Normal neurogenesis and scrapie pathogenesis in neural grafts lacking the prion protein homologue Doppel

Abstract

The agent that causes prion diseases is thought to be identical to PrPSc, a conformer of the normal prion protein PrPC. Recently a novel protein, termed Doppel (Dpl), was identified that shares significant biochemical and structural homology with PrPC. To investigate the function of Dpl in neurogenesis and in prion pathology, we generated embryonic stem (ES) cells harbouring a homozygous disruption of the Prnd gene that encodes Dpl. After in vitro differentiation and grafting into adult brains of PrPC-deficient Prnp0/0 mice, Dpl-deficient ES cell-derived grafts contained all neural lineages analyzed, including neurons and astrocytes. When Prnd-deficient neural tissue was inoculated with scrapie prions, typical features of prion pathology including spongiosis, gliosis and PrPSc accumulation, were observed. Therefore, Dpl is unlikely to exert a cell-autonomous function during neural differentiation and, in contrast to its homologue PrPC, is dispensable for prion disease progression and for generation of PrPSc.

Fig. 1. Generation of Dpl-deficient ES cells. (A) Schematic representation of the targeting strategy to generate ES cells harbouring a heterozygous mutation of Prnd. Exons of the Prnd gene are represented by rectangles; thin lines represent intronic regions of the Prnd locus. The Dpl ORF is represented as a dark gray rectangle. The neomycin resistance gene (PGK-Neo) and the diphtheria toxin alpha gene (DTα) are represented by light gray rectangles; loxP sites are shown as black triangles. The location of the ApaI restriction sites, the ApaI restriction fragments, and the PCR primers used for genotyping are indicated. (B) Schematic representation of the gene conversion event generating Prnd homozygous mutant ES cells selected for with high G418 concentration. (C) PCR analysis of genomic DNA from the parental ES cell line E14.1 (Prnd^+/+ CTR), one targeted Prnd^+/neo clone (Prnd^+/neo CTR) and four ES cell clones that had undergone selection with 4 mg/ml G418 (Prnd^+/neo and Prnd^neo/neo). (D) Southern blot analysis of genomic DNA from the parental ES cell line E14.1 (Prnd^+/+), one Prnd^+/neo and one Prnd^neo/neo ES cell clone.

Fig. 2. Histological characterization of non-infected and Scrapie-infected Prnd^neo/neo neuroectodermal grafts. Histological analysis of non-infected Prnd^+/+ (A–D), Prnd^+/neo (E–H) grafts and Prnd^neo/neo grafts (I–L). Intraventricularly or intraparenchymally placed ES cell-derived grafts of all genotypes showed no difference in their differentiation pattern, as they contained astrocytes (GFAP immunostain, B, F, J) and showed regular immunoreactivity for synaptophysin (C, G, K) and MAP-2 (D, H, L). Inoculation with mouse prions (185 days) resulted in typical histopathological hallmarks of scrapie, such as vacuolation (M) and astrogliosis (N) but not yet significant loss of neuropil, as indicated by normal staining pattern for synaptophysin and MAP-2. Scale bar: 500 µm (A, E, I, M) and 200 µm (all other panels).

Fig. 3. Quantitation of cell populations in Prnd^+/+, Prnd^+/neo and Prnd^neo/neo grafts. Neurons, astrocytes and oligodendrocytes were counted on an area of 400 × 400 µm (as described in Benninger, 2000). There was no apparent difference in distribution, ratio and number of these cell types in grafts of either genotype.

Fig. 4. Detection of disease-associated PrP^C in intraparenchymal grafts derived from Prnd^+/neo and Prnd^neo/neo ES cells. (A–D) Frozen sections with intraparenchymally located grafts (outlined with asterisks). Histoblots of these grafts without (E–H) and with (I–L) proteinase K treatment. After short incubation time (52 days), minute amounts of proteinase resistant PrP were detectable in both, Prnd^neo/neo and Prnd^+/neo grafts, while prolonged incubation time (195–203 days) led to strong accumulation of proteinase resistant PrP^Sc. The central irregular shaped region in the Prnd^+/neo graft (H, L) results from regressive changes and calcifications which are occasionally observed in ES cell-derived neuroectodermal grafts of all genotypes.