Asis [5]. The only ubiquitination target of MGRN1 identified to date is tumor susceptibility gene 101 (TSG101), a component of the endocytic trafficking machinery that sorts membrane proteins into multivesicular bodies [6,7]. Loss of MGRN1-dependent ubiquitination disrupts endo-lysosomal trafficking, leading to accumulation of activated epidermal growth factor receptor (EGFR) and alterations in the morphology of early endosomes, late endosomes and lysosomes. PrP is normally secreted and tethered to the plasma membrane by a GPI anchor, but ER stress and some pathogenic mutations in PRNP can induce AZP-531 mislocalization of PrP to the cytosol and induce non-transmissible neurotoxicity [8]. A recent study demonstrated that cytosolically exposed forms of PrP can bind to and sequester MGRN1 in HeLa cells [9], resulting in similar abnormalities in endo-lysosomal trafficking to those observed in cells in which Mgrn1 was knocked down by siRNA. Over-expressing MGRN1 rescued the trafficking defects. Reduced immunostaining for MGRN1 was observed in the brains of MedChemExpress AZP-531 transgenic mice expressing a transmembrane form of PrP, along with an age-dependent increase in lysosome size/number (based on Cathepsin D staining) in Purkinje cells. These data suggested that disrupted MGRN1dependent endo-lysosomal trafficking could be the cellularMGRN1 Levels Do Not Influence Prion DiseaseTable 1. Brain Mgrn1 expression.MiceMgrn1md-nc (null) mutant mice and Tg(Mgrn1I)C3Tmg transgenic (hereafter referred to as Tg+) mice, which express wild-type Mgrn1 isoform I from the human ?actin promoter, were described previously [4,13]. The Tg(Mgrn1I)C3Tmg transgenic line completely rescue all aspects of the Mgrn1 null mutant phenotype, including spongiform degeneration of the CNS. Mgrn1 null mutant (Mgrn1md2nc) mice are maintained by breeding heterozygotes with their homozygous mutant siblings. A wild-type control line was established by inbreeding +/+ animals that were generated by intercrossing Mgrn1md2nc/+ mice; this line is re-generated from Mgrn1 heterozygotes every 3 years. Tg+; Mgrn1 null mutant mice were backcrossed to wild-type mice to generate Tg+ and Tg2 Mgrn1md2nc/+ and wild-type (Mgrn1+/+) mice. Mice were genotyped for the Mgrn1md2nc mutation and the transgene as previously described [13].Genotype Tg2; Mgrn1md2nc/+ Tg2; Mgrn1+/+ Tg+; Mgrn1md2nc/+ Tg+; Mgrn1+/+aMgrn1 relative quantification value (range)0.42 (0.22?.79) 1.00 (0.82?. 21) 1.41 (0.76?.61)b 4.43 (1.59?2.32)p valuea 0.04 n/a 0.21 0.Student’s t-test against Tg2; Mgrn1+/+ value, p,0.05 significant. Student’s t-test against Tg2; Mgrn1mdnc/+ yields p = 0.04. doi:10.1371/journal.pone.0055575.tbmechanism underlying spongiform neurodegeneration in prion diseses. Cytosolically-exposed PrP has been proposed to contribute to the pathogenesis of inherited and transmissible spongiform encephalopathies [8]. Mutations in the hydrophobic domain of the prion gene that lead to increased production of a transmembrane form with its N-terminal domain exposed to the cytosol cause neurodegeneration with pathology reminiscent of prion disease in transgenic mice [10]. Similar transmembrane forms of prion protein have been detected in both genetic and transmitted prion diseases [10?2]. The relationship between cytosolic PrP and CNS vacuolation is unclear. We tested whether functional sequestration of MGRN1 by cytosolic PrP contributes to transmissible prion disease 1407003 by inoculating mice expressing reduced or elevated levels of Mgrn1 with Rock.Asis [5]. The only ubiquitination target of MGRN1 identified to date is tumor susceptibility gene 101 (TSG101), a component of the endocytic trafficking machinery that sorts membrane proteins into multivesicular bodies [6,7]. Loss of MGRN1-dependent ubiquitination disrupts endo-lysosomal trafficking, leading to accumulation of activated epidermal growth factor receptor (EGFR) and alterations in the morphology of early endosomes, late endosomes and lysosomes. PrP is normally secreted and tethered to the plasma membrane by a GPI anchor, but ER stress and some pathogenic mutations in PRNP can induce mislocalization of PrP to the cytosol and induce non-transmissible neurotoxicity [8]. A recent study demonstrated that cytosolically exposed forms of PrP can bind to and sequester MGRN1 in HeLa cells [9], resulting in similar abnormalities in endo-lysosomal trafficking to those observed in cells in which Mgrn1 was knocked down by siRNA. Over-expressing MGRN1 rescued the trafficking defects. Reduced immunostaining for MGRN1 was observed in the brains of transgenic mice expressing a transmembrane form of PrP, along with an age-dependent increase in lysosome size/number (based on Cathepsin D staining) in Purkinje cells. These data suggested that disrupted MGRN1dependent endo-lysosomal trafficking could be the cellularMGRN1 Levels Do Not Influence Prion DiseaseTable 1. Brain Mgrn1 expression.MiceMgrn1md-nc (null) mutant mice and Tg(Mgrn1I)C3Tmg transgenic (hereafter referred to as Tg+) mice, which express wild-type Mgrn1 isoform I from the human ?actin promoter, were described previously [4,13]. The Tg(Mgrn1I)C3Tmg transgenic line completely rescue all aspects of the Mgrn1 null mutant phenotype, including spongiform degeneration of the CNS. Mgrn1 null mutant (Mgrn1md2nc) mice are maintained by breeding heterozygotes with their homozygous mutant siblings. A wild-type control line was established by inbreeding +/+ animals that were generated by intercrossing Mgrn1md2nc/+ mice; this line is re-generated from Mgrn1 heterozygotes every 3 years. Tg+; Mgrn1 null mutant mice were backcrossed to wild-type mice to generate Tg+ and Tg2 Mgrn1md2nc/+ and wild-type (Mgrn1+/+) mice. Mice were genotyped for the Mgrn1md2nc mutation and the transgene as previously described [13].Genotype Tg2; Mgrn1md2nc/+ Tg2; Mgrn1+/+ Tg+; Mgrn1md2nc/+ Tg+; Mgrn1+/+aMgrn1 relative quantification value (range)0.42 (0.22?.79) 1.00 (0.82?. 21) 1.41 (0.76?.61)b 4.43 (1.59?2.32)p valuea 0.04 n/a 0.21 0.Student’s t-test against Tg2; Mgrn1+/+ value, p,0.05 significant. Student’s t-test against Tg2; Mgrn1mdnc/+ yields p = 0.04. doi:10.1371/journal.pone.0055575.tbmechanism underlying spongiform neurodegeneration in prion diseses. Cytosolically-exposed PrP has been proposed to contribute to the pathogenesis of inherited and transmissible spongiform encephalopathies [8]. Mutations in the hydrophobic domain of the prion gene that lead to increased production of a transmembrane form with its N-terminal domain exposed to the cytosol cause neurodegeneration with pathology reminiscent of prion disease in transgenic mice [10]. Similar transmembrane forms of prion protein have been detected in both genetic and transmitted prion diseases [10?2]. The relationship between cytosolic PrP and CNS vacuolation is unclear. We tested whether functional sequestration of MGRN1 by cytosolic PrP contributes to transmissible prion disease 1407003 by inoculating mice expressing reduced or elevated levels of Mgrn1 with Rock.
