Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 1. PPX induces autophagy through a DRD3-dependent mechanism in mouse striata and HEK cells. (A) Western blot and densitometric analysis for LC3 and p62 in WT and drd3KO mice treated with 0.15 mg/kg/d for six days (n = 5). PPX promotes a decline in LC3-II and p62 in WT but not in drd3KO mice. (B–D) Autophagic flux in HEK (n = 7–8; (B)), DRD2-HEK (n = 10; (C)), and DRD3-HEK (n = 8–19; (D)) cells treated with the autophagosome–lysosome fusion blocker chloroquine (CQ, 20 µM, 1 h; lanes 3 and 4) and 0.1 µM PPX (lanes 5 and 6) or 10 µM PPX (lanes 7 and 8) for 4 h. CQ was added after PPX. Western blot for LC3 revealed that autophagic flux (LC3-II levels in CQ + PPX vs. CQ) was increased after PPX treatment in DRD3-HEK cells (D) but not in HEK (B) or DRD2-HEK cells (C). Statistical analyses were performed using the unpaired t-test for mice and Mann–Whitney test for cells. n = number of mice per experimental group or number of experimental repeats in cells; ns = non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, MANN-WHITNEY

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 3. DRD3-induced autophagy is prolonged in cells expressing a pathogenic form of polyQ-HTT. The effects of PPX (0.1 µM) on LC3-II levels were analyzed in the absence (lanes 3 and 4) and presence (lanes 7 and 8) of the autophagosome–lysosome fusion blocker chloroquine (CQ, 20 µM, 1 h; lanes 5 and 6). (A–C) After 4 h of PPX treatment (n = 5–9), the autophagic flux (LC3-II levels in CQ + PPX vs. CQ) was increased in DRD3- (A), Q23-DRD3- (B), and Q74-DRD3-HEK (C) cells. (D–F) After 24 h of PPX treatment (n = 6), the autophagic flux remained increased in Q74-DRD3-HEK cells (D) but not in DRD3-HEK (E) or Q23-DRD3-HEK cells (F). Statistical analyses were performed using the Mann–Whitney test. n = number of experimental repeats; ns = non-significant; * p < 0.05.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Expressing, MANN-WHITNEY

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 5. PPX promotes Q74 clearance and protects DRD3-HEK cells against its genotoxic effect. (A) Western blot for polyQ in Q74-DRD3- (n = 12), Q74- (n = 7), and Q23-DRD3-HEK (n = 7) cells treated with PPX (6 h within 12 h after transfection). The densitometric analysis showed that PPX promotes a significant decrease in Q74 but not Q23 in DRD3-HEK cells, nor Q74 in HEK cells (Kruskal– Wallis followed by Dunn’s multiple comparison test). (B–E) Fluorescent labeling and quantitative analysis of the number of pyknotic nuclei (B,D) and immunofluorescent intensity of polyQ- and γH2AX expression in Q74-DRD3- and Q23-DRD3-HEK cells treated with PPX (C,E). PPX reduced the number of pyknotic nuclei (B(iv,v),D) and polyQ and γH2AX immunofluorescent intensity (C(iv,v),E) in Q74-DRD3-HEK cells without affecting Q23-DRD3-HEK cells. Statistical analyses were performed using ANOVA followed by Bonferroni’s (number of pyknotic nuclei and H2AXγ labeling intensity) and Brown–Forsythe tests, followed by Sidak’s (polyQ labeling intensity) multiple comparison tests. Arrows in (B) indicate pyknotic nuclei. A.U., arbitrary units. Bar: in (B(v)) (for (B(i–v))): 50 µm; in (C(v)) (for (C(i–v))): 10 µm. n = number of experimental repeats; *** p < 0.001. ns, not significant.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, Transfection, Comparison, Labeling, Expressing

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 6. Short treatment with PPX (0.15 mg/kg, 6 days) induces mTORC1 inhibition through a DRD3- dependent mechanism. Western blot and densitometric analysis for the total and phosphorylated forms of p70S6K at Thr389 (A), AMPKα at Thr172 (B), and ULK1 at Ser at 757 and Ser555 (C) in striatal extracts of WT and drd3KO mice. PPX promoted a decrease in Thr389-p70S6K and Ser757-ULK phosphorylation in WT mice without affecting Thr172-AMPKα and Ser555-ULK phosphorylation. No phosphorylation changes were detected in drd3KO mice. n = 5–9 mice per experimental group, unpaired t-test; ns = non-significant; * p < 0.05; ** p < 0.01.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Inhibition, Western Blot, Phospho-proteomics

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 8. DRD3 promotes Q74 clearance through AMPK. Western blot and densitometric analysis of the total and phosphorylated forms of AMPKα at Thr172 (A) and Q74 in Q74-DRD3-HEK cells (B). PPX (0.1 µM, 6 h) induces Thr172-AMPKα phosphorylation ((A), lanes 3 and 4), rescues Thr172- AMPKα dephosphorylation induced by the AMPK inhibitor compound C (CC; 2 µM, 6 h; (A), lanes 7 and 8), and reduces Q74 levels in Q74-DRD3-HEK cells ((B), lanes 4 and 5) and Q74-DRD3-HEK cells treated with CC ((B), lanes 8 and 9). Lane 1 in (B), polyQ-untransfected DRD3-HEK cells. n = 6–8 experimental repeats. Statistical analyses were performed using ANOVA followed by the Fisher LSD test in the study of AMPKα phosphorylation and ANOVA followed by the Holm–Sidak multicomparison test in the study of Q74 levels. * p < 0.05. Analysis of S2488-mTOR was inconclusive since no differences among treatments resulted from either mTOR-independent AMPK activation or the recovery of transient mTOR inhibition in non-Q74 transfected cells.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Western Blot, Phospho-proteomics, De-Phosphorylation Assay, Activation Assay, Inhibition, Transfection

Journal: Cells

Article Title: Dopamine Receptor D3 Induces Transient, mTORC1-Dependent Autophagy That Becomes Persistent, AMPK-Mediated, and Neuroprotective in Experimental Models of Huntington's Disease.

doi: 10.3390/cells14090652

Figure Lengend Snippet: Figure 10. Schematic view of signaling pathways involved in transient and persistent DRD3-medited autophagy in WT and R6/1 mice, respectively. In WT mice (left), autophagy is transiently induced by mTORC1 inhibition with the subsequent inhibition of p70S6K and the activation of ULK1. In R6/1 mice (right), where mTORC1 is hyperphosphorylated, autophagy is persistently activated through AMPK with the direct activation of ULK1, promoting an effective clearance of soluble mHTT. In persistent autophagy, the mTORC1-p70S6K branch of mTORC1 signaling is also inhibited, but mTORC1-ULK1 signaling is not activated. In addition, the crosstalk between mTORC1-p70S6K and MAPK-p90S6K pathways is activated and rpS6 activity is preserved. Blue arrows and blocked red lines indicate the activation or inhibition of downstream targets, respectively. Dashed red lines indicate putative pathways of mTORC1 inhibition in persistent autophagy.

Article Snippet: An additional group of R6/1 mice was treated with the selective DRD3 antagonist NGB2904 (DRD2 Ki 217 nM, DRD3 Ki 1.4 nM; 0.5 mg/kg, i.p.; Tocris, Bristol, UK; #2635) or vehicle (2.5% w/v 2-hydroxypropylβ-cyclodextrin; Sigma-Aldrich; #H107) 30 min before PPX.

Techniques: Protein-Protein interactions, Inhibition, Activation Assay, Activity Assay