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Prion. 2009 Apr-Jun; 3(2): 110–117.
PMCID: PMC2712608

The cellular prion protein and its role in Alzheimer disease

Abstract

The cellular prion protein (PrPC) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrPSc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrPC is needed for the establishment and further evolution of prions.

The present work compares the expression and localization of PrPC between healthy human brains and those suffering from Alzheimer disease (AD).

In both situations we have observed a rostrocaudal decrease in the amount of PrPC within the CNS, both by immunoblotting and immunohistochemistry techniques. PrPC is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrPC in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands.

With regards to amyloid plaques, those present in AD cases were positively labeled for PrPC, a result which is further supported by the presence of PrPC in the amyloid plaques of a transgenic line of mice mimicking AD.

The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.

Key words: cellular prion protein, Alzheimer disease, transgenic mice

Introduction

The cellular prion protein (PrPC) is a cell membrane-bound glycoprotein with a molecular weight of 33–35 kDa. Present in various organs, it is especially abundant in the central nervous system (CNS).1,2 One of the first studies on the inmunorreactivity of PrPC in the human brain is that of Esiri et al.3 Little is known about the physiology of PrPC. PrPC involvement in phenomena such as adhesion, neuroprotection and cellular signalling has been noted.4

The scrapie prion protein (PrPsc) is an isoform of PrPC. PrPsc is responsible for transmissible spongiform encephalopathies (TSEs); the presence of PrPC is necessary for its establishment and evolution. After inoculation with PrPsc, PrPC knockout mice do not develop TSEs.5 Both PrP isoforms are coded by the same gene, showing the same aminoacid sequence, but with differences in their secondary structure. Given this special structure, different isoforms have different conformation, so that PrPC can be digested by proteinase K, while PrPSc is only partially digested, generating a 27–30 kDa fragment. PrPsc is only present in the prionopathies, and its brain concentration is 10 to 20 times higher than that of PrPC in healthy brains. TSEs are characterized by three neuropathological features: spongiosis, neuronal loss and gliosis. Sometimes PrP accumulations appear in the form of plaques.6,7

Based on previous studies carried out in various animals, and knowing that extrapolation is legitimate, a retrograde spread of modified prion proteins from the zone of entry into the nervous system is proposed here. For some authors, such transmission is anterograde;8 others see it as retrograde911 and still others posit a mixed form.12 However, an increasing number of researchers have concluded that the spread is retrograde,1315 in line with our hypothesis.9,11,16

Alzheimer disease (AD) is one of the most common neurodegenerative diseases, characterized by the presence of an accumulation of abnormal proteins, and may be categorized as a proteinopathy. It has been postulated that progressive and diffuse neuronal loss may be due to cell damage, as a result of the formation of free radicals (cause unknown). AD and human TSEs overlap in some cases, such as Gerstmann-Sträussler-Scheinker disease (GSS); this latter shares a significant number of pathological features with AD including the presence of β-amyloid plaques and neurofibrilar tangles, caused by hyperphosphorilation of tau protein which is responsible for the stability of the neuronal cytoskeleton. According to Kovacs and Budka,17 the elderly are more susceptible than the young people to presenting a prionopathy.

The idea that PrP and A β amyloids may somehow interact is an old one put forward by Gajdusek.18 Ferrer et al.19 have observed PrPC in AD plaques. It has been assumed that PrPC plays an important role in the pathogenesis of AD.18,19 So, the analysis of the location of PrPC in this clinical entity may provide clues for a better understanding of the role that PrPC plays in the development of AD. In addition, Ferreiro et al.22 and Soto et al.23 have observed a pathophysiological relationship between AD and prionopathies.

The aim of this work is to study the localization and expression of PrPC in healthy human brains, as compared with cases of AD, bearing in mind the findings from experimental studies previously carried out on laboratory animals at our facility. The secondary objective of this project is to provide a comparative analysis of human cases in relation to healthy and transgenic mice.

Results

Inmunohistochemistry.

(1) Control, healthy humans (Figs. 1 and and2).2). PrP immunopositivity was observed in cortical areas, mainly in deep layers. Cortical pyramidal cells were intensely labeled for PrP (Fig. 1A–C). PrP immunopositivity was less intense in the occipital cortex than in other neocortical areas. The hippocampus also contained PrP-immunopositive cells (Fig. 1D).

Figure 1
PrP immunoreactivity in healthy human brains. (A) frontal cortex (case H4); (B) temporal cortex (case H4); (C) occipital cortex (case H7); (D) hippocampus (case H4).
Figure 2
PrP immunoreactivity in a healthy human brain case (H7). (A) thalamus; (B) caudate-putamen; (C) cerebellum; (D) pons; (E) Medulla oblongata.

Similarly, there was immunopositivity for PrP in the thalamus (Fig. 2A), area in which a few positive neurons were noticed. A higher amount of them were detected in the striatum (Fig. 2B).

Within the cerebellum, Purkinje cells were remarkably positive for PrP, drawing row-like formations in which cells were labeled on an alternant fashion (Fig. 2C).

In the brainstem, PrP immunopositivity was lower than in the rostral areas of the encephalon (Fig. 2D and E).

(2) AD-affected humans and senile humans (Figs. 3 and and4).4). PrP immunopositivity was observed in the same encephalic areas previously outlined, with the particularity that the cerebellum in AD cases (Fig. 4B) was similar to control cases, whereas the other brain structures showed a wide neuronal disorganization, neuronal loss and amyloid plaques which were indeed positive for PrP.

Figure 3
Amyloid plaques and PrP immunoreactivity in an AD case (H2, which showed also a Down syndrome), and a senile case (H3). (A) frontal cortex (H3); (B) prefrontal cortex (H2); (C) orbitary cortex (H2); (D) Temporal cortex (H2); (E) occipital cortex (H3); ...
Figure 4
PrP immunoreactivity in an AD case (H2, which showed also a Down syndrome), and a senile case (H3). (A) thalamus (H2); (B) cerebellum (H3); (C) inferior olive (H3).

(3) Transgenic mice (Figs. 5 and and6).6). Amyloid plaques were also immunolabeled for PrP in transgenic mice.

Figure 5
Amyloid plaques showing PrP immunoreactivity (green) and Beta-amiloid (red), in a case of AD plus Down syndrome (H2).
Figure 6
(A) PrP positive amyloid plaques in the hippocampus of a transgenic mouse. (B) Hippocampus of the same animal (red: amyloid plaques; green: PrP immunoreactivity). (C) frontal cortex of the same animal (red: amyloid plaques; green: PrP immunoreactivity). ...

Western blot.

(1) PrPC. Healthy humans and humans affected by AD (Fig. 7). A rostrocaudal shift was observed in the intensity of PrP immunostaining, being higher in the most rostral areas. We focused our attention on the frontal and temporal cortices and the hippocampus.

Figure 7
Western blot in healthy (control) humans and AD cases. Frontal cortex, temporal cortex, hippocampus. Bottom: quantitative study.

We observed a tendency of a diminution of the amount of PrP in our AD cases in comparison with healthy (control) cases and especially in the hippocampus. On the other hand, in our AD cases the intensity of the unglycosylated band was higher than in control cases.

(2) Neuronal loss and astrogliosis in the hippocampus. Healthy humans and humans affected by AD (Fig. 8).

Figure 8
PrP western blot along with NeuN and GFAP immunopositivity of hippocampus in healthy (control) humans and AD cases. Right: correlation between PrP and neuronal loss and astrogliosis.

There was a neuronal loss and astrogliosis in our AD cases, in comparison with control cases. The ratios can be observed in Figure 8.

(3) Comparative study in mice (Fig. 9). The western blot of hippo-campus of healthy mice showed that the amount of PrP was greater in old specimens than in younger ones. And there was a tendency of an increasing of the intensity of the unglucosylated band in oldest mice.

Figure 9
Comparative study of western blot of healthy (9A) and transgenic AD (9B) mice of 7 and 17 months old.

Discussion

The present work demonstrates a great ubiquity of PrPC within the CNS, although it is noteworthy that the presence of PrPC is higher in some brain areas (Figs. 14). On the one hand, there is a clear decrease of PrPC along the rostrocaudal axis. The higher presence of PrPC in several brain areas of the CNS might mean that PrPSc would be more intensely replicated within these areas, and prions would be more intensely transmitted from these brain regions. On the other side, the fact that PrPC is especially abundant in areas that project to the thalamus (deep layers of the cerebral cortex, for instance), supports hypothesis stating that prions follow a retrograde pathway within the brains.9,11 Laffont-Proust et al. (2007)24 found out how during development, the amount of PrPC increases in the supra and infragranular layer of the visual cortex of primates. Interestingly, these layers also send projections to the thalamus.

With regards to the central topic of this work, both the global amount and intensity of PrPC immunostaining and immunoblotting is lower in our AD cases than in control cases (Figs. 7 and and8).8). Moreover, within the former cases the number of neurons is lower and their structure shows a deep disorganization when compared with control cases. And there was a clear astrogliosis in AD cases (Fig. 8). In addition to these results, amyloid plaques of AD patients were immunopositive for PrP (Figs. 3 and and4).4). Taken together, all these findings suggest that, even though the amount of PrPC is globally lower in AD cases than in control cases, the existing PrPC expressed in AD cases may suggest a protective role in Alzheimer disease.19,2529 However, other authors2,20 suggest that PrPC plays an important role in the pathogenesis of AD. In agreement with our results, Rezai et al.30 observed that within the frontal cortex of both AD and control cases, the expression of PrPC is higher than in the occipital cortex. Schwarze-Eicker et al.31 observed how amyloid plaques of transgenic mice were accumulated in higher amounts within the telencephalon than in any other region of the CNS.

The higher intensity of the lowest molecular band (the unglycosylated isoform) compared to the other bands present in the immunoblotting in AD cases would support the presence of an isoform of PrPC which, at least in humans, might have a special role in AD. As a matter of fact, in some prionopaties the intensity of the non glycolisated protein band is lower than that of the other bands. Perhaps this finding would support the hypothesis that the diminution of the amount of that protein could be facilitator of the cellular death in such entities.

Finally, if we compare the hippocampus of humans and mice (Figs. 59), we observe that there are some differences, but not many. In fact, the hippocampus is a crucial structure in the development of AD. The third band of the western blot is more intense in old healthy and transgenic mice than in younger healthy and transgenic ones.

Material and Methods

Brains of healthy humans (control cases, n = 8) and of patients with AD (n = 3), one of which showed the pathology together with a Down syndrome (case H2) were subjected to immunohistochemical analysis. Several cortical areas, thalamus, hippocampus, caudate nucleus, putamen, cerebellum and brainstem were sampled.

Tissue was fixed by immersion in fixatives that allowed a good antigenic preservation. Tissue was paraffin-embedded and cut at 3 µm thick with the aim of a rotational microtome in order to obtain the slides for the immuhistochemical detection of PrP. The antibodies used were those yielding the best immunostaining results (polyclonal antibodies, Anti Prion Protein 91511, Assay Dessigns Inc., and ARP-01-8634, American Research Products).

Western blot techniques were also performed on the control and AD cases (in frontal and temporal cortices and hippocampus). Fresh, unfixed brains were dissected in the areas of interest and samples were rapidly frozen on dry ice and stored at −80°C until quantification of the total protein content in each region. Proteins were further separated on 12%-acrylamide gels and transferred to a nitrocellulose membrane which was incubated with a commercially available antibody to PrP (mAb 6H4, Prionics). Once the optimal conditions were reached, blots were measured by densitometry and compared with a control to show the equal amount of total proteins loaded in the gel in order to analyze and quantify the areas and encephalic nuclei expressing PrP. We carried out ratios of the three isoforms of PrP. We also studied de neuronal loss, utilizing a neuronal marker, such as NeuN. And we carried out a control of glial cells (utilizing GFAP). And we studied the correlation of PrP with neuronal loss and astrogliosis.

A comparative study between samples from three control human cases and two AD patients was performed.

With regards to the comparative study of control and transgenic mice, tissue was provided by Dr. Cuadrado, from the Center for Applied Medical Research (CIMA) of the University of Navarra.

Acknowledgements

Supported by PIUNA 2006–2008 and BMH4-CT96-856 (EU). Human samples were provided by Dra. Cristina Caballero (Anatomía Patológica, Hospital de Navarra, Pamplona, Spain).

Footnotes

Previously published online as a Prion E-publication: http://www.landesbioscience.com/journals/prion/article/9135

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