Here, we report that motor behavioral deficits in Tg(PG14) mice emerge before neurodegeneration and are associated with defective depolarization-induced glutamate exocytosis from cerebellar granule neurons (CGNs). Altered calcium influx due to inefficient membrane delivery of VGCCs accounts for the exocytosis Navitoclax manufacturer defect and is causally linked to intracellular retention of mutant PrP. Confirming this, alterations in VGCC transport and glutamate exocytosis are also found in cells and in Tg mice expressing a mouse PrP homolog of the D178N mutation linked to inherited CJD. These results provide new insights into the mechanism of neuronal dysfunction

in genetic prion diseases. The Tg(PG14) mice used in

this study express mutant PrP at a level similar to endogenous PrP in wild-type mice; they develop ataxia, kyphosis, and foot clasp reflex at ∼240 days of age and die prematurely at ∼450 days (Chiesa et al., 1998 and Chiesa et al., 2000). To find out the earliest appearance of motor dysfunction, Tg(PG14) mice were tested on the accelerating Rotarod. This motor behavioral task requires the mice to walk on an accelerating rotating rod with latency to fall as readout, and is a sensitive indicator of cerebellar abnormalities (Hilber and Caston, 2001 and Yamamoto et al., 2003). Tg(PG14) mice performed well between small molecule library screening 19 and 25 days of age (see Figure S1A available online), confirming normal postnatal development of the cerebellum (which is completed in the third week of life) and motor learning (Chiesa et al., 2000). From 45 days on,

however, the mice showed a significantly shorter latency to fall than non-Tg littermates Phosphoprotein phosphatase and Tg(WT) mice expressing wild-type PrP (Chiesa et al., 1998); their performance worsened with aging until they became virtually unable to perform the task (Figure 1A). We used MRI to see whether the emergence of the motor deficit was associated with cerebellar degeneration. Tg(PG14) mice were examined at ∼50 and ∼250 days of age, with age-matched non-Tg and Tg(WT) controls. At 50 days the cerebellar volume of Tg(PG14) mice was comparable to controls (Figures 1B and 1C). In older Tg(PG14) mice there was a significant atrophy of the cerebellum (Figures 1B and 1C), consistent with previous histological analysis showing age-dependent cerebellar degeneration (Chiesa et al., 2000). To investigate changes in cerebellar synaptic structures, we immunostained the brains of young and old animals with an antibody against the vesicular glutamate transporter VGLUT1, which specifically labels glutamatergic projections of CGNs in the molecular layer of the cerebellum (Fremeau et al., 2001). There was no difference in VGLUT1 immunostaining between young non-Tg and Tg(PG14) mice, whereas a significant decrease was seen in older Tg(PG14) mice (Figures S1B–S1D).