Journal: Genes & Diseases

Article Title: TFE3-mediated neuroprotection: Clearance of aggregated α-synuclein and accumulated mitochondria in the AAV-α-synuclein model of Parkinson's disease

doi: 10.1016/j.gendis.2024.101429

Figure Lengend Snippet: TFE3 overexpression transcriptionally up-regulates Parkin, promoting the removal of accumulated mitochondria in the AAV-α-Syn model. (A, C) Immunofluorescence (A) and Western blot (C) analysis for Parkin in dopaminergic neurons of the SN or ventral midbrain homogenates from mice injected with AAV-EGFP and AAV-TFE3. Immunofluorescence: n = 6 mice per group. Scale bars, 50 μm. (B) Quantitative analysis of the fluorescence results shown in (A). (D) Quantification of Western blot bands corresponding to Parkin normalized to β-actin. n = 6 mice per group. (E) Quantitative reverse-transcription PCR analysis of Prkn mRNA in ventral midbrain from mice injected with AAV-EGFP and AAV-TFE3. n = 4 mice per group. The data were presented as mean ± standard error of the mean. Statistical significance was determined using a two-tailed student's t -test. ∗ P < 0.05, ∗∗∗∗ P < 0.0001. (F) Western blot analysis for Parkin expression in ventral midbrain homogenates from mice injected with AAV-Flag, AAV-α-Syn, and AAV-α-Syn/TFE3. (G) Quantification of Western blot bands corresponding to Parkin normalized to β-actin. n = 4 mice per group. The data were presented as mean ± standard error of the mean. Statistical significance was determined using one-way analysis of ANOVA followed by Tukey's multiple comparisons test. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (H, I) Immunofluorescence analysis for Tom20 (H) and VDAC1 (I) in dopaminergic neurons of the SN from mice injected with AAV-Flag, AAV-α-Syn, and AAV-α-Syn/TFE3. n = 3–5 mice per group. Scale bars, 10 μm. (J, K) Immunofluorescence analysis for Tom20 (J) and VDAC1 (K) in dopaminergic neurons of the SN from mice injected with AAV-Flag, AAV-α-Syn, and AAV-α-Syn/Parkin. n = 4 mice per group. Scale bars, 10 μm. TFE3, transcription factor binding to IGHM enhancer 3; α-Syn, α-synuclein; AAV, adeno-associated virus; SN, substantia nigra; Parkin, Parkin RBR E3 ubiquitin protein ligase; Tom22, outer mitochondrial membrane protein; VDAC1, voltage-dependent anion channel 1.

Article Snippet: Free-floating 20 μm-thick sections were rinsed in PBS and then incubated in a blocking solution at room temperature for 1 h. Primary antibodies, including TH (1:1,000, # ab76442, Abcam, Cambridge, UK), TFE3 (1:500, #ab93808, Abcam), α-Syn (1:500, #ab138501, Abcam), p-α-Syn Ser129 (1:500, #ab51253, Abcam), Lamp1 (lysosomal associated membrane protein 1; 1:500, #1D4B–C, DSHB), p62 (1:1000, # 18420 -1-AP, Proteintech, Illinois, USA), LC3 (microtubule-associated protein light chain 3; 1:100, #2775, Cell Signaling Technology), Parkin (1:100, #2132, Cell Signaling Technology), Tom20 (outer mitochondrial membrane protein; 1:500, # 11802-1-AP, Proteintech), VDAC1 (voltage-dependent anion channel 1; 1:300, # 55259-1-AP, Proteintech), PGC1-α (1:200, # 66369-1-Ig, Proteintech), and TFAM (1:200, # 22586-1-AP, Proteintech) were diluted in 1% bovine serum albumin in 1× TBST (0.3% Triton X-100) and applied to the sections overnight at 4 °C.

Techniques: Over Expression, Immunofluorescence, Western Blot, Injection, Fluorescence, Reverse Transcription, Two Tailed Test, Expressing, Binding Assay, Virus, Ubiquitin Proteomics, Membrane