Abstract
Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that mediates the retrograde transport of cargo proteins from endosomes to the trans-Golgi network. Mutations such as D620N in the VPS35 gene have been identified in patients with autosomal dominant Parkinson’s disease (PD). However, it remains poorly understood whether and how VPS35 deficiency or mutation contributes to PD pathogenesis; specifically, the studies that have examined VPS35 thus far have differed in results and methodologies. We generated a VPS35 D620N mouse model using a Rosa26-based transgene expression platform to allow expression in a spatial manner, so as to better address these discrepancies. Here, aged (20-months-old) mice were first subjected to behavioral tests. Subsequently, DAB staining analysis of substantia nigra (SN) dopaminergic neurons with the marker for tyrosine hydroxylase (TH) was performed. Next, HPLC was used to determine dopamine levels, along with levels of its two metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in the striatum. Western blotting was also performed to study the levels of key proteins associated with PD. Lastly, autoradiography (ARG) evaluation of [3H]FE-PE2I binding to the striatal dopamine transporter DAT was carried out. We found that VPS35 D620N Tg mice displayed a significantly higher dopamine level than NTg counterparts. All results were then compared with that of current VPS35 studies to shed light on the disease pathogenesis. Our model allows future studies to explicitly control spatial expression of the transgene which would generate a more reliable PD phenotype.
Highlights
Parkinson’s disease (PD) is one of the most common neurodegenerative disorders globally, with the prevalence of PD being reported to be approximately 1% in people 60 years old and older, and increasing to 1–3% in those80 years old and older [1]
Ishizu’s group first investigated the Vacuolar protein sorting 35 (VPS35) D620N gene in vivo using D620N knock-in (KI) mice, and they found neither homozygous nor heterozygous VPS35 D620N KI mice had suffered premature death or had developed clear signs of neurodegeneration at up to 15 months of age [14]. These results suggest that the VPS35 D620N allele is still functional and does not cause obvious dopamine neuron loss; they suggest that the VPS35 D620N allele is a partial loss-of-function allele, and genetic predisposition and age-related alterations in the nigrostriatal dopamine system cooperatively influence the pathogenesis of VPS35
Generation of VPS35 D620N transgenic mice The generation of this VPS35 D620N mouse model started with the microinjection of the entire VPS35D620N construct into pronuclei fertilized oocytes of pseudopregnant C57BL/6j females
Summary
Parkinson’s disease (PD) is one of the most common neurodegenerative disorders globally, with the prevalence of PD being reported to be approximately 1% in people 60 years old and older, and increasing to 1–3% in those. 80 years old and older [1]. PD is largely an idiopathic disease, approximately 5 to 10% of cases are hereditary, with mutations in at least 13 genes being shown to cause familial PD; one of these is the vacuolar protein sorting 35 (VPS35, PARK17) gene [4, 5]. The VPS35 gene encodes a key component of the retromer complex, which operates by packaging particular endosomal cargoes into tubules and vesicles and transporting these cargoes either to the trans-Golgi network or to the plasma membrane [8]. The retromer consists of two subprotein complexes: the cargo-selective complex, which contains a VPS35-VPS29-VPS26 trimer [9], and the membrane deformation complex, which consists of a sorting nexin dimer [10]
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