Journal: International Journal of Molecular Sciences

Article Title: Choline-Sigma-1R as an Additional Mechanism for Potentiation of Orexin by Cocaine

doi: 10.3390/ijms22105160

Figure Lengend Snippet: OXA (17-33) A increases [Ca 2+ ] i via choline-Sigma-1R-dependent mechanism. ( A ) Illustration of average Ca 2+ transients (± SD) induced by OXA (17-33) (10 nM) alone (left) and in the presence of VU0155056 (1 μM), PLD inhibitor (middle), and BD1047 (50 µM) or NE-100 (5 µM), Sigma-1R antagonists (right). ( B ) Comparison of the amplitude of the increase in [Ca 2+ ] i (average + SD) in each condition. Inhibition of PLD or antagonism of Sig-1R reduces the Ca 2+ response elicited by OXA (17-33) (10 nM); p < 0.05 as compared to amplitude of [Ca 2+ ] i increase produced by OXA (17-33) (*) or produced in the presence of the inhibitors ( # ) ( n = 6 neurons/condition).

Article Snippet: VU0155056, a PLD inhibitor [ ], was purchased from Avanti Polar Lipids (Alabaster, AL, USA).

Techniques: Comparison, Inhibition, Produced

Journal: JCI insight

Article Title: Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

doi: 10.1172/jci.insight.165841

Figure Lengend Snippet: Figure 3. Inhibition of AEP blunts cholestanol-induced α-syn aggregation in vitro. α-Syn–HEK293 cells were pretreated with CP11 (2 μM), followed by transduction with α-syn PFFs (1 μg/mL) and cholestanol (5 μM). (A) AEP activity assay (n = 7 independent experiments). (B) Colocalization of pS129 and α-syn aggregates. The histogram shows the percentage of cells with aggregated α-syn (n = 5 independent experiments). Scale bars: 20 μm. (C) Western blot analysis of AEP-generated α-syn N103 fragment, p-α-syn, total α-syn, Bcl-2, and Bax. (D) Quantitative analysis of protein expression levels (n = 5). (E) CP11 attenuates the phosphorylation of α-syn in primary neurons. The histogram shows the ratio of the pS129 area to the MAP2 area (n = 5 independent experi- ments). Scale bar: 20 μm. Data are presented as mean ± SD. **P < 0.01, ***P < 0.005, ****P < 0.001; Student’s t test. AFU, arbitrary fluorescence unit.

Article Snippet: 1 1 R E S E A R C H A R T I C L E JCI Insight 2023;8(21):e165841 https://doi.org/10.1172/jci.insight.165841 catalog DBI-2043); DAPI (BioFroxx); CP11 (Santa Cruz Biotechnology, catalog sc-319780); AEP substrate Z-Ala-Ala-Asn-AMC (Bachem); NP-40 lysis buffer (Beyotime, catalog P0013F); Nissl Stain Kit (methyl violet method) (Solarbio, catalog G1432), RIPA lysis buffer (Fdbio Science, catalog FD009), DMSO (Sigma, catalog D5879), and Triton X-100 (Sigma-Aldrich, catalog T8787).

Techniques: Inhibition, In Vitro, Transduction, Activity Assay, Western Blot, Generated, Expressing, Phospho-proteomics, Fluorescence

Journal: JCI insight

Article Title: Cholestanol accelerates α-synuclein aggregation and spreading by activating asparagine endopeptidase.

doi: 10.1172/jci.insight.165841

Figure Lengend Snippet: Figure 5. CP11 ameliorates cholestanol-induced α-syn pathology and motor deficits. The mice were injected with CP11 i.p. or vehicle control, fed a cholestanol diet, and then were stereotaxically injected with α-syn PFFs or PBS. (A) Rotarod test (n = 6). (B) Pole test (n = 6). (C) Beam-walking test (n = 6). (D) Representative pS129 immunostaining in the cortex, striatum, substantia nigra, and hippocampus of mice sacrificed 6 months after intrastriatal α-syn PFF or PBS injection. The histogram shows the percentage of neurons containing α-syn aggregates (n = 4 mice/group). Scale bar: 20 μm. (E) Double immunostaining of pS129 and TH in the substantia nigra. The histogram shows the ratio of the pS129 area to the TH area (n = 4 mice/group). Scale bar: 20 μm. (F) Western blot analysis of p-α-syn, total α-syn, TH, and DAT (n = 3 mice/group). (G and H) Representative images of TH-positive fibers in the striatum. The histogram shows the density of TH-positive fibers in the striatum (n = 5 mice per group). Scale bar: 200 μm. (I and J) Representative images of TH-positive neurons in the SNpc. The histogram shows the number of TH-positive neurons in the SNpc (n = 4 mice/ group). Scale bar: 100 μm. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001; compared by 1-way ANOVA with Tukey’s multiple comparison test. Ctx, cortex; ST, striatum; SN, substantia nigra; Hip, hippocampus.

Article Snippet: 1 1 R E S E A R C H A R T I C L E JCI Insight 2023;8(21):e165841 https://doi.org/10.1172/jci.insight.165841 catalog DBI-2043); DAPI (BioFroxx); CP11 (Santa Cruz Biotechnology, catalog sc-319780); AEP substrate Z-Ala-Ala-Asn-AMC (Bachem); NP-40 lysis buffer (Beyotime, catalog P0013F); Nissl Stain Kit (methyl violet method) (Solarbio, catalog G1432), RIPA lysis buffer (Fdbio Science, catalog FD009), DMSO (Sigma, catalog D5879), and Triton X-100 (Sigma-Aldrich, catalog T8787).

Techniques: Injection, Control, Immunostaining, Double Immunostaining, Western Blot, Comparison