, 2006, Cunningham et al., 2003 and Mallucci et al., 2007). Our results indicate that defective glutamate release in the cerebellum of Tg(PG14) mice is due to inefficient VGCC function in CGNs, and that mutant PrP is directly responsible. Lentivirus-mediated knockdown of PG14 PrP restored the depolarization-evoked calcium rise, and transfection

of a plasmid encoding PG14 PrP impaired the calcium response in DAPT molecular weight wild-type neurons. The latter observation indicates that PG14 PrP alters calcium dynamics with a dominant effect over wild-type PrP, consistent with a gain-of-toxicity mechanism. However, a loss of a putative PrP function in governing VGCC activity (see below) may also be involved. Previous analyses suggested that accumulation of mutant PrP in the secretory pathway might be critical in neuronal find more dysfunction, possibly due to interference with transport and delivery of essential cargo molecules to synapses (Dossena et al., 2008, Massignan et al., 2010 and Medrano et al., 2008). Here, we found that intracellular retention of mutant PrP was required for perturbing neuronal calcium

dynamics, and identified the α2δ-1 subunit of VGCCs as a target molecule. We documented a physical interaction between PrP and α2δ-1 by coimmunoprecipitation, and the two proteins colocalized in transfected cells, consistent with analysis of the native PrP interactome, which identified α2δ subunits

as candidate PrP interactors (Rutishauser et al., 2009). The α2δ subunits play a vital role in intracellular trafficking of the pore-forming CaVα1 subunits of the CaV1 and CaV2 classes, and boost calcium current amplitude by increasing the number of channels on the cell surface (Cantí et al., 2005). α2δ interacts with CaVα1 during biosynthetic maturation, and promotes the transport of the heteromeric channel complex to correct presynaptic sites (Bauer et al., 2010, Cantí et al., 2005, Hendrich et al., Tryptophan synthase 2008 and Saheki and Bargmann, 2009). We found that α2δ-1 and CaVα1A were weakly expressed on the cell surface and localized mainly in the ER and Golgi in mutant PrP-expressing cells, suggesting impaired secretory transport. We also found smaller amounts of α2δ-1 and CaVα1A in cerebellar synaptosomal fractions of Tg(PG14) mice, and reduced colocalization with synaptic markers, consistent with inefficient targeting of the channel complex to axonal terminals of granule neurons. Thus, the smaller depolarization-evoked calcium rise in cerebellar synaptosomes and in primary CGNs can be explained by the fact that there are fewer functional channels on the plasma membrane.