Fig. 2

Characterization of 20-months-old VPS35 D620N Tg mice. a VPS35 D620N is highly expressed in the CNS. Western blot analysis showed VPS35 D620N is abundantly expressed in the brain, spinal cord and, to a lesser extent, in the kidney, lung and stomach. No expression is observed in the liver or spleen. β-actin is included as a loading control. HA is used to detect VPS35 D620N. b Quantification of HA (VPS35 D620N). Expression levels are relative to brain. c VPS35 D620N expression in the various brain regions. From the western blot analysis, VPS35 D620N is determined to be expressed in the olfactory bulb, cerebellum, brainstem, midbrain, striatum, hippocampus, and cortex of Tg mice, while no expression is observed in NTg mouse brains. β-tubulin is included as a loading control. d Quantification of HA (VPS35 D620N). Expression levels are relative to cortex (most abundant). e Immunofluorescence analysis of NTg and Tg mouse brains. Red staining indicates HA (VPS35 D620N), green indicates NeuN, and blue indicates DAPI. VPS35 D620N is abundantly found in Tg mouse brains, specifically in the olfactory bulb, striatum, cortex, hippocampus, cerebellum, and brainstem, but is largely absent in NTg mouse brains. ×4 magnification. Scale bar 1 mm. f IHC analysis of TH and HA (VPS35 D620N) in dopaminergic neurons from SN. Green staining indicates TH, blue indicates DAPI, and red indicates HA (VPS35 D620N). There is an absence of HA (VPS35 D620N) in NTg mice, while TH and HA (VPS35 D620N) are colocalized in dopaminergic neurons from Tg mice. Column 1: ×60 magnification. Scale bar 20 μm. Columns 2–5: ×60 magnification. ×5 zoom. Scale bar 5 μm. g Evaluation of transgenic mutant VPS35 D620N and endogenous VPS35. QPCR analysis. VPS35 mRNA was extracted from the striatum, hippocampus and cortex of 20-months-old NTg and Tg mice, and quantified (normalized against NTg group). There was a significant increase in VPS35 mRNA in the Tg mice (n = 3) than in age-matched NTg counterparts (n = 3), across the striatum (P = 0.0067), hippocampus (P = 0.00029) and cortex (P = 0.00054); **p < 0.01, ***p < 0.001, Student’s t-test. h Western blot analysis. VPS35 protein from the striatum, hippocampus and cortex of 20-months-old Tg mice was analyzed. There were two VPS35 bands present for all Tg mice: endogenous VPS35 (lower band) and transgenic mutant VPS35 D620N (upper band). There was a significant increase in transgenic mutant VPS35 D620N compared to endogenous VPS35 across the striatum (P = 0.0014), hippocampus (P = 0.0012) and cortex (P = 0.0031); n = 6. **p < 0.01, Student’s t-test