Evidence of presynaptic location and function of the prion protein

Abstract

The prion protein (PrP(C)) is a copper-binding protein of unknown function that plays an important role in the etiology of transmissible spongiform encephalopathies. Using morphological techniques and synaptosomal fractionation methods, we show that PrP(C) is predominantly localized to synaptic membranes. Atomic absorption spectroscopy was used to identify PrP(C)-related changes in the synaptosomal copper concentration in transgenic mouse lines. The synaptic transmission in the presence of H(2)O(2), which is known to be decomposed to highly reactive hydroxyl radicals in the presence of iron or copper and to alter synaptic activity, was studied in these animals. The response of synaptic activity to H(2)O(2) was found to correlate with the amount of PrP(C) expression in the presynaptic neuron in cerebellar slice preparations from wild-type, Prnp(0/0), and PrP gene-reconstituted transgenic mice. Thus, our data gives strong evidence for the predominantly synaptic location of PrP(C), its involvement in the regulation of the presynaptic copper concentration, and synaptic activity in defined conditions.

Fig. 1.

PrP^C is preferentially localized to areas of high synaptic density. Histoblots of wild-type (A), Prnp^0/0(B), and PrP^C-overexpressing reconstituted Prnp^0/0 (tg35) (C) mice. The blots were probed with anti-PrP antiserum Ra5. No PrP^C was detected in Prnp^0/0 mice (B) or in wild-type mouse brains (A). D and F show enlargements from boxes in C.E and G show consecutive hematoxilin and eosin stains. D, E, PrP^C is strongly expressed in areas of high synaptic density of the hippocampal stratum orients (1), stratum radiatum (3), stratum lacunosum moleculare (4), and the molecular layer (5). No signal was detected in the hippocampal pyramidal cell layer (2) or the granular layer of the dentate gyrus (6). F,G, High PrP^C expression in the cerebellar molecular layer (7). Scale bars:A, 1 mm; B, C, 2.5 mm;D–G, 250 μm.

Fig. 2.

Laser scanning confocal images of PrP^C in the cerebellar cortex and retina of wild-type and PrP^C-overexpressing Prnp^0/0-reconstituted mice. A,B, Cerebellar cortex of tg35 and tg20 animals analyzed using PrP antibody 3B5. In tg35 (A), PrP^C was detected over the granule cell layer (gcl), the Purkinje cell bodies (pcl), and the molecular layer (ml). In tg20 (B), a strong PrP^C expression was observed over the molecular layer and granule cell layer but not in the Purkinje cell layer.C–F, Retina of wild-type (C) and PrP^C-overexpressing tg20 (D–F) mice. Using PrP antibody 3B5, no signal was detected in wild-type mice (C), whereas the tg20 retina (D) showed a strong PrP^C expression in the inner plexiform layer (ipl), as well as the outer plexiform layer (opl). In tg20 mice, synaptophysin (E) was found to be coexpressed with PrP^C in the outer and inner plexiform layers (F). PrP^C expression is low in the outer (onl) and inner nuclear layers (inl). Scale bars: A–F, 150 μm.

Fig. 3.

Enrichment of PrP^C in the synaptic plasma membrane fraction but not in the postsynaptic density fraction isolated from mouse brains. Equal amounts (100 μg/lane) of subcellular fractions from wild-type mouse brain and prion protein-deficient mouse brain (Prnp^0/0) were analyzed by immunoblotting with anti-PrP antibody 3B5 (A) and antiserum Ra5 (B). For comparison, subcellular fractions of PrP^C-overexpressing tg 35 mice (30 μg/lane) were also examined using antibody Ra5 (C). The immunostaining for synaptotagmin (D) and NMDA receptor subunit NMDA-R1 (E) was used as a control for synaptic vesicle proteins and postsynaptic membrane proteins, respectively. Subcellular fractions are designated as follows: H, homogenate; SV, crude synaptic vesicle fraction; CS, cytosolic synaptosomal fraction; SPM, synaptic plasma membranes.F, In the PSD fraction, PrP^C is not detectable (Western blot analysis in homogenates and PSD with antibody 3B5; 30 μg of protein were loaded in each lane). G, Immunostaining for NMDA-R1 was used as a control for postsynaptic membrane proteins.

Fig. 4.

Copper concentration in synaptosomes correlates with PrP^C expression. The copper concentration in whole-brain homogenates (A) and synaptosomal fractions (B) of wild-type (WT), Prnp^0/0, and Prnp-reconstituted Prnp^0/0 mice (tg20) was studied by atomic absorption spectroscopy. The copper concentration was not significantly different in the whole-brain homogenates of the three lines tested. The synaptosomal preparations reveal a significantly reduced copper concentration in Prnp^0/0 mice compared with wild-type mice (Student’s t test;p < 0.05). In PrP^Cgene-reconstituted Prnp^0/0 mice (tg20), the synaptosomal copper concentration was similar to wild-type mice. Shown are the mean and SE for four to six independent preparations of five age- and sex-matched brains. Asterisks indicate that the observed differences were found to be statistically significant.

Fig. 5.

Hydrogen peroxide enhances inhibitory synaptic activity in wild-type (A–G), but not Prnp^0/0 (H–N) mouse Purkinje cells. Samples of the continuous recording of inhibitory synaptic currents before (A, H) and after (B, I) bath perfusion with 0.01% H₂O₂ at time points indicated inC and J. C andJ show plots of the number of sIPSCs detected in 30 sec sample intervals as a function of time. The barindicates the time of H₂O₂ application.D and K, as well as E andL, show the mean amplitudes and the mean charge of sIPSCs, calculated for 30 sec intervals, as a function of time.F and M show the amplitude histograms for the time periods (2.5 min) indicated in C andJ. G and N show the cumulative amplitude distributions of the histograms shown inF and M. No shift in the amplitude distribution was observed in either the wild type (G) or Prnp^0/0(N). The holding potential was −70 mV in this and all experiments shown in the following figures.

Fig. 6.

Hydrogen peroxide enhances inhibitory synaptic activity in two lines of Prnp-reconstituted Prnp^0/0mice. PrP^C is overexpressed in all neurons in tg35 mice (A–C) and in all neurons except Purkinje cells in tg20 mice (D–F). A,D, Plots of the sIPSC frequency in 30 sec sample intervals against time in tg35 (A) and tg20 (D) mice. The bar indicates the time of H₂O₂ application. B,E, Plots of the mean sIPSCs amplitude, calculated for 30 sec intervals, as a function of time in tg35 (B) and tg20 (E) mice. C,G, Cumulative amplitude distributions for the time periods indicated in A and B. Whereas in tg35 (C) no significant shift can be observed, there is a shift to higher values in tg20 (F).

Fig. 7.

Pooled data of the effects of 0.01% H₂O₂ on the frequency and mean amplitude of Purkinje cell inhibitory synaptic currents in wild-type (●), Prnp^0/0 (○), tg35 (■), and tg20 (▵) mice. Each point represents the mean ± SEM of frequency or amplitude of sIPSCs in 30 sec intervals normalized to the values before H₂O₂ application. Because of the tendency for amplitudes and frequencies of sIPSCs to decay slowly during the control period, only the last 3 min before the application were chosen for calculating the baseline (broken line at the 100% level). The threshold for detection of sIPSCs was set to −30 pA.A, Plots of the mean values of sIPSC frequency in wild-type (n = 14) and Prnp^0/0(n = 21) mouse Purkinje cells, calculated for 30 sec intervals, as a function of time. The bar indicates the time during which H₂O₂ was applied. Significant differences between the two mouse strains are marked byasterisks. p < 0.01;t test. B, C, Plots of the mean values of the frequency of sIPSCs in tg35 (B;n = 15) and tg20 (C;n = 4) compared with wild-type and Prnp^0/0 mouse data. D, Plots of the mean sIPSC amplitudes normalized to values before H₂O₂ application in wild-type, Prnp^0/0, tg35, and tg20 mouse Purkinje cells. The amplitudes did not change in wild-type and tg35 cells after the application of H₂O₂. It increased slightly in tg20 mouse Purkinje cells and decreased significantly in Prnp^0/0 mouse cells.