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. Author manuscript; available in PMC: 2015 Sep 10.
Published in final edited form as: Cell Rep. 2015 Aug 28;12(10):1631–1643. doi: 10.1016/j.celrep.2015.08.001

Figure 7. Restoration of MFN2-reduction and DA neuron loss by suppressing MUL1 expression in VPS35+/− SNpc.

Figure 7

(A) Schematic of AAV injection strategy. AAV5-shR-MUL1-GFP were stereotactically injected into VPS35+/+ or Vps+/− mice brains at 6-M old of age. Two months post injection, coronal brain sections, ventral midbrain or STR homogenates were collected and subjected to immunohistochemical staining, Western blot and HPLC analysis. (B) AVV5 infect the DA neurons in coronal brain sections. Contralateral (CON) and ipsilateral (shR-MUL1) substantia nigra (SNpc) are indicated (Scale bar = 1mm). Right insets show magnified images of the marked squares (Scale bar = 50μm). (C) Representative Western blots of homogenates of ventral midbrain derived from the virus ipsilateral (shR-MUL1) and contralateral (CON) hemispheres. (D) Quantification of data from C. Relative MUL1 and MFN2 protein levels were normalized by un-incetion side of VPS35+/+. Means ± SEM (n = 3) were shown. *p < 0.05. (E–F) Stereological analysis of TH-positive dopaminergic neurons in the SNpc. E, Representative images of TH immunostaining analysis in the virus injected SNpc (Scale bar = 200 μm). Insets show magnified images (Scale bar = 10 μm) and arrows indicate axon swelling. Quantification analysis of TH+ or NeurN+ cells in each hemisphere (mean ± SEM, n = 3) were presented in F. *p < 0.05 by one-way ANOVA with post hoc analysis. (G–H) Distribution of TH immunoreactive fibers in the STR. G, Representative images of TH staining analysis in adjacent sections of the injected STR (Scale bar = 400 μm). Insets show magnified images (Scale bar = 10 μm). G, Quantification of TH-immunoreactive intensity in the STR. Data are expressed as the percentage of the TH intensity of VPS35+/+ CON side. Mean ± SEM, n = 3. *p < 0.05 by one-way ANOVA with post hoc analysis. (I) Dopamine levels in the VPS35+/+ and VPS35+/− STR were measured by HPLC analysis. Isoproterenol was added as internal control, n=3, *p < 0.05 by one-way ANOVA with post hoc analysis.