Prion replication alters the distribution of synaptophysin and caveolin 1 in neuronal lipid rafts

Abstract

The main event in the pathogenesis of prion diseases is the conversion of the cellular prion protein (PrP(C)) into the abnormal, protease-resistant prion protein (PrP(res)). PrP(C) is a GPI-anchored protein located in lipid rafts or detergent-resistant membranes (DRMs). Here we describe the association of PrP with DRMs in neuronal cell bodies and axons during the course of murine scrapie and its relation with the distribution of the PrP-interacting proteins caveolin 1 and synaptophysin. Scrapie infection triggered the accumulation of PrP(res) in DRMs from retinas and optic nerves from early stages of the disease before evidence of neuronal cell loss. Most of the PrP(res) remained associated with lipid rafts throughout different stages in disease progression. In contrast to PrP(res), caveolin 1 and synaptophysin in retina and optic nerves shifted to non-DRM fractions during the course of scrapie infection. The accumulation of PrP(res) in DRMs was not associated with a general alteration in their composition, because no change in the total protein distribution across the sucrose gradient or in the flotation characteristics of the glycosphingolipid GM1 or Thy-1 were observed until advanced stages of the disease. However, an increase in total cholesterol levels was observed in optic nerve and retinas. Only during late stages of the disease was a decrease in the number of neuronal cell bodies observed, suggesting that synaptic abnormalities are the earliest sign of neuronal dysfunction that ultimately results in neuronal death. These results indicate that prion replication triggers an abnormal localization of caveolin 1 and synaptophysin, which in turn may alter neuronal function.

Figure 1

Accumulation of PrP in DRMs from retinas and optic nerves at early stages of prion disease in mice. The distribution of PrP along buoyant sucrose gradient after Triton X-100 extraction at different times after scrapie infection was analyzed in retinas (A) and optic nerves (B) by Western blotting. The ratio between the PrP-immunoreactive signal and the total protein concentration in each fraction was calculated at different times after infection in retinas (C) and optic nerves (D). D-, M-, and N- correspond to the di-, mono-, and nonglycosylated forms of PrP, respectively. wpi, weeks postinjection. Data were statistically analyzed by Student’s t-test; *, P < 0.05.

Figure 2

PrP^res in retinas and optic nerves. A: Western blot showing the PrP profile in retina (top) and optic nerve (bottom) before and after PK digestion in control (Ct) and scrapie-infected animals (Sc). B: The graph illustrates the proportion of PrP after PK digestion (PrP^res) related to the amount of total PrP before digestion, considered as 100%, in retinas and optic nerves (ON). D-, M-, and N- correspond to the di-, mono-, and nonglycosylated forms of PrP. Data were statistically analyzed by Student’s t-test; *, P < 0.05.

Figure 3

Distribution of total proteins, GM1, tubulin, and Thy-1 in retinas and optic nerves after 0.1% Triton X-100 extraction. Total protein content in different fractions of the sucrose gradient was measured by silver staining of the gels from samples obtained from retinas (A) and optic nerves (B) of controls and scrapie-infected animals at advanced stages of the disease. None of the differences was statistically significant, as evaluated by Student’s t-test. The distribution of the DRM-resident ganglioside GM1 was measured by dot blotting in different fractions of samples from retina (C) and optic nerve (D). The distribution of tubulin and Thy-1 were also studied in samples from retina (E and G) and optic nerve (F and H).

Figure 4

Dissociation of caveolin 1 from DRMs in retinas and optic nerves during scrapie infection in mice. Distribution of caveolin 1 along the sucrose gradient after Triton X-100 extraction at different times after scrapie injection in retinas (A) and optic nerves (B). The ratio between the amount of caveolin 1 and total protein in each fraction was calculated at different times after infection in whole homogenates from retinas (C) and optic nerves (D). Also, the ratio between the total amount of caveolin 1 and the total amount of proteins in retinas (E) and optic nerves (F) was calculated. Data were statistically analyzed by Student’s t-test; *, P < 0.05. wpi, weeks postinjection.

Figure 5

Dissociation of synaptophysin from DRMs in retinas during scrapie infection in mice. Synaptophysin distribution along the buoyant sucrose gradient after Triton X-100 extraction at different survival times after scrapie infection in retinas (A) and optic nerves (B). C: The ratio between the amount of synaptophysin and total protein in each fraction was calculated at different times after infection. D: The ratio between the total amount of synaptophysin and the total amount of proteins in retinas was calculated. The inset shows Western blotting of synaptophysin in controls and scrapie-infected mice at different survival times. Data were statistically analyzed by Student’s t-test; *, P < 0.05. E: Western blotting from retina (left) and optic nerve (right) after cytosol and membrane separation in the absence of detergent.

Figure 6

Degeneration of retinal ganglion cells at advanced stages of scrapie. The photomicrographs illustrate the retinal ganglion cell layer in flat-mounted retinas stained by cresyl violet. A: Control mice. B: Retina from mice at 20 weeks after infection. C: Retina from mice at advanced stage of the disease. D: Graph illustrating the number of neurons per mm² in the retinal ganglion cell layer after prion infection. The arrows indicate the retinal ganglion cells and the arrowheads indicate the displaced amacrine cells. Data were statistically analyzed by Student’s t-test; *, P < 0.05.

Figure 7

Altered distribution of caveolin 1 and synaptophysin in the cortex of scrapie-infected mice. A: Eight animals per group were injected in the hippocampus with 1 μl of 10% brain homogenate from normal (Ct) or 139A scrapie-infected mice. Muscle strength was determined by measuring the time during which animals were able to cling to an inverted grill. Values represent the percentage of animals in each group that fell within 1 minute after inversion. Inset: Western blot analysis of PrP^res in PK-digested cortex brain homogenates from infected animals at 12, 16, and 20 weeks after injection. In cortex brain homogenates PrP (B), caveolin 1 (C), and synaptophysin (D) distribution along the buoyant sucrose gradient after Triton X-100 extraction at different survival times after scrapie infection.