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. 2021 Feb 3;29(5):1862–1882. doi: 10.1016/j.ymthe.2021.01.033

Figure 6.

Figure 6

UPRplus protects dopaminergic neurons in a preclinical model of PD

(A) Experimental design to evaluate the effects of UPRplus in a pharmacological PD model. Animals were injected with AAV particles expressing UPRplus or control vectors into the substantia nigra pars compacta (SNpc) using brain stereotaxis. Two weeks later, animals were exposed to 6-OHDA into the striatum followed by behavioral and histological analyses 1 week later. (B) The expression of UPRplus was monitored in the brain using immunohistochemistry with an anti-HA antibody (scale bars, 200 μm). (C) Immunofluorescence analysis of tyrosine hydroxylase (TH; red) and BiP (green) was performed in brain tissue derived from AAV-mock-injected (upper) or AAV-UPRplus-injected (bottom) animals. Hoechst staining of the nucleus was also performed. The third panel shows merged images of the three stainings (scale bars, 200 μm). Right panel shows high magnification of the white square region of merged images. (D) Three-month-old WT mice were injected with AAV-UPRplus, AAV-ATF6f, AAV-XBP1s, or AAV-mock (control) into the SNpc and then exposed to 6-OHDA as described in (A). Quantification of the percentage of contralateral touches relative to total touches (both forepaws) obtained before and 1 week after the injection of 6-OHDA (before and after 6OHDA) is indicated. Data represent the mean and standard error of six to eight animals per group. (E) Immunohistochemistry analysis was performed in striatal sections from animals described in (D) to quantify 6-OHDA-induced denervation in both injected (6-OHDA) and non-injected (control) hemispheres (scale bar, 1 mm) (left panel). The integrated density of pixel intensity was calculated from images of anti-TH immunohistochemistry covering the entire striatum and expressed as the percentage of TH loss relative to the control side. Data represent the mean and standard error from six to eight animals per group (right panel). (F) Immunohistochemistry analysis was performed in midbrain sections from mice described in (D) to quantify 6-OHDA-induced dopaminergic neuronal loss in both injected (6-OHDA) and non-injected (control) sides (scale bar, 1 mm) (left panel). The total content of TH-positive somas was measured in midbrain sections covering the entire SNpc, in the non-injected (control) and injected (6-OHDA) side, for each group. Data represent the mean and standard error from 6 to 10 animals per group (right panel). (G) The expression of BiP was monitored in the brain obtained from AAV-UPRplus-, AAV-ATF6f-, AAV-XBP1s-, or AAV-mock-injected animals for 6 months (upper panel) or 1 year (bottom panel) using immunohistochemistry with an anti-BiP antibody. (scale bars, 100 μm). (H) Experimental design to evaluate the effects of UPRplus in an idiopathic PD model. Animals were injected with PFF α-synuclein into the striatum using brain stereotaxis. 1.5 months later, animals were injected with UPRplus or control vectors into the SNpc followed by histological analysis 1.5 months later. (I) Transmission electronic microscopy of sonicated α-synuclein-aggregated fibrils (scale bar, 100 nm). (J) Immunohistochemical analysis of the phosphorylated α-synuclein (p-α-syn) levels in the SNpc region (scale bars, 100 μm) (left panel). Quantification of the p-α-syn levels (integrated density) covering SNpc region. Values are expressed as the average and standard error. Mock, n = 9; UPRplus, n = 9; XBP1s, n = 9; ATF6f, n = 6 (right panel). Statistical analysis was performed using Dunnett’s multiple comparison test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.