Abstract
Background/Objective: PrPc has been suggested to play a role in AD pathophysiology. CSF concentrations of PrPc have been shown to be reduced in AD compared with healthy controls. Furthermore, serum levels of PrPc have recently been reported to be associated with the cognitive status of healthy elderly subjects. Therefore, we hypothesized that CSF levels of PrPc could be associated with cognitive function of AD patients at the time of diagnosis.
Methods: AD patients (n = 114) included into an observational study underwent CERAD testing and lumbar puncture at time of diagnosis / study inclusion. CSF PrPc was determined. Generalized linear models were fitted to assess the associations of PrPc plus a variety of possible confounding factors and CERAD subscale measures.
Results: No association of CSF PrPc and cognitive status could be established, while other factors (i.e., use of antipsychotic drugs, use of anti-dementia drugs, female sex, pre-progression time) were related to worse cognitive function in some domains.
Conclusion: CSF PrPc appears not to be a useful biochemical surrogate of cognitive status in AD at the time of diagnosis. Follow-up analyses will examine possible associations with the speed of cognitive decline.
Keywords: :
Introduction
Alzheimer disease (AD) is going to be one of the most important public health problems of the decades to come. In our aging societies it will affect ever-growing numbers of elderly people.Citation1 Therefore, research efforts aiming to elucidate the pathogenesis of the disease as well as trials testing potential therapeutic agents have increased almost exponentially.
In animal studies conducted during the last years it has become more and more evident that the cellular Prion protein (PrPc) may be involved in the pathophysiological processes underlying Alzheimer disease. Parkin et. al demonstrated that the Prion protein can act as a regulator of the β-secretase and therefore may influence the Amyloid β (Aβ) production.Citation2 Especially PrPc‘s role as a potential Aβ oligomer receptor and their conjoint impact on long-term potentiation (LTP), synaptic plasticity and neuronal death have been discussed controversially.Citation3-Citation6 Although the overall expression of PrPc in the brain appears to be similar in healthy controls and AD patientsCitation7 its concentration in the CSF appears to be lower in subjects suffering from AD.Citation8
Lot is known about diseases involving misfolding of PrPc into its pathological form PrPsc. Still, the physiological role of PrPc itself is not completely understood to date. PrPc is already expressed at the stages of embryogenesis. The highest concentrations in adults can be found in the central nervous system while it is also expressed in a variety of non-neuronal tissues.Citation9 It is mainly localized within the cellular membrane in lipid rafts but also underlies endocytosis and rapid recycling.Citation10 Excretion into the CSF happens via exosomal transfer.Citation11 PrPc appears to have a multitude of functions. It has been demonstrated to be involved in inflammatory reactions, cell proliferation and differentiation, apoptotic pathways, synapse formation as well as protection against oxidative stress.Citation12,Citation13 Besides these mostly cyto-protective functions it furthermore appears to be part of various signaling mechanisms. Linden and colleagues describe PrPc as “a dynamic cell surface platform for the assembly of signaling modules, based on which selective interactions with many ligands and transmembrane signaling pathways translate into wide-range consequences upon both physiology and behavior.”Citation12
In animal experiments PrPc has been associated with learning processes.Citation14,Citation15 Very recently, Breitling and coworkers have demonstrated that CSF concentrations of PrPc are correlated with cognitive function of healthy subjects.Citation16 Those with higher Prion protein concentrations in the serum featured worse cognitive functioning. Except the study of MeyneCitation8 mentioned above, studies relating CSF PrPc to cognitive status are lacking.
Considering these molecular and functional links between PrPc and cognition as well as PrPc and Alzheimer disease the question arises, whether CSF PrPc levels might also be associated with cognitive function in Alzheimer disease patients. Herein we analyzed CSF levels of PrPc in a cohort of AD patients from an observational study and fitted generalized linear models (GZLM) to unveil possible associations with cognitive status as measured with the CERAD test battery.
Results
The size of the patient group was n = 114. As to expect for AD a majority of subjects was female (58%). The mean age was 68 y. At time of diagnosis, the median MMSE was 20 pts. Detailed baseline data (sociodemographic, medical, cognitive) are given in . All relevant CSF biomarkers were altered as necessary for the diagnosis of AD: (values are given as: median (IQR)) Aβ42 = 428(216) (Cut-Off 450) [pg/ml]; Aβ40 = 6399(4430) (Cut-Off 450) [pg/ml]; Aβ-Ratio: 0.6(0.3) (Cut-Off 1); Tau: 474(530) (Cut-Off 450) [pg/ml]; pTau: 83(56) (Cut-Off 61) [pg/ml]). The median PrPc concentration in the CSF at baseline was 45 (IQR 31) [ng/ml]. The ApoE genotype was determined with 55% of the patients possessing at least one E4 allele as well as the PRNP C129 genotype with 40% being methionine homozygous, 19% being valine homozygous and 41% heterozygous.
Table 1. Baseline sociodemographic, medical and neuropsychological data
Pre-analyses aimed to uncover associations of CSF PrPc and other CSF biomarkers as well as possible influencing factors. GZLMs were applied using PrPc concentration as the dependent variable and the following as independent: age, gender, PPT, presence of diabetes mellitus, nicotine use, PRNP and ApoE genotypes, Aβ42, Aβ40, Tau, pTau. In an optimized GZLM (AICC 572) age, Aβ42, Aβ40 and Tau were included into the model. CSF PrPc was significantly associated with age (B = 0.56, p = 0.008), Aβ40 (B = 0.002, p = 0.029) and Tau levels (B = 0.016, p < 0.001).
The main analyses with scores on cognitive scales as the outcome variables consisted of ten GZLMs. At first, all variables were entered into the model, as explained above in the “methods” section. Those independent variables not associated with the outcome variable were subsequently excluded if this resulted in a reduction of the AICC indicating a better model fit. These optimized models are depicted in in detail. CSF PrPc, use of nicotine and the PRNP C129 genotype were not associated with the cognitive measures at all. Associations with many of the CERAD subscales could be seen for the pre-progression time (PPT; the longer, the worse the cognitive status), gender (females performed worse) and use of neuroleptics (worse cognitive performance). Patients using anti-dementia drugs also featured a lower status in some cognitive domains. shows the results in detail and p values are indicated. For steady variables coefficients (B) and standard errors (in brackets) are given indicating the association while possible effects of all other independent variables are controlled. For categorical data estimated marginal means and standard errors (in brackets) are shown. These are the estimated mean values of the dependent variables after the effects of all other independent variables are mathematically “eliminated” by the model.
Table 2. Fitted GZLMs
Discussion
Various studies have suggested a role of the Prion protein (PrPc) regarding cognitive function as well as in Alzheimer disease. Most of these data have originated in animal experiments conducted with rodents, while studies using primate or human tissue have been rather rare. In knockout mice potential neuroprotective effects of the physiological Prion protein have been demonstrated.Citation14,Citation17,Citation18 As mentioned above, PrPc’s role as a β-secretase regulator has been described thus linking PrPc to Aβ generation and Alzheimer disease.Citation2 Even more recently, evidence of PrPc acting as an Aβ oligomer receptor has emerged.Citation3-Citation6 Yet, the potential impact on physiologic processes associated with learning and memory such as long-term potentiation and synaptic plasticity have been discussed rather controversially. By acting as an Aβ oligomer receptor, initiation of cascades leading to neuronal injury and cell death have been proposed.Citation4,Citation19 Electrophysiologic studies also impute that PrPc mediates effects of Aβ assemblies.Citation20 In animal experiments it was possible to counteract detrimental effects of Aβ and rescue parts of cognitive function by applying antibodies directed against PrPc.Citation19,Citation21-Citation23 Considering all these findings, it stands to reason that further research on this topic is necessary, since PrPc might be useful as an AD biomarker as well as a potential therapeutic target.Citation24
Put into a clinical perspective, Meyne et al. were able to demonstrate levels of CSF PrPc to be decreased in various neurodegenerative diseases including AD.Citation8 Moreover, Breitling et al. discovered inverse associations of serum PrPc concentrations and cognitive status of healthy elderly subjects.Citation16 In this study we strived to reveal associations between concentrations of Prion protein in the CSF and cognitive status of Alzheimer patients at the time of diagnosis.
In our analysis, we were not able to find any association between CSF levels of PrPc and cognitive status as measured with the CERAD test battery in AD patients. These findings join the controversial results of MeyneCitation8 and BreitlingCitation16: On the one side, levels of PrPc appeared decreased in AD while on the other side lower concentrations of PrPc were associated with better cognitive function. Of course, these results are not entirely comparable, since different categories of subjects (healthy and AD) have been examined as well as PrPc levels in different compartments (plasma and CSF). Eventually, it is still not clear if and how these concentrations really reflect CSF levels in the brain tissue, let alone PrPc function. Considering these different compartments, it has to be kept in mind, that PrPc is expressed in the CNS as well as in peripheral tissues.Citation9 Transfer from brain to CSF is mediated by exosomal processes.Citation11 To cross the blood brain barrier, bi-directional transcytotic transport mechanisms have been shown to exist.Citation25
While it was not possible to establish a link between CSF PrPc levels, other associations were found. As to expect, the pre-progression time (a concept established by Doody et al.Citation26) was related to cognitive function: the longer the patients had already suffered from cognitive decline before the diagnosis of AD was formally established, the worse they performed on CERAD testing. Also expectable was the association of anti-dementia drug intake and worse cognitive function at time of diagnosis: It appears conceivable that before formal diagnosis was made some primary care physicians already had prescribed these medications in case patients were affected more severely. This explanation would correspond well to observations from the daily clinical routine. Furthermore, neuroleptic drug use was linked to worse cognitive function. This finding is consistent with previous reports.Citation27,Citation28 Yet, it can be interpreted in different ways—either as a risk factor for worsening of cognitive function or vice versa in terms of worse cognitive function being associated with more neuropsychiatric symptoms necessitating use of neuroleptic drugs. So is the observation of women presenting with a lower cognitive status here.Citation29,Citation30 Many more potentially influential variables have been included into the model, which did not feature associations with CERAD values. Incorporating these variables into the generalized linear models assured control of possible confounding effects. Still, residual confounding cannot be excluded entirely since other factors, which are unknown might exist and therefore have not been entered into the model.
Considering limitations and strengths of this analysis, first the sample size has to be mentioned. Approximately 100 patients is not a very large group in epidemiologic terms. Yet, it has to be considered, that although CSF biomarker analyses should have entered the routine of dementia diagnostics, the reality is different, thus rendering CSF of AD patients utilizable for scientific analyses a rare good. This relativizes the small sample size. Moreover, the use of updated research criteria requiring determination of CSF biomarkers to support the diagnostic process should have increased the certainty of the diagnoses as another strength of this study.
Methods
Patients included into this analysis were enrolled in the rpAD-study (rapidly progressive Alzheimer disease). The rpAD-study is a prospective, observational study comprising a broad spectrum of slowly as well as rapidly declining AD cases. The diagnoses were established using updated research criteria suggested by Dubois et al.Citation31 Essentially, these criteria resemble the classic McKhann criteriaCitation32 in many aspects but make use of supportive diagnostic tools such as CSF biomarker analyses and neuroimaging. At the time of diagnosis all patients underwent detailed neuropsychological testing (CERAD-NP plus Trailmaking Test ACitation33), neurological examinations and neuroimaging (CT or MRI). Blood samples were taken and a lumbar puncture was performed as part of the routine diagnostic process. CSF Aβ1–40 and Aβ1–42 as well as Tau and hyperphosphorylated Tau (pTau) were determined by standard methods using commercially available kits (Innogenetics). CSF PrPc was quantified using “BetaPrion EIA” kits by AJ Roboscreen.
All patients or legal caregivers provided informed consent. The study has been approved by the local ethics committee (Georg August University, votum No 9/6/08).
For the statistical analyses CERAD subscale raw values as well as a CERAD composite value were used as dependent variables. The CERAD subscales comprised: Minimental Status examination (MMSE), phonematic fluency (PhF), semantic fluency (SeF), Boston Naming Test (BNT), word learning (WL), word recall (WR), drawing (copying figures) (DC), drawing recall (figure recall) (DR) and Trailmaking Test A (TMT-A). A CERAD “composite” value was calculated using principal component analyses including all CERAD subscales. With this technique a single variable has been extracted from all CERAD subscale variables reflecting most of the variance observed in the CERAD subscale variables. The following variables were used as independent variables: PrPc concentration in the patients’ CSF, age, pre-progression time (PPT) (estimated time from first clinical manifestation of the disease until establishment of the diagnosisCitation34), gender, use of nicotine, presence of diabetes mellitus, use of benzodiazepines, use of antidepressants, use of neuroleptic drugs, use of anti-dementia drugs, Prion Protein Gene (PRNP) genotype, ApoE genotype and level of education (years of formal / institutional education).
Generalized linear models (GZLM) were fitted for each dependent variable. For pre-analyses all independent variables were included into the models. Subsequently, the models were fitted using only a number of selected independent variables that constituted an optimal subset in terms of model fit (backward selection). Akaike’s corrected information criterion (AICC) was used as an indicator of model fit (the lower, the better). The p values below 0.05 were considered significant. The reported p values were adjusted for multiple testing by means of the sequential Sidak procedure. SPSS version 20 (IBM corporation) was used to calculate the statistics.
Conclusion and Outlook
In summary, we were not able to establish an association between CSF levels of PrPc of Alzheimer disease patients at the time of diagnosis and their cognitive function. Thus, CSF PrPc in these cases appears not to be a useful biochemical surrogate for the cognitive status under the given circumstances. Still, these results do certainly not imply that CSF PrPc might not be useful as a diagnostic biomarker for AD. Yet, to evaluate this requires a different study approach including healthy controls. However, further research is necessary to put our results and the—on first glance converse—findings of BreitlingCitation16 into a broader perspective. Moreover, it could still be possible that CSF PrPc concentrations of AD patients may be associated with dynamic parameters such as the velocity of cognitive decline. Because the rpAD study, from which the patient sample analyzed here originated, is designed prospectively, follow-up data will be available in the near future. According analyses are going to be conducted.
| Abbreviations: | ||
| Aβ | = | Amyloid beta |
| AD | = | Alzheimer disease |
| APOE | = | apolipoprotein E gene |
| BNT, CERAD subscale | = | Boston Naming Test |
| CSF | = | cerebrospinal fluid |
| DC, CERAD subscale | = | Drawing (copying figures) |
| DR, CERAD subscale | = | Drawing recall (figure recall) |
| f | = | female |
| GZLM | = | generalized linear model |
| IQR | = | interquartile range |
| LTP | = | long term potentiation |
| m | = | male |
| MMSE, CERAD subscale | = | Minimental Status examination |
| PhF, CERAD subscale | = | Phonematic Fluency |
| PPT | = | pre-progression time |
| PRNP C129 | = | Prion Protein Gene Codon 129 |
| PrPc | = | physiological form of the Prion protein |
| pTau | = | hyperphosphorylized Tau |
| pts | = | points |
| SD | = | standard deviation |
| SE | = | standard error |
| SeF, CERAD subscale | = | Semantic Fluency |
| TMT-A, CERAD subscale | = | Trailmaking Test A |
| WL, CERAD subscale | = | Word Learning |
| WR, CERAD subscale | = | Word Recall |
Acknowledgment
This work was supported by a Bundesministerium für Bildung und Forschung grant within the German Network for Degenerative Dementia (KNDD-2, 2012–2015, determinants for disease progression in AD, grant No. 01GI1010C).
Disclosure of Potential Conflicts of Interest
The authors have nothing to disclose. No conflict of interest is reported.
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