Soon after PrPSc was proposed to be the causative agent of prion diseases, Prnp knockout mice lacking PrPC were generated in order to answer the question whether the loss of physiological PrPC function would lead to neurodegeneration in prion diseases. The first Prnp null mouse strain, designated Prnp -/-, or Zurich I (ZrchI, Prnp ZH1/ZH1), was produced in a mixed C57BL/6 J × 129/Sv(ev) background [28] and a second line of PrPC-deficient mice, known as Npu or Edinburgh (Edbg), was produced with a pure 129/Ola genetic background [29]. In a first round of characterization, these mice were not found to show any clear abnormality except for their resistance to prion infection [30]. They developed and bred normally, and although they displayed subtle alterations in behavior [28], their otherwise apparent normality seemed to rule out a physiological function of PrPC that is essential for life. If there is one, it is highly redundant or it can be compensated for. PrPC-deficient mice from which the entire Prnp gene was removed [31,32,33,34] develop progressive cerebellar ataxia, which was originally attributed to the loss of PrPC but was later discovered to be due to the deletion of a splice acceptor site in exon 3 of Prnp [35]. This led to aberrant overexpression of the PrPC paralogue gene (Prnd) encoding Doppel (Dpl) [36, 37], causing selective neurodegeneration of cerebellar Purkinje cells. Notably, the reintroduction of Prnp in mice overexpressing Prnd in the brain rescued the phenotype, suggesting a functional link between the two proteins [38].

Later, using the Cre-loxP system, conditional PrPC knockout NFH-Cre/tg37 mice were generated to examine the effects of acute PrPC depletion on neuronal viability and function in the brain of 9-week-old adults. This approach was thought to avoid compensatory mechanisms active at the embryonal stage that would have masked PrPC loss of function phenotypes [39]. Again, depleting neuronal PrPC in adult mice did not result in neurodegeneration or histopathological changes, but it led to subtle electrophysiological abnormalities in the hippocampus (Table 1). A closer look at different neuronal and other cell functions in PrPC-ablated mice revealed a number of differences from wild-type mice that were attributed to the physiological function of PrPC. While some of these studies were consistent among different PrPC-deficient lines, others yielded contradictory results depending on methodologies and the mouse models that were used (Table 2).

Table 1 Lines of PrPC-ablated mice covered in this review Full size table

Table 2 Proposed physiological roles of cellular prion protein Full size table

A genetic confounder has been shown to underlie some of these inconsistencies [40, 41]. For many years, knockout alleles were usually created in embryonic stem cells from the Sv129 strain of mice, and the resulting mice were backcrossed to C57BL/6 mice [42]. This practice typically leads to variable, poorly controlled Mendelian segregation of polymorphic alleles whose distribution depends on their genetic linkage to the knockout allele. All Prnp knockout mouse lines have been generated in this way with the exception of the “Edinburgh” mouse, which was maintained in a pure 129 background [42]. Even after more than 12 generations of backcrossing, a small part of the chromosome around the Prnp locus still stems from the 129 strain, raising the question whether any observed phenotypes were actually due to polymorphisms in genes flanking Prnp. Indeed, we found that SIRPα, a polymorphic Prnp-flanking gene, is actually responsible for an alleged Prnp -/- phenotype: the inhibition of macrophage phagocytosis of apoptotic cells that was observed in PrPC-deficient mice with mixed genetic background but not in co-isogenic Prnp -/- mice [40]. Recently, a new PrPC-deficient mouse strain, Prnp ZH3/ZH3, was produced in our lab using TALEN-mediated genome editing in fertilized mouse oocytes and maintained in a pure C57BL/6 J genetic background [42]. These strictly co-isogenic C57BL/6 J-Prnp ZH3/ZH3 mice differ from wild-type mice only by eight deleted nucleotides in the Prnp reading frame. In an effort to improve the quality of studies on the function of the cellular prion protein, we are distributing Prnp ZH3/ZH3 mice without requesting any kind of Material Transfer Agreement, hence enabling better-controlled future studies. In view of the broad availability of Prnp ZH3/ZH3 mice, we contend that the use of mixed-background PrPC-deficient mice is obsolete and liable to artifacts.

Do further mammal species teach us more about the function of PrPC? The gene encoding PrPC has been ablated experimentally in cattle [43] and goats [44], and a naturally occurring Prnp knockout goat has been reported [45]. While no pathological phenotypes were reported in any of these animals, it may be rewarding to perform specific investigations of these animals, e.g., concerning the integrity of the peripheral nervous system in advanced age.

A large set of human genomic data was analyzed to quantify the penetrance of variants of the human PrPC gene (PRNP) in prion disease [46]. Surprisingly, heterozygous loss-of-function variants were identified in three individuals. These individuals in their 50s and 70s are probably healthy, and no evidence of any neurological defect or peripheral neuropathy was documented. This result suggests that heterozygous loss of PRNP in humans may not be haploinsufficient. It remains to be assessed, however, whether homozygous deletion and therefore complete loss of PrPC may create a disease in humans.