Unaltered prion protein expression in Alzheimer disease patients

Abstract

The suggested role of cellular prion protein (PrP^C) in mediating the toxic effects of oligomeric amyloid β peptide (Aβ) in Alzheimer disease (AD) is controversial. To address the hypothesis that variable PrP^C expression is involved in AD pathogenesis, we analyzed PrPC expression in the frontal and temporal cortices and hippocampus of individuals with no cognitive impairment (NCI), amnestic mild cognitive impairment (aMCI), mild AD (mAD), and AD. We found that PrP^C expression in all brain regions was not significantly altered among the various patient groups. In addition, PrP^C levels in all groups did not correlate with expression of methionine (M) or valine (V) at codon 129 of the PrP gene, a polymorphism that has been linked in some studies to increased risk for AD, and which occurs in close proximity to the proposed binding region for the oligomeric Aβ peptide. Our results indicate that, if PrP^C is involved in mediating the toxic effects of the oligomeric Aβ peptide, these effects occur independently of steady state levels of PrP or the codon 129 polymorphism.

Introduction

For many years, the overlapping clinical, pathological and biochemical characteristics of Alzheimer and prion diseases suggested a shared pathogenic mechanism. The prion protein (PrP) and the Aβ peptide, derived from the amyloid precursor protein (APP), both undergo structural transitions associated with a gain of toxic function leading to neurodegeneration.1 APP and PrP are directly or indirectly associated on the cell surface,2 and there are similarities in the post-translational processing of both proteins. Analogous to the α-secretase cleavage of APP, PrP^C undergoes proteolytic cleavage at amino acids 110/111 to produce a 17-kDa carboxyl-terminal fragment referred to as C13 by the possible action of TNFα-converting enzyme (TACE) or members of the ADAM (α disintegrin and metalloprotease) family. PrP cleavage following residue 89 results in the formation of an approximately 21 kDa carboxy-terminal C2 fragment, which, in infected brains, is resistant to protease digestion and appears to be facilitated by calpain.4 These two cleavage sites flank the amino acid charge cluster in the central domain where oligomeric Aβ has been proposed to bind PrP^C.5 Both proteins contain conserved histidine metal-binding domains, GxxxG transmembrane recognition motifs and histidine-based high-affinity metal-binding sites, which favor the binding of transition metals. Related to this, oxidative stress has been implicated in AD6 and prion disease.7

There is now convincing evidence that amplification of Aβ aggregates occurs by a prion-like mechanism.8–10 Moreover, prion infection is accelerated and formic acid-extractable Aβ1-42 peptide levels are higher, in transgenic mice expressing mutant APP (Tg2576 mice) compared to non-transgenic controls,11 suggesting that cross-seeding of the two abnormally conformed proteins may occur. In line with this observation, the brains of diseased scrapie-infected wild type mice also contain increased levels of the Aβ1-42 peptide.12 Conformational templating also appears to occur in other protein misfolding diseases13 involving tau,14 α-synuclein15 and polyglutamine proteins.16

Although soluble oligomeric forms of the Aβ peptide, derived from APP, are proposed as key mediators of synaptic and cognitive dysfunction in AD, the mechanisms by which these events occur remain unclear. Several recent studies suggest a direct mechanistic link between PrP and the Aβ peptide in AD pathogenesis. The studies of Strittmatter and co-workers implicated PrP^C as a major receptor for synthetic soluble Aβ1-42 oligomers, and indicated that PrP^C mediates the deleterious effects of oligomeric Aβ1-42 on synaptic function.5 Soluble Aβ1-42 oligomer binding was shown to occur at amino-acid residues 95–110 of PrP, a region close to the PRNP methionine (M)/valine (V) codon 129 polymorphism, which has numerous important influences on human prion diseases, and is also implicated as a risk factor for early-onset AD.17,18 In subsequent studies, this group demonstrated a requirement for PrP expression for axonal degeneration, loss of synaptic markers, early death and learning and memory deficits in AD transgenic mice.19

While the foregoing findings implicate PrP as a potential therapeutic target in AD, other studies have been less supportive of this concept. In independent transgenic mouse studies, ablation or overexpression of PrP^C had no effect on impairment of hippocampal synaptic plasticity.20 Moreover, while the interaction of Aβ1-42 oligomers with PrP^C was confirmed in separate studies, both PrP-expressing and PrP knockout mice were equally impaired in hippocampal dependent behavioral tests following intracerebroventricular injections of synthetic Aβ1-42 oligomers.21 Finally, other studies in which overexpression of PrP in vitro was shown to negatively regulate β-secretase12 would appear to be consistent with a model in which high levels of PrP result in low levels of Aβ1-42 oligomers.

Here we performed an extensive analysis of PrP levels in AD and pre-AD patients to address the hypothesis that variable PrP^C expression is involved in AD pathogenesis.

Results and Discussion

Table 1 summarizes patient characteristics by diagnostic group. Included in our studies were 13 individuals with no cognitive impairment (NCI), 7 patients with amnestic mild cognitive impairment (aMCI), 6 with mild AD (mAD) and 11 with AD. Braak and CERAD scores were determined for all samples (Tables 2 and 3). Age, post-mortem interval (PMI) and brain weight were similar among the various groups (Table 1). Differences in mini-mental state examination (MMSE) scores were highly significant between groups [F(3,33) = 88.853, p < 0.0001]. Post hoc comparisons using the student Newman-Keuls test showed statistically significant differences between NCI and aMCI (p < 0.05), between aMCI and mAD (p < 0.05), and between mAD and AD groups (p < 0.05). All dementia groups showed lower MMSE scores than NCI. Both AD groups showed lower scores than aMCI patients, while the AD group showed lower scores than mAD (Table 1).

In addition to its important influence on human prion diseases,22 the methionine (M)/valine (V) polymorphism at codon 129 of the prion protein gene (PRNP) has also been implicated as a risk factor for early-onset AD,17,18 although its influence as a risk factor for AD has been challenged.23 Analysis of this polymorphism is also important given its close proximity to the proposed region of Aβ1-42 oligomer binding.5 We analyzed codon 129 genotypes by treating polymerase chain reaction (PCR) amplified patient genomic DNA samples with NspI or MaeII restriction endonucleases (Fig. 1A and B) to distinguish PRNP coding sequence restriction fragment length polymorphisms (RFLPs) produced by the M and V codons. The PRNP M129 and V129 allele frequencies were approximately 77 and 23% respectively in the NCI group compared to 71 and 29% in the cognitively impaired groups. Valine homozygosity was not observed in the NCI group (Table 4). A previous association of 129V homozygosity and increased risk of early-onset AD was reported in a Dutch population.24 The mean age in each AD group in the current study is over 80 (Table 1). The period of follow up for patients was up to 14 years with a mean of 8 years approximately, raising the possibility that some cases may have been younger onset. Nonetheless, patient numbers in our study are too small to draw definitive conclusions.

To examine a possible relationship between cognitive decline and PrP^C expression, homogenates were prepared from frozen samples of hippocampus, superior frontal cortex (BA9) and superior-middle temporal cortex (BA21-22) of NCI, aMCI, mAD and AD patients, and immunoblots of samples containing equivalent total protein levels were probed with either anti-PrP monoclonal antibodies (mAb) 6H4 or 3F4. Levels of PrP in these preparations were determined by densitometric analysis of western blots using actin levels as an internal control in each sample. While we observed a tendency of diminished PrP levels in rostral compared to caudal areas when immunblots were probed with mAb 6H4, regional differences were not significant, and rostrocaudal decreases in PrP^C were not confirmed when the experiment was repeated using mAb 3F4 (Fig. 2).

Western blot analysis using both mAb 6H4 and 3F4 showed that expression of PrP^C was not significantly altered among NCI compared to aMCI, mAD or AD in either the frontal and temporal cortices or the hippocampus (Fig. 3). Levels of unglycosylated PrP were not higher in AD compared to control cases, although there was a tendency for higher levels of aglycosyl PrP^C levels in the temporal cortex of aMCI patients compared to other study groups, and when measured with mAb 3F4 levels were significantly higher in temporal cortex of aMCI compared to NCI and AD patients (Fig. 4). PrP^C expression levels were also independent of PRNP codon 129 genotype (Fig. 5).

Our results appear to be in general disagreement with the central topic of recently published studies by Velayos and co-workers29 which indicated a tendency for lower steady state PrP^C levels in the brains of AD patients compared to controls,29 especially in the hippocampus, an outcome apparently inconsistent with previous reports of increased PrP^C immunoreactivity in the temporal cortex, hippocampus (CA2) and subiculum in AD patients compared to controls.30 Although not discussed in the context of their data, the report of Velayos and co-workers was significant in light of recent studies implicating PrP^C as a major receptor that mediates the deleterious effects of oligomeric Aβ1-42 on synaptic function.5 Here we observed no differences in total PrP^C levels in AD compared to NCI controls, nor did we observe a graded decrease in PrP levels with increasing cognitive impairment. The minor observed differences between PrP^C levels probed with mAb 6H4 and mAb 3F4 could be related to the sites of binding of these mAbs to PrP. The epitope for mAb 6H4 has been mapped to the sequence DYEDRYYRE, corresponding to PrP residues 144–152,25 while the epitope for 3F4 is located in the KTNMKHM, corresponding to residues 106–112.26–28 Thus, while mAb 6H4 can detect the proteolytically processed sub-fragment of PrP^C, referred to as C1,3 mAb 3F4 cannot. Because di-glycosylated C1 fragments overlap with mono- and non-glycosylated forms of full-length PrP^C, this could potentially affect quantification of PrP^C levels and explain the discrepancy between the two mAbs.

What could explain the discrepancies between our study and the previous report? Velayos and coworkers studied three patients with AD. No clinical information was provided, except that one AD patient also had Down syndrome. Western blotting of PrP^C was recorded in three control human cases and two AD patients. In the present study, we analyzed PrP^C expression in a larger cohort comprised of 37 individuals including 11 AD cases, six mAD, seven aMCI patients and 13 non-demented controls. All patients were well characterized by diagnostic group, and all samples had short post-mortem intervals (Tables 1–3). Our analyses also included actin as an internal loading control for all samples. In addition to mAb 6H4, which was also used in the previous study, we used a second thoroughly characterized mAb (3F4) with reactivity against human PrP^C. The high numbers of well-characterized patients, and the rigorous analysis of regional PrP^C expression allow for meaningful associations (or lack of association in this case) to be made between levels of PrP and the presence of AD.

Velayos and coworkers also reported a shift in the profile of PrP glycosylation, with the unglycosylated form predominating in AD patients compared to controls. While we were also unable to confirm the finding of increased levels of aglycosyl PrP^C in AD or mAD patients, we did observe significantly higher levels of unglycosylated PrP in the temporal cortex of aMCI patients compared to NCI and AD (but not mAD) patients. Whether this change corresponds to a specific role for PrP expressing cells in this critical brain region at early stages of the development of AD, prior to the onset of significant neurodegeneration and neuronal loss, remains to be determined.

In summary, we conclude that, if PrP^C is involved in mediating the toxic effects of oligomeric Aβ, then this occurs by a mechanism that does not involve modulation of steady state levels of PrP. This would appear to be an important insight given recent associations of PrP in AD pathogenesis that implicate PrP as a potential therapeutic target.

Materials and Methods

Patients.

Frozen samples of hippocampus, superior frontal cortex (BA9) and superior-middle temporal cortex (BA21-22) were obtained from the University of Kentucky Rapid Autopsy Program of the Alzheimer Disease Clinical Center (UKADC). Tissue samples from 37 individuals with a mean age of 86.7 ± 7.6 years (Table 1) were examined. These individuals were part of a longitudinal clinical-pathologic study of aging and AD at the UKADC.31,32 The Human Investigations Committee at the University of Kentucky College of Medicine approved the studies. Individuals included in these studies agreed to annual clinical evaluation and brain donation at the time of death. For all subjects, cognitive test scores were available within the last year of life; the average interval from last evaluation to time of death was 7.0 ± 3.6 months, with no differences among the three diagnostic groups (p < 0.1). Subjects were categorized as NCI (n = 13), aMCI (n = 7), mAD (n = 6) or AD (n = 11),33 based on cognitive testing prior to death. The NCI subjects were without a history of dementia or other neurological disorders. Standard criteria for exclusion were the presence of (1) significant cerebral stroke regardless of antemortem date, (2) large cortical infarcts identified in the postmortem neuropathologic evaluation, (3) significant trauma within 12 months before autopsy, (4) individuals on a respirator longer than 12 h before death, (5) individuals in coma longer than 12 h immediately before death, (6) individuals currently undergoing radiation therapy for CNS tumor or (7) individuals with Lewy bodies.

Details of the UKADC have been published elsewhere in reference 32. All subjects have detailed mental status testing annually, and have neurologic and physical examinations annually. Subjects had been followed for 1–14 years (median 8.2 years). Once a subject transitioned to having aMCI or mAD, they received the mental status test battery and neurologic evaluation every 6 to 9 months. The 7 subjects with aMCI, 6 with mAD and 11 with AD were initially normal on enrollment into the longitudinal study and later developed aMCI and AD during follow-up. All aMCI subjects were amnestic without multi domain involvement. The diagnosis of aMCI, mAD, AD and NCI were defined by consensus conference. Histological examination of NCI subjects showed only age-related changes and Braak stage score of 0–II, meeting the NIA-RI low-likelihood criteria for the histopathologic diagnosis of AD. The clinical criteria for diagnosis of aMCI included (1) memory complaints, (2) intact activities of daily living, (3) objective memory impairment for age and education, (4) failure to meet criteria for dementia and (5) a clinical dementia rating (CDR) scale score of 0.5. The Braak stage scores had a range of I–V. Clinical progression to AD was diagnostically characterized by (1) a decline in cognitive functions from a previous higher level, (2) decline in one or more areas of cognition in addition to memory, (3) impaired activities of daily living, (4) a CDR score between 0.5–1 and (5) a clinical evaluation that excludes other causes of dementia. The criteria for mAD subjects included the above clinical progression plus a histopathologic diagnosis that included a Braak stage score of II–VI. For an AD categorization, subjects demonstrated a more progressive intellectual decline as described above, a MMSE less than that of the mAD cohort and Braak scores of II–VI. None of the mAD subjects were considered to be at the end-stage of the disease progression.

Transgenic mice.

Tg(HuPrP-M129)6816^+/− mice and Tg(HuPrP-V129)7823^+/− mice express human PrP encoding either M or V at codon 129, referred to as HuPrP-M129 and HuPrP-V129, respectively.34 Transgenic lines were maintained by breeding with Prnp^0/0 mice maintained on an FVB background (FVB/Prnp^0/0) and transgenic offspring were identified by tail biopsy and extraction of genomic DNA using a Beckman Biomek FX robotics station followed by PCR screening for the presence of the transgene. Approximately 1 cm of tail tissue was digested overnight at 55°C with proteinase K (0.5 mg/ml final concentration) in 50 mM Tris pH 8.0, 100 mM EDTA, 0.5% SDS, the DNA extracted with phenol and chloroform and concentrated by ethanol precipitation.

Analysis of the PRNP codon 129 polymorphism.

Genomic DNA was extracted from brain homogenates of the 37 individuals. The sense primer (5′-ATG GCG AAC CTT GGC TGC TGG ATG C-3′) and antisense primer (5′-GTG GTT GTG GTG ACC GTG TGC TGC TTG AT-3′) were used to amplify the PRNP cording sequence using PCR. The PRNP codon 129 polymorphism was assessed by digestion of the amplicons with restriction endonucleases NspI (New England Biolabs. Inc.,) and MaeII (HpyCH4IV, New England Biolabs. Inc.,), and the digested products were analyzed on 1.2% agarose gels.

Western blotting.

10% brain homogenates were prepared in sterile phosphate buffered saline (PBS) lacking Ca²⁺ and Mg²⁺ ions. The concentration of a total protein in each sample was determined by bicinchoninic acid assay and standardized for each lane (5 µg per lane). Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride Immobilon-FL membranes (Millipore). The transferred membrane was blocked with 5% non-fat milk in 0.5% Tween-20 in Tris-buffered saline (TBST) and immunoprobed with mouse monoclonal antibody anti-PrP 6H4 (Prionics) or 3F4 (Covance) followed by horseradish peroxidase-conjugated anti-mouse secondary antibody. Proteins were visualized using ECL Plus (GE Healthcare) in an FLA-5000 scanner (Fujifilm Life Science). Anti-actin (Pan) Ab-5 monoclonal antibody (NeoMarkers) was used as an internal control.

Statistical analysis.

Statistical analysis of western blot data from the frontal and temporal cortices and hippocampus of NCI, aMCI, mAD and AD was performed using a one-way ANOVA for each region separately. When appropriate, differences between groups were probed using a Newman-Keuls post hoc test. Each histogram of PrP^C in western blots was read by MultiGauge (Fujifilm Life Science) and the values were standardized against the histogram value of actin. All data were analyzed with GraphPad Prism 4 and values were expressed as mean standard deviation. Differences with p < 0.05 was considered to be a significant and indicated with asterisks.

Abbreviations

PrP	=	prion protein
PrPC	=	cellular prion protein
PrPSc	=	scrapie form of the prion protein
Aβ	=	amyloid β peptide
AD	=	Alzheimer disease
NCI	=	no cognitive impairment
aMCI	=	amnestic mild cognitive impairment
mAD	=	mild AD
M	=	methionine
V	=	valine
APP	=	amyloid precursor protein
kDa	=	kilodalton
TACE	=	TNFα-converting enzyme
ADAM	=	αdisintegrin and metalloprotease
PRNP	=	gene encoding human prion protein
CERAD	=	consortium to establish a registry for Alzheimer disease
MMSE	=	mini-mental state examination
PCR	=	polymerase chain reaction
RFLP	=	restriction fragment length polymorphism
mAb	=	monoclonal antibody
PMI	=	post mortem interval

Figures and Tables

Figure 1 Representative restriction fragment length polymorphism (RFLP) analyses. Lanes 1 and 2, brain extracts from Tg(HuPrP-M129)6816^+/− mice expressing human PrP-M129; lanes 3 and 4, brain extracts from and Tg(HuPrP-V129)7823^+/− mice expressing human PrP-V129; lanes 5 and 6, equal mixture of brain extracts from transgenic mice expressing human PrP-M129 and transgenic mice expressing human PrP-V129; lanes 7–16, five representative human samples. Odd numbers, undigested PCR samples; even numbers, digested PCR samples. (A) PCR amplified samples treated with restriction enzyme NspI, which cleaves PRNP encoding methionine at codon 129. Methionine carriers produce a 363 bp fragment; valine carriers produce a 438 bp fragment. (B) PCR samples treated with MaeII, which cleaves PRNP encoding valine at codon 129. Valine carriers produce a 359 bp fragment.

Figure 2 Rostrocaudal analysis of PrP^C levels in NCI and AD patients. For all samples, levels of total PrP^C and actin were measured by densitometric scanning of western blots. Each PrP value was normalized to its actin value. Normalized PrP^C levels were compared among the frontal (F) and temporal (T) cortices and hippocampus (H) in NCI and AD groups. Error bars represent standard deviations from the means.

Figure 3 Levels of PrP^C in different brain regions of patients with differing levels of cognitive impairment. Representative western blot analyses of brain homogenates from NCI, aMCI, mAD and AD patients are shown. Anti-PrP 6H4 (A–C) and 3F4 (D–F) antibodies were used to compare the levels of PrP^C among the groups. Actin levels are shown below the 6H4 and 3F4 immunoblots. NCI: lanes 1–4. aMCI: 5–8. mAD: 9–12. AD: 13–16. Three brain regions including the frontal (A, D, G and J), temporal (B, E, H and K) cortices and hippocampus (C, F, I and L) were analyzed. Molecular markers indicated are 53, 36, 28 and 19 kDa. Graphs in (G–L) include analyses of all samples from each group. For each sample, levels of total PrP^C were measured by densitometry. For each sample, levels of actin were also assessed. Each PrP value was normalized to its actin value. Means and standard deviations were calculated for each patient group and expressed as the percent value relative to the NCI group.

Figure 4 Unaltered levels of unglycosylated PrP^C in AD patients compared to NCI individuals. Levels of unglycosylated PrP^C (∼molecular weight 25–30 kDa) were measured by densitometric analysis of western blots. Means and standard deviations were calculated for samples from each patient group. y-axis values are arbitrary densitometric units. Asterisks indicate p > 0.05.

Figure 5 Levels of PrP^C do not correlate with human PrP 129 polymorphism in AD patients. Levels of PrP^C were measured by densitometry. For each sample, levels of actin were also assessed. Each PrP value was normalized to its actin value. These values were compared among individuals with various PRNP genotypes (M/M, M/V, V/V). (A–C) represent the data analyzed with 6H4; (D–F) represent the data analyzed with 3F4. Three brain regions including the frontal (A and D) and temporal (B and E) cortices and hippocampus (C and F) were analyzed.

Table 1 General demographics of subjects

	NCI	aMCI	mAD	AD	ANOVA
Group size	13	7	6	11
Male	3	4	3	5
Female	10	3	3	6
Mean age ± SD	87.4 ± 7.6	91.1 ± 5.0	85.5 ± 7.2	83.9 ± 8.7	F = 1.416
Range	70–101	84–99	77–94	68–99	p > 0.1
PMI (hours)
Mean ± SD	3.0 ± 0.8	2.9 ± 0.7	3.2 ± 0.9	3.5 ± 0.9	F = 0.988
Range	2.3–5	2–3.5	2–4.5	2–5	p > 0.1
Brain weight (g)					F = 0.232
Mean ± SD	1177 ± 166	1181 ± 155	1170 ± 57	1137 ± 110	p > 0.1
MMSE Mean ± SD	28.7 ± 1.7	24.7 ± 2.4a	22.2 ± 1.7ab	13.4 ± 3.4abc	F = 88.853
Range	26–30	20–27	20–25	7–19	p < 0.0001

a p < 0.05 compared to NCI.

b p < 0.05 compared to aMCI.

c p < 0.05 compared to mAD.

Table 2 Braak scores by clinical diagnosis

Group (N)	0–II	III–IV	V–VI
NCI (13)	7	5	1
aMCI (7)	2	3	2
mAD (6)	1	2	2
AD (11)	1	0	10

Table 3 CERAD classification by clinical diagnosis

Group (N)	None	Possible	Probable	Definite
NCI (13)	9	0	3	1
aMCI (7)	0	2	3	2
mAD (6)	1	0	1	4
AD (11)	0	0	2	9

Table 4 Codon 129 PRNP allele frequency and genotype of subjects

	NCI		aMCI		mAD		AD
	N	%	N	%	N	%	N	%
Allele
M	20	77	10	71	8	67	16	73
V	6	23	4	29	4	33	6	27
Total	26		14		12		22
Genotype
MM	7	54	4	57	3	50	6	55
VV	0	0	1	14	1	17	1	9
MV	6	46	2	29	2	33	4	36
Total	13		7		6		11

Acknowledgments

This study was supported by grants 5P30 AG028383 and 1P01AI077774-015261 to GCT from the National Institute on Aging (NIA), and the National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH); AG027219 to SWS from the NIA, NIH, and by the Sanders Brown Center on Aging, University of Kentucky Medical Center, Lexington, KY.