PMC

PrP and α₂δ-1 Are Coimmunoprecipitated from Brain Extracts
(A) Cerebellar protein extracts (500 μg) from Tg(WT), Tg(PG14), and Prnp^0/0 (KO) mice were incubated with uncoated magnetic beads (lanes 1–3) or magnetic beads coated with anti-PrP monoclonal antibody 94B4 (lanes 4–6). The immunoprecipitated proteins were analyzed by western blot with an anti-α₂δ-1 antibody (upper panel) or anti-PrP polyclonal antibody P45-66 (lower panels). The input (lanes 7–9) is 25 μg of total protein.
(B) Cerebellar protein extracts (500 μg) from the same animals as in (A) were incubated with uncoupled magnetic beads (lanes 1–3) or magnetic beads coated with anti-α₂δ-1 polyclonal antibody (lanes 4–6). The immunoprecipitated proteins were analyzed by western blot with an anti-α₂δ-1 monoclonal antibody (upper panel) or anti-PrP monoclonal antibody 12B2 (lower panels). The input (lanes 7–9) is 25 μg of total protein.

Targeting PrP to the ER or Golgi Induces Intracellular Accumulation of α₂δ-1
HeLa cells were cotransfected with plasmids carrying cDNAs encoding PrP-ER or PrP-Golgi and the VGCC subunits α_1A, Ca_vβ4, and α₂δ-1 (0.5:3:2:2 ratio).
(A) After 48 hr, cells were fixed, permeabilized, and stained with anti-PrP monoclonal antibody 98A3 (green), and polyclonal antibodies against protein disulfide isomerase (PDI) or giantin to stain the ER or Golgi (red).
(B) Transfected cells were stained with anti-PrP polyclonal antibody R505 (green) and an anti-α₂δ-1 monoclonal antibody (red). Cells were reacted with DAPI (blue) to stain the nuclei.
Scale bars, 20 μm.

Tg(PG14) Mice Develop Deficits in Motor and Balance Coordination before Cerebellar Degeneration
(A) Groups of 6–15 Tg(WT), 7–12 Tg(PG14) mice, and 6–17 non-Tg littermates were tested on a Rotarod at the ages indicated. Each mouse was tested three times. Bars indicate the mean ± SEM latency to fall (s); ^∗∗p < 0.01, ^∗∗∗p < 0.001 versus Tg(WT) by one-way analysis of variance (ANOVA), Bonferroni's post hoc test.
(B) Brain anatomy of non-Tg, Tg(WT), and Tg(PG14) mice at the ages indicated. Representative T2-weighted images (TE/TR = 50/2,500 ms).
(C) Cerebellar volumes (mm³) of non-Tg, Tg(WT), and Tg(PG14) mice at the ages indicated. Mean ± SD. The number of animals is given in parentheses. ^∗∗∗∗p < 0.0001 versus non-Tg and Tg(WT) by two-way ANOVA, Bonferroni's post hoc test.

Defective Calcium Influx in CGNs Is Due to PG14 PrP Expression and Accumulation in Secretory Organelles
(A) Primary CGNs from C57BL/6J and Tg(PG14) mice were transduced with 1–5 × 10⁶ TU/ml of lentiviruses encoding anti-PrP shRNA (LV-MW1 or LV-MW2), or with a lentivirus lacking shRNA (LV-E). The depolarization-induced [Ca²⁺]_i rise was measured in lentivirus-exposed CGNs and expressed as ΔF. Each bar indicates the mean ± SEM of cells transduced by the LV-E (40 C57BL/6J and 20 PG14 cells), LV-MW1 (19 C57BL/6J and 13 PG14 cells), and LV-MW2 (9 C57BL/6J and 27 PG14 cells). ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by two-way ANOVA, Bonferroni's post hoc test.
(B) Primary CGNs from C57BL/6J mice were transfected with pBUD-GFP plasmid carrying the wild-type, PG14, PG14/ΔHC, or ΔHC PrP cDNA. The K⁺-induced [Ca²⁺]_i rise was measured in GFP-positive cells and expressed as ΔF. Each bar is the mean ± SEM of 330 wild-type, 235 PG14, 299 PG14/ΔHC, and 192 ΔHC PrP-expressing cells. ^∗∗∗p < 0.001 versus the corresponding value of Tg(WT) by one-way ANOVA, Bonferroni's post hoc test.

α₂δ-1 Is Retained in Intracellular Compartments of Cells Expressing PG14 PrP
(A–I) HeLa cells were cotransfected with plasmids encoding wild-type, PG14, or PG14/ΔHC PrP-EGFP fusion protein, and the VGCC subunits Ca_Vα_1A, Ca_Vβ₄, and α₂δ-1 (0.5:3:2:2 ratio). After 48 hr, cells were fixed, permeabilized, stained with anti-α₂δ-1 monoclonal antibody followed by Alexa 594 (red)-conjugated anti-mouse IgG secondary antibody, and reacted with DAPI (blue) to stain the nuclei. Scale bar, 20 μm. The fluorescent density of PrP-EGFP (J) and α₂δ-1 (K) on the cell surface was measured and expressed as a percentage of the total. Each bar indicates the mean ± SEM of 28 wild-type, 48 PG14, and 8 PG14/ΔHC transfected cells. ^∗∗p < 0.01 and ^∗∗∗p < 0.001 by one-way ANOVA, Dunn's post hoc test. n.s., not significant.

Cerebellar Tg(PG14) Synaptosomes Show Impaired Depolarization-Evoked Glutamate Release and Calcium Influx
(A) Cerebellar synaptosomes from 230- to 250-day-old mice were preloaded with [³H]D-aspartate and stratified on filters in a 20 chamber superfusion apparatus. After equilibration, synaptosomes were exposed to 15 mM KCl for 1.5 min. The K⁺-induced [³H]D-aspartate overflow was measured in the collected fractions. Each error bar indicates the mean ± SEM of 5–20 replicate chambers from 2 to 4 independent experiments. Data are expressed as percentages of the values of Tg(WT) mice. ^∗p < 0.05 versus corresponding value in Tg(WT) by Student's t test.
(B) K⁺-induced release was determined in cerebellar synaptosomes from Tg(WT) and Tg(PG14) mice of the ages indicated. Each bar indicates the mean ± SEM of 5–14 replicates from 1 to 3 independent experiments. ^∗p < 0.05 and ^∗∗p < 0.01 by Student's t test.
(C) Depolarization-induced release was determined in synaptosomes from the cerebral cortex of the same Tg(WT) and Tg(PG14) mice used in (B). Each bar indicates the mean ± SEM. ^∗p < 0.05 versus corresponding value of Tg(WT) by Student's t test.
(D) Cerebellar synaptosomes were preloaded with fura-2 AM and depolarized with 50 mM KCl. The K⁺-induced [Ca²⁺]_i rise is expressed as percent ΔF (difference of the fluorescence ratio F340/380 before and after the stimulus) of K⁺-induced calcium responses in synaptosomes of Tg(WT) mice. Each bar indicates the mean ± SEM of 9–21 replicates from 2 to 4 independent synaptosomal preparations pooling 13 Tg(WT), 12 Tg(PG14), 4 Prnp^0/0, and 4 Prnp^+/+ mice of 230–250 days. ^∗p < 0.05 and ^∗∗∗p < 0.001 versus corresponding value for Tg(WT) by Student's t test.

The D177N PrP Mutation Alters α₂δ-1 Trafficking and Impairs Depolarization-Induced Calcium Influx and Glutamate Release
(A–F) HeLa cells were cotransfected with plasmids encoding D177N or D177N/ΔHC PrP-EGFP fusion protein, and the VGCC subunits Ca_Vα_1A, Ca_Vβ₄, and α₂δ-1 (0.5:3:2:2 ratio). After 48 hr, cells were fixed, permeabilized, stained with anti-α₂δ-1 monoclonal antibody followed by Alexa 594 (red)-conjugated anti-mouse IgG secondary antibody, and reacted with DAPI (blue) to stain the nuclei. Scale bar, 20 μm. The fluorescent density of PrP-EGFP (G) and α₂δ-1 (H) on the cell surface was measured and expressed as a percentage of the total. Each bar indicates the mean ± SEM of 11 D177N and 19 D177N/ΔHC transfected cells. ^∗∗∗p < 0.001 by Mann-Whitney U test.
(I) Primary cultures of CGNs from C57BL/6J (wild-type) and Tg(CJD) mice were preloaded with 10 μM fura-2 AM and depolarized with 30 mM KCl. The K⁺-induced [Ca²⁺]_i rise was measured in single cells and expressed as ΔF. Values are the mean ± SEM of 540 cells from C57BL/6J and 607 cells from Tg(CJD) mice, from 3 independent experiments. ^∗∗∗p < 0.001 by Student's t test.
(J) Depolarization-induced [³H]D-aspartate release was determined in cerebellar synaptosomes from 489- to 520-day-old Tg(WT) and Tg(CJD) mice. Each bar indicates the mean ± SEM of 2 independent analyses (18 superfusion chambers per experimental group) of synaptosomes from 5 Tg(WT) and 5 Tg(CJD) mice. ^∗∗p < 0.01 versus the corresponding values of Tg(WT) by Student's t test.
(K) Groups of 9–20 C57BL/6J (wild-type) and 3–12 Tg(CJD) mice were tested on a Rotarod at the ages indicated. Each mouse was tested three times, and the mean latency to fall was calculated. Bars indicate the mean ± SEM latency to fall (s). ^∗p < 0.05, ^∗∗∗p < 0.001 by Student's t test.

Depolarization-Evoked [³H]D-Aspartate Release, Calcium Currents, and Glutamatergic Neurotransmission Are Impaired in Primary CGNs from Tg(PG14) Mice
(A) K⁺-induced [³H]D-aspartate release from primary cultures of CGNs from Tg(WT) and Tg(PG14) mice. Each value represents the mean ± SEM of 13–20 replicates from 3 independent experiments. ^∗p < 0.05 by Student's t test.
(B) CGNs preloaded with 10 μM fura-2 AM were depolarized with 30 mM KCl. K⁺-induced [Ca²⁺]_i rise was measured in single cells and expressed as ΔF. Values are the mean ± SEM of 129 cells from Tg(WT) and 286 cells from Tg(PG14) mice. ^∗∗∗p < 0.001 by Student's t test.
(C) Representative pseudocolor images of fura-2 AM-loaded CGNs stimulated with 30 mM KCl. The images were taken before the stimulus (a and b), at an intermediate (c and d), and at the peak response (e and f). The color scale is shown on the left. Scale bar, 50 μm.
(D) Representative whole-cell VGCC inward Ba²⁺ currents in response to 10 mV increment step depolarizations from −80 to 60mV recorded in CGNs from Tg(WT) and Tg(PG14).
(E) Mean IV relationship of peak I_Ba recorded in CGNs from Tg(WT) and Tg(PG14), in response to 250 ms voltage pulses in 10mV increments from −80 to 60mV. I_Ba current densities (pA/pF) are shown as mean ± SEM; n = 17 for Tg(WT) and n = 10 for Tg(PG14).
(F) Voltage-dependent activation curves of VGCC currents generated in Tg(WT) (n = 8) and Tg(PG14) (n = 6) CGNs by measuring tail currents stimulated by a repolarization to −40mV, normalizing to the largest tail current in the series and then plotting against the prepulse voltage.
(G) Cultured CGNs from wild-type (C57BL/6J) and Tg(PG14) mice were voltage clamped at a holding potential of −70mV, and EPSCs were evoked by pairs of 1 ms depolarizing pulse to 30mV at 50 ms interpulse interval. Representative sample traces are shown.
(H) Amplitude of the first evoked EPSC in wild-type and PG14 neurons.
(I) The PPR value was calculated as the ratio of the amplitudes of the second to the first EPSC.
Bars in (H) and (I) indicate the mean ± SEM of 10 wild-type and 11 PG14 cells. ^∗p < 0.05, ^∗∗∗p < 0.001 by Mann-Whitney U test.