Prion protein accumulation and neuroprotection in hypoxic brain damage

Abstract

The function of the normal conformational isoform of prion protein, PrP(C), remains unclear although lines of research have suggested a role in the cellular response to oxidative stress. Here we investigate the expression of PrP(C) in hypoxic brain tissues to examine whether PrP(C) is in part regulated by neuronal stress. Cases of adult cerebral ischemia and perinatal hypoxic-ischemic injury in humans were compared with control tissues. PrP(C) immunoreactivity accumulates within neuronal processes in the penumbra of hypoxic damage in adult brain, and within neuronal soma in cases of perinatal hypoxic-ischemic injury, and in situ hybridization analysis suggests an up-regulation of PrP mRNA during hypoxia. Rodents also showed an accumulation of PrP(C) in neuronal soma within the penumbra of ischemic lesions. Furthermore, the infarct size in PrP-null mice was significantly greater than in the wild type, supporting the proposed role for PrP(C) in the neuroprotective adaptive cellular response to hypoxic injury.

Figure 1

Immunohistochemistry on the adult CI cases. a: H&E staining of an ischemic lesion, case A3. The same lesion was probed for PrP using 3F4 (b) and βAPP using MAB348 (c). d: Another lesion probed with 3F4 demonstrating both axonal- and punctate-type staining patterns (case A1). e: A higher power view of the axonal PrP staining seen in several cases (case A7). f and g: Case A4 (f) and case A12 (g) show typical patterns of intense punctate PrP deposition in penumbra of ischemic damage. h and i: Case A6 demonstrates the widespread punctate PrP staining that was seen in all cases adjacent to ischemic lesions, with h being the no primary antibody control of the same region for contrast. j: The similar staining pattern of PrP deposition seen in the vicinity of a gray matter lesion (case A1). k: A small cluster of neurons strongly positive for PrP protein in the gray matter of an affected case (case A1). l: A normal piece of tissue stained for PrP using 3F4 for comparison. Scale bars: 500 μm (d); 250 μm (a–c, f, g, j, l); 100 μm (e); 50 μm (h, i, k).

Figure 2

Immunohistochemistry and in situ hybridization on cases of HII. a and b: PrP staining of hypoxic neurons using 3F4 on cases P1 and P3, respectively. c: The in situ hybridization signal seen for PrP mRNA in case P3 coinciding with cells positive for PrP protein. d: The in situ hybridization sense control probe. Scale bars, 50 μm.

Figure 3

Serial slices through the infarcted brain after staining with tetrazolium blue chloride. Serial slices from co-ordinates F +1.8 to F −1.6. The damaged regions can be seen as pale areas on the left-hand hemisphere. It can be seen that the damage in the PrP^0/0 mice is more widespread than the other two lines. Note that the full range of the damaged area has not been shown. The data shown in Table 4 includes all slices with damage. Left, PrP^+/+; middle, PrP^+/0; and right, PrP^0/0.

Figure 4

Vascular structure determination using latex filling. The circle of Willis and the left and right MCA in three strains of the 129Ola mouse after the vascular system was filled with latex colored with Indian ink. Four mice were analyzed of each genotype. The vascular structure relating to the MCA is closely similar in the three strains analyzed. Left, PrP^+/+; center, PrP^+/0; and right, PrP^0/0.

Figure 5

Analysis of MCA occlusion in the mouse. a: The PrP signal from the ischemic region of the mouse brain showing large amounts of protein deposition. b: The contralateral control region treated with the same antibodies. c: A cresyl violet stain (CV) showing the ischemic lesion as a pale area (left) and the in situ hybridization signal for PrP mRNA from the cells in the penumbra of the lesion (right). Scale bars: 100 μm (a, b); 200 μm (c).