Ablation of Prion Protein in Wild Type Human Amyloid Precursor Protein (APP) Transgenic Mice Does Not Alter The Proteolysis of APP, Levels of Amyloid-β or Pathologic Phenotype

The cellular prion protein (PrPC) has been proposed to play an important role in the pathogenesis of Alzheimer’s disease. In cellular models PrPC inhibited the action of the β-secretase BACE1 on wild type amyloid precursor protein resulting in a reduction in amyloid-β (Aβ) peptides. Here we have assessed the effect of genetic ablation of PrPC in transgenic mice expressing human wild type amyloid precursor protein (line I5). Deletion of PrPC had no effect on the α- and β-secretase proteolysis of the amyloid precursor protein (APP) nor on the amount of Aβ38, Aβ40 or Aβ42 in the brains of the mice. In addition, ablation of PrPC did not alter Aβ deposition or histopathology phenotype in this transgenic model. Thus using this transgenic model we could not provide evidence to support the hypothesis that PrPC regulates Aβ production.


Introduction
Alzheimer's disease (AD) is the most common form of dementia affecting 30 million individuals world-wide [1,2]. Age is the greatest risk factor for AD, with the incidence doubling every 5 years after age 65. Therefore, with our ageing population, AD is placing immense financial and social pressure on society. Currently there are no treatments that either cure or halt the progression of this neurodegenerative disease [3]. The majority (>95%) of AD cases have no underlying genetic mutation and are referred to as sporadic or late-onset AD [4]. In a small proportion of cases, mutations in the genes encoding the amyloid precursor protein (APP) or presenilin (PS) 1 or PS2 give rise to early onset, familial AD [4]. The disease is characterised by the deposition in the brain of extracellular plaques of amyloid-β (Aβ) which is derived from the proteolytic processing of APP [5], along with intracellular neurofibrillary tangles of hyperphosphorylated tau protein [6]. APP is cleaved first by the β-secretase, β-site APP cleaving enzyme-1 (BACE1), and then by the PS-containing γ-secretase complex to release Aβ, the predominant forms of which are 40 or 42 amino acids in length (Aβ 40 and Aβ 42 , respectively) [5].
Cleavage of APP by BACE1 is the rate-limiting step in Aβ production [7] and various cellular proteins have been reported to influence this step, including the cellular form of the prion protein (PrP C ) [8]. PrP C inhibited the action of BACE1 on wild type APP (APP WT ) in various cellular models, in part through glycosaminoglycan-mediated interaction at the cell surface and in part through retaining the pro-domain containing form of BACE1 in the early secretory pathway [8,9]. In the brains of PrP C null mice there was a significant increase in the amount of endogenous murine Aβ [8], consistent with PrP C having a role in regulating the production of Aβ from APP in vivo. Together with the observation that PrP C was decreased in the brains from sporadic AD individuals and that the amount of PrP C inversely correlated with BACE1 activity, soluble and insoluble Aβ and Braak stage in the human brain [10,11], led us to propose that PrP C may function to protect against AD and that loss of PrP C would lead to the earlier onset of AD [12,13].
The inhibitory effect of PrP C on the BACE1 cleavage of APP was only apparent on APP WT and was lost on APP with the Swedish double point mutation adjacent to the BACE1 cleavage site (APP Swe ) [9]. APP Swe is subject to BACE1 cleavage in the secretory pathway [14,15] rather than in the endosomal pathway as for APP WT [16,17]. As PrP C interacted directly with the prodomain of the immature Golgi-localised form of BACE1, decreasing the amount of BACE1 at the cell surface and in endosomes, this provided a mechanism to explain the differential inhibitory effect of PrP C towards APP WT and APP Swe [9]. In transgenic mice expressing human APP Swe.Ind we [9] and others [18][19][20] have reported that upon genetic ablation of PrP C there is no alteration in APP processing, Aβ levels or plaque pathology, consistent with this differential inhibitory mechanism. Therefore, in this study we aimed to determine whether ablation of PrP C in transgenic mice expressing human APP WT results in increased Aβ and subsequently causes the premature appearance of plaque pathology.

Transgenic Animals
Transgenic APP WT mice over expressing human wild-type APP (line I5) or APP Swe,Ind mice over expressing human APP with the Swedish (K670N/M671L) and Indiana (V717F) familial AD mutations (line J20) [21] were obtained from The Jackson Laboratory, (Line B6.Cg-Tg (PDGFB-APP)5Lms/J, stock number 004662 and B6.Cg-Tg(PDGFB-APP Swe,Ind )20Lms/2J, stock number 006293, respectively) and The J. David Gladstone Institutes, San Francisco, CA 94158) and crossed with inbred PrP knockout mice (129Ola PrP -/-) [22]. The genetic background of all mice used in this study was mixed B6/129Ola and only female mice were used. Operators were blinded to genotype and animals were randomly assigned to cages of n = 4 and given access to food and water ad libitum. During housing, animals were monitored daily for health status and no adverse effects were noted. All the transgenic mice used in this study were genotyped. DNA was prepared from ear punch tissue using a DNeasy Kit (Qiagen). PCR was performed using the protocol specific for these mice from The Jackson Laboratory. At end point, animals were culled by cervical dislocation and brain hemispheres were either frozen at -80°C for biochemical analysis or fixed in 10% formol saline for histopathological analysis. These experiments were approved by The Roslin Institute's Animal Welfare and Ethical Review Board and were conducted according to the regulations of the UK Home Office Animals (Scientific Procedures) Act 1986. All efforts were made to minimise suffering.

Homogenisation
Brain hemispheres were homogenised using a two-step extraction protocol [23]. Briefly, initial homogenisation (120 mg/ml wet weight) was carried out using an electrical homogeniser in 2% (w/v) SDS containing protease inhibitor cocktail (Roche Diagnostics GMbH, Germany) and PhosSTOP phosphatase inhibitor (Roche Diagnostics GMbH, Germany), followed by centrifugation at 100,000 g for 1 h at 4°C. The resultant supernatant (containing 'soluble' Aβ) was collected and analysed as described below. The pellet was extracted in 70% (v/v) formic acid in dH 2 O followed by centrifugation at 100,000 g for 1 h at 4°C. The supernatant (containing 'insoluble' Aβ) was collected and analysed as described below.

Statistical Analysis
Densitometric analysis was performed using the advanced image data analyser (AIDA) programme (Raytest Scientific Ltd). The Kolmogorov-Smirnov test was used to determine that the data in each group was normally distributed. Following this the Levene's test was used to ensure that the data sets were of equal variance. In samples where the data met the criteria of a normal distribution and equal variance, the parametric independent t-test was used to calculate significance. In the samples which were not normally distributed the non-parametric twotailed Mann-Whitney U test was used to compare two independent samples. The data were analysed using the Statistical Package for Social Sciences (SPSS 19) program (Chicago, USA).

Results and Discussion
In order to investigate whether ablation of PrP C in transgenic mice expressing human APP WT results in increased Aβ, mice expressing human APP WT [21] were crossed with PrP C -null 129/Ola mice [22]. Immunoblot analysis of mice at 32 and 75 weeks of age confirmed the absence of PrP C in the APP WT /PrP -/mice and that the lack of PrP C had no effect on APP expression (Fig 1A). The amounts of the soluble APP species, sAPPα and sAPPβ, were no different between the APP WT /PrP -/and APP WT /PrP +/+ mice at either 32 or 75 weeks of age ( Fig  1B), indicating that deletion of PrP C had no effect on either the αor β-secretase cleavage of APP. There was a significant increase in sAPPα in both genotypes at 75 weeks of age compared to 32 weeks of age ( Fig 1B) which resulted in an increased sAPPα:sAPPβ ratio (Fig 1C). The levels of soluble (SDS-extracted) Aβ 38 , Aβ 40 and Aβ 42 were investigated. At both 32 and 75 weeks of age there was no difference in the amount of soluble Aβ 38 , Aβ 40 or Aβ 42 in the brain homogenates of the APP WT /PrP -/mice compared to the APP WT /PrP +/+ mice (Fig 1D). The amount of Aβ 40 and Aβ 42 was higher at 75 weeks of age than at 32 weeks of age regardless of Prnp genotype (Fig 1D), with a proportionately larger increase in Aβ 42 such that the Aβ 42 : Aβ 40 ratio increased in both the APP WT /PrP -/and APP WT /PrP +/+ mice with age ( Fig 1E). The levels of Aβ peptides in the insoluble (formic acid extracted) fraction from either the APP WT /PrP -/or the APP WT /PrP +/+ mice were below the limit of detection in the assay. These data indicate that lack of PrP C in transgenic mice expressing human APP WT does not alter the proteolysis of APP or the levels of Aβ.
To investigate whether lack of PrP C in the APP WT /PrP -/mice affected the deposition of Aβ in the brain, histopathological analysis of the hippocampus was carried out at 32 weeks of age and compared to APP WT /PrP +/+ mice. APP Swe,Ind /PrP +/+ mice, which are known to accumulate limited Aβ-positive plaques at this age, were also analysed [9,21]. APP WT /PrP +/+ and APP WT /PrP -/mice at 32 weeks of age showed no obvious neuropathologic alterations (data not shown). To explore whether histopathologic alterations were detected in older animals, we also examined mice at 75 weeks of age. Histopathological analysis of WT, APP WT /PrP +/+ and APP WT /PrP -/mice at 75 weeks of age showed similar histopathologic features; i.e. absence of obvious neuronal loss or other obvious alterations in the hippocampus proper or dentate gyrus (Fig 2A, 2D and 2G) or 4G8 positive Aβ plaque formation (Fig 2B, 2E and 2H) in these mice. We investigated whether the ablation of PrP C affected the level of Aβ fibrils and fibrillar oligomers by using the OC antibody [25]. We observed that WT, APP WT /PrP +/+ and APP WT /PrP -/mice did not accumulate OC immunopositive deposits as shown by immunohistochemistry (Fig 2C, 2F and 2I). As expected, in the APP Swe,Ind /PrP +/+ mice at 75 weeks of age there was disruption of the hippocampal structure with accumulation of 4G8 and OC positive Aβ deposition forming numerous uni and multicentric plaques (Fig 2J, 2K and 2L), as seen previously [9]. Synapses are particularly vulnerable to the toxic effect of protein oligomers. Thus, we performed immunostaining for the detection of the presynaptic protein synaptophysin and observed disruption of immunoreactivity in 75 week old APP Swe,Ind /PrP +/+ mice but not in APP WT /PrP +/+ or APP WT /PrP -/mice (Fig 3A, 3D and 3G). Glial markers (GFAP and Iba1) showed similar intensity and pattern of reactivity in APP WT /PrP +/+ and APP WT /PrP -/mice (Fig 3B, 3C, 3E and 3F). In contrast, gliosis was observed particularly in the vicinity of amyloid deposits in APP Swe,Ind /PrP +/+ mice (Fig 3H and 3I). Thus, we did not detect any effect of PrP C ablation on Aβ deposition using immunohistochemistry.
A comparison of the Aβ levels between the APP WT /PrP +/+ mice at 75 weeks of age with APP Swe,Ind /PrP +/+ mice at 5 and 10 weeks (before significant Aβ deposition occurs) and at 40 weeks (after Aβ deposition in plaques appears) revealed that at 75 weeks of age the amount of Aβ peptides in the soluble (SDS extracted) fraction of the APP WT /PrP +/+ mice was comparable to the amount of each peptide in the 5 week old APP Swe,Ind /PrP +/+ mice (Fig 4A). Although Aβ 40 and Aβ 42 were readily detectable in the insoluble (formic acid extracted) fraction from the APP Swe,Ind /PrP +/+ mice (Fig 4B), no Aβ was detected in the insoluble fraction from the APP WT /PrP +/+ mice. The soluble Aβ 42 :Aβ 40 ratio increased with age in both APP WT /PrP +/+ and APP Swe,Ind /PrP +/+ mice (Fig 4C) but was significantly higher in the APP Swe,Ind /PrP +/+ mice than in the APP WT /PrP +/+ mice consistent with the presence of the Indiana mutation increasing production of Aβ 42 as observed previously [21]. Even at 75 weeks of age the amount of Aβ 42 in the APP WT /PrP +/+ mice is significantly lower than that in the APP Swe,Ind /PrP +/+ mice at 5, 10 and 40 weeks of age (Fig 4A). Thus in the APP WT /PrP +/+ mice even at 75 weeks of age the low level of Aβ 42 is likely below the threshold required for deposition to occur.

Conclusion
In cellular models PrP C differentially affected the activity of the β-secretase BACE1 towards APP WT and APP Swe , inhibiting the processing of the former but having no effect on the cleavage of the latter [8,9]. Thus we undertook this study in order to investigate the effect of genetic ablation of PrP C on human APP WT processing in vivo. In contrast to the cellular models, lack of PrP C had no effect on the αand β-secretase proteolysis of APP or on the amount of Aβ peptides in the brains of mice expressing human APP WT . Also we did not detect any Aβ deposition as shown by immunohistochemistry in the mice lacking PrP C as compared to those with a normal level of the protein. In the human brain the amount of PrP C inversely correlated with BACE1 activity, soluble and insoluble Aβ and Braak stage [11], consistent with levels of PrP C affecting APP processing and Aβ production. However, we could find no evidence that loss of PrP C affected APP proteolysis, Aβ levels or plaque pathology in vivo in transgenic mice expressing human APP WT . Whether this reflects a difference in the role of PrP C in regulating APP processing and Aβ production between this transgenic mouse model and the situation in the human brain will require further study.