No abstract

SummaryHow mutant prion protein (PrP) leads to neurological dysfunction in genetic prion diseases is unknown. Tg(PG14) mice synthesize a misfolded mutant PrP which is partially retained in the neuronal endoplasmic reticulum (ER). As these mice age, they develop ataxia and massive degeneration of cerebellar granule neurons (CGNs). Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective glutamate exocytosis from granule neurons due to impaired calcium dynamics. We found that mutant PrP interacts with the voltage-gated calcium channel α2δ-1 subunit, which promotes the anterograde trafficking of the channel. Owing to ER retention of mutant PrP, α2δ-1 accumulates intracellularly, impairing delivery of the channel complex to the cell surface. Thus, mutant PrP disrupts cerebellar glutamatergic neurotransmission by reducing the number of functional channels in CGNs. These results link intracellular PrP retention to synaptic dysfunction, indicating new modalities of neurotoxicity and potential therapeutic strategies.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a SNARE protein that regulates neurotransmission by the formation of a complex with syntaxin 1 and synaptobrevin/VAMP2. SNAP-25 also reduces neuronal calcium responses to stimuli, but neither the functional relevance nor the molecular mechanisms of this modulation have been clarified. In this study, we demonstrate that hippocampal slices from Snap25 ؉/؊ mice display a significantly larger facilitation and that higher calcium peaks are reached after depolarization by Snap25 ؊/؊ and Snap25 ؉/؊ cultured neurons compared with wild type. We also show that SNAP-25b modulates calcium dynamics by inhibiting voltage-gated calcium channels (VGCCs) and that PKC phosphorylation of SNAP-25 at ser187 is essential for this process, as indicated by the use of phosphomimetic (S187E) or nonphosphorylated (S187A) mutants. Neuronal activity is the trigger that induces the transient phosphorylation of SNAP-25 at ser187. Indeed, enhancement of network activity increases the levels of phosphorylated SNAP-25, whereas network inhibition reduces the extent of protein phosphorylation. A transient peak of SNAP-25 phosphorylation also is detectable in rat hippocampus in vivo after i.p. injection with kainate to induce seizures. These findings demonstrate that differences in the expression levels of SNAP-25 impact on calcium dynamics and neuronal plasticity, and that SNAP-25 phosphorylation, by promoting inhibition of VGCCs, may mediate a negative feedback modulation of neuronal activity during intense activation. S ynaptosomal-associated protein of 25 KDa (SNAP-25) belongs to the SNARE superfamily of membrane proteins that participate in the regulation of neuronal exocytosis. SNAP-25 is present in two isoforms, a and b, resulting from alternative splicing of the exon 5 of the gene, which is differentially expressed during development. SNAP-25a is expressed at the embryonic stage, and SNAP-25b becomes the major isoform postnatally (1-3). SNAP-25 is anchored to the cytosolic face of membranes by palmitoyl side chains located in the central region of the molecule and contributes two ␣-helices to the exocytotic fusion complex, together with syntaxin-1 and synaptobrevin/ VAMP2 (4-5). SNAP-25 also interacts with the synaptic vesicle protein, synaptotagmin I (6), a major calcium sensor that regulates neurotransmitter release (7,8). Interaction of synaptotagmin with SNAP-25 is essential for the calcium-dependent triggering of membrane fusion (9) and for the control of fusion pore during the final steps of exocytosis (10). Furthermore, the C terminus of SNAP-25 is a target of G protein ␤-and ␥-subunits that mediate presynaptic inhibition (11). Therefore, SNAP-25 represents a multifunctional protein involved in the control of secretion by multiple interactions. Besides its well characterized role in regulating exocytosis, there is increasing evidence that SNAP-25 modulates various ion channels (12, 13). In particular, SNAP-25 physically interacts with different types of voltagegated calcium chann...