Ebellum; Wm: white matter of cerebellum; Bst: brainstem. Severity 15900046 scores: NSL (no significant lesions), average Title Loaded From File vacuolation score ,5; Mild, average vacuolation score 5?9; Mod (moderate), average vacuolation score 20?9; na: brain region not examined. doi:10.1371/journal.pone.0055575.tMGRN1 Levels Do Not Influence Prion Diseaseloss of MGRN1-dependent cellular processes is not the underlying cause of spongiform encephalopathy caused by RML prions. Furthermore, our data suggest that either cytosolic PrP is not produced in this disease model or it does not play a significant role in the pathogenesis of transmissible prion diseases. We cannot, however, rule out the possibility that functional sequestration of MGRN1 may contribute to the neurotoxicity associated with cytosolic PrP. Our results are consistent with other studies that have suggested cytosolic PrP does not make a significant contribution to prion disease, particularly the pathogenesis of CNS vacuolation. For example, transgenic mice expressing cytosolic PrP did not develop spongiform change, even when the transgene was expressed on 18325633 a Prnp null background and the mice were inoculated with RML prions, nor did the inoculated mice get sick or accumulate Title Loaded From File protease-resistant PrP [16]. Spongiform change was not observed a patient carrying a truncation mutation in PRNP (Q160X) that has been shown to lead to significant cytoplasmic retention of PrP [17,18]. In several studies, accumulation of cytosolic PrP was shown to not be toxic to human or mouse neurons in primary culture or to N2a cells [19?1], and in two of those studies, the presence of cytosolic PrP was in fact associated with protection against apoptosis. Together, these data suggest that the presence of cytosolic prion protein is not sufficient to cause prion disease and is not functionally equivalent to loss of MGRN1. An alternative mechanism for vacuolar degeneration of neurons and their synapses is the accumulation of PrPSc in neuronal cellular membranes. At least 80 of PrPSc formed accumulates in the neuronal plasma membranes, especially in synaptic regions [22], and most vacuoles occur in pre- and post-synaptic structures [23]. In experimental scrapie and sporadic CJD, PrPSc accumulation and vacuolation begin focally in the brain and progress by axonal transport of PrPSc to different regions of the central nervoussystem [22]. The brain regions affected in the terminal stages of prion disease are determined by the strain of prions (PrPSc) [24]. Neuronal dysfunction and morphological changes (vacuolation) appear to be caused directly by accumulation of PrPSc in plasma membranes [22,25] and are related to the great effect of PrPSc has on membrane functions [26?8]. Dendritic degeneration, which is an additional abnormal step in synapse pathobiology, is caused specifically by PrPSc activation of Notch-1 signaling in the neuronal plasma cell membrane [29,30]. Therefore the effects of PrPSc on membrane pathobiology cannot be ignored. PrPSc accumulates to a lesser degree by endocytosis into lysosomes and by phagocytosis into autophagosomes that release PrPSc into the extracellular space [25], and ingestion of PrPSc by activated microglia causes release of cytokines from microglia that cause nerve cell death [31]. The similar disease progression of Mgrn1 null mutant mice, transgenic mice that over-express Mgrn1, and controls inoculated with RML prions indicates that MGRN1-dependent processes are not necessary for the pathogenesis of.Ebellum; Wm: white matter of cerebellum; Bst: brainstem. Severity 15900046 scores: NSL (no significant lesions), average vacuolation score ,5; Mild, average vacuolation score 5?9; Mod (moderate), average vacuolation score 20?9; na: brain region not examined. doi:10.1371/journal.pone.0055575.tMGRN1 Levels Do Not Influence Prion Diseaseloss of MGRN1-dependent cellular processes is not the underlying cause of spongiform encephalopathy caused by RML prions. Furthermore, our data suggest that either cytosolic PrP is not produced in this disease model or it does not play a significant role in the pathogenesis of transmissible prion diseases. We cannot, however, rule out the possibility that functional sequestration of MGRN1 may contribute to the neurotoxicity associated with cytosolic PrP. Our results are consistent with other studies that have suggested cytosolic PrP does not make a significant contribution to prion disease, particularly the pathogenesis of CNS vacuolation. For example, transgenic mice expressing cytosolic PrP did not develop spongiform change, even when the transgene was expressed on 18325633 a Prnp null background and the mice were inoculated with RML prions, nor did the inoculated mice get sick or accumulate protease-resistant PrP [16]. Spongiform change was not observed a patient carrying a truncation mutation in PRNP (Q160X) that has been shown to lead to significant cytoplasmic retention of PrP [17,18]. In several studies, accumulation of cytosolic PrP was shown to not be toxic to human or mouse neurons in primary culture or to N2a cells [19?1], and in two of those studies, the presence of cytosolic PrP was in fact associated with protection against apoptosis. Together, these data suggest that the presence of cytosolic prion protein is not sufficient to cause prion disease and is not functionally equivalent to loss of MGRN1. An alternative mechanism for vacuolar degeneration of neurons and their synapses is the accumulation of PrPSc in neuronal cellular membranes. At least 80 of PrPSc formed accumulates in the neuronal plasma membranes, especially in synaptic regions [22], and most vacuoles occur in pre- and post-synaptic structures [23]. In experimental scrapie and sporadic CJD, PrPSc accumulation and vacuolation begin focally in the brain and progress by axonal transport of PrPSc to different regions of the central nervoussystem [22]. The brain regions affected in the terminal stages of prion disease are determined by the strain of prions (PrPSc) [24]. Neuronal dysfunction and morphological changes (vacuolation) appear to be caused directly by accumulation of PrPSc in plasma membranes [22,25] and are related to the great effect of PrPSc has on membrane functions [26?8]. Dendritic degeneration, which is an additional abnormal step in synapse pathobiology, is caused specifically by PrPSc activation of Notch-1 signaling in the neuronal plasma cell membrane [29,30]. Therefore the effects of PrPSc on membrane pathobiology cannot be ignored. PrPSc accumulates to a lesser degree by endocytosis into lysosomes and by phagocytosis into autophagosomes that release PrPSc into the extracellular space [25], and ingestion of PrPSc by activated microglia causes release of cytokines from microglia that cause nerve cell death [31]. The similar disease progression of Mgrn1 null mutant mice, transgenic mice that over-express Mgrn1, and controls inoculated with RML prions indicates that MGRN1-dependent processes are not necessary for the pathogenesis of.