Cytosolic prion protein in neurons

Localizing the cellular prion protein (PrPC) in the brain is necessary for understanding the pathogenesis of prion diseases. However, the precise ultrastructural localization of PrPC still remains enigmatic. We performed the first quantitative study of the ultrastructural localization of PrPC in the mouse hippocampus using high-resolution cryoimmunogold electron microscopy. PrPC follows the standard biosynthetic trafficking pathway with a preferential localization in late endosomal compartments and on the plasma membrane of neurons and neuronal processes. PrPC is found with the same frequency within the synaptic specialization and perisynaptically, but is almost completely excluded from synaptic vesicles. Unexpectedly, PrP is also found in the cytosol in subpopulations of neurons in the hippocampus, neocortex, and thalamus but not the cerebellum. Cytosolic PrP may have altered susceptibility to aggregation, suggesting that these neurons might play a significant role in the pathogenesis of prion diseases, in particular those mammals harboring mutant PrP genes

Notch-1 activation and dendritic atrophy in prion disease

In addition to neuronal vacuolation and astrocytic hypertrophy, dendritic atrophy is a prominent feature of prion disease. Because increased Notch-1 expression and cleavage releasing its intracellular domain (NICD) inhibit both dendrite growth and maturation, we measured their levels in brains from mice inoculated with Rocky Mountain Laboratory (RML) prions. The level of NICD was elevated in the neocortex, whereas the level of β-catenin, which stimulates dendritic growth, was unchanged. During the incubation period, levels of the disease-causing prion protein isoform, PrP(Sc), and NICD increased concomitantly in the neocortex. Additionally, increased levels of Notch-1 mRNA and translocation of NICD to the nucleus correlated well with regressive dendritic changes. In scrapie-infected neuroblastoma (ScN2a) cells, the level of NICD was elevated compared with uninfected control (N2a) cells. Long neurofilament protein-containing processes extended from the surface of N2a cells, whereas ScN2a cells had substantially shorter processes. Transfection of ScN2a cells with a Notch-1 small interfering RNA decreased Notch-1 mRNA levels, diminished NICD concentrations, and rescued the long process phenotype. These results suggest that PrP(Sc) in neurons and in ScN2a cells activates Notch-1 cleavage, resulting in atrophy of dendrites in the CNS and shrinkage of processes on the surface of cultured cells. Whether diminishing Notch-1 activation in vivo can prevent or even reverse neurodegeneration in prion disease remains to be established

Convection-Enhanced Delivery of AAV2-PrPshRNA in Prion-Infected Mice

Prion disease is caused by a single pathogenic protein (PrPSc), an abnormal conformer of the normal cellular prion protein PrPC. Depletion of PrPC in prion knockout mice makes them resistant to prion disease. Thus, gene silencing of the Prnp gene is a promising effective therapeutic approach. Here, we examined adeno-associated virus vector type 2 encoding a short hairpin RNA targeting Prnp mRNA (AAV2-PrP-shRNA) to suppress PrPC expression both in vitro and in vivo. AAV2-PrP-shRNA treatment suppressed PrP levels and prevented dendritic degeneration in RML-infected brain aggregate cultures. Infusion of AAV2-PrP-shRNA-eGFP into the thalamus of CD-1 mice showed that eGFP was transported to the cerebral cortex via anterograde transport and the overall PrPC levels were reduced by ∼ 70% within 4 weeks. For therapeutic purposes, we treated RML-infected CD-1 mice with AAV2-PrP-shRNA beginning at 50 days post inoculation. Although AAV2-PrP-shRNA focally suppressed PrPSc formation in the thalamic infusion site by ∼ 75%, it did not suppress PrPSc formation efficiently in other regions of the brain. Survival of mice was not extended compared to the untreated controls. Global suppression of PrPC in the brain is required for successful therapy of prion diseases