LabsCat. #T-8040In vivo experiments (1 mg/kg, i.p.)
Chemical compound, drugTrehaloseMerck-CalbiochemCat. #90210In vivo experiments (10 g/L in drinking water)
Chemical compound, drugHU-210TocrisCat. #0966In vitro experiments (10 nM)
Chemical compound, drugHydroxychloroquineMerckCat. #509272In vitro experiments (0.1 mM)
Chemical compound, drugE64dEnzo Life SciencesCat. #BML-PI107-0001In vitro experiments (0.1 μM)
Chemical compound, drugPepstatin AEnzo Life SciencesCat. #ALX-260–085 M005In vitro experiments (10 ng/ml)
Software, algorithmGraph Pad
Prism 8.0
GraphPad Software IncRRID:SCR_002798Descriptive analysis and statistics
Software, algorithmIBM SPSSIBM CorporationRRID:SCR_002865Statistical power analysis
Software, algorithmImage JNIHRRID:SCR_003070Western blot and immune-microscopy image analysis
Software, algorithmTCS-SP8
Leica Application Suite X, LASX
LeicaRRID:SCR_013673SP8 AOBS confocal microscopy image capture
Software, algorithmACTITRACKUPG V2.7PanlabCat. #76–0610Motor activity analysis
OtherDAPI stainInvitrogenCat. #D1306;
RRID:AB_2629482
IF (1 µg/mL)
OtherRotaRod LE8200Harvard ApparatusCat. #LE8200 (76–0237)Motor coordination testing
OtherIR actimeter
(ActiTrack)
PanlabCat. #76–0127, #76–0131, #76–0134, #76–0125Motor activity testing
Author response table 1
Motor coordination (time to fall, s)
Raw DataVehicleTHC
Pre-treatmentPost-treatmentPost-treatment/Pre-treatmentPre-treatmentPost-treatmentPost-treatment/Pre-treatment
931011.08113710.63
911311.431131120.99
791221.541011051.05
721301.8164480.75
1001431.4480801.00
Mean871251.4694830.88
SEM5.056.960.129.6611.620.08
CI72.98106.101.1467.3850.920.66
101.00144.701.78121.00115.501.11

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