PCSK9 Concentrations in Cerebrospinal Fluid Are Not Specifically Increased in Alzheimer’s Disease

Abstract

The role of PCSK9 in Alzheimer’s disease (AD) is controversial. We compared cerebrospinal fluid (CSF) PCSK9 concentrations in 36 AD and 31 non-AD patients. CSF PCSK9 levels did not differ between AD and non-AD groups (2.80 versus 2.62 ng/mL). However, PCSK9 CSF levels were increased in AD and non-AD patients with other neurodegenerative process (non-AD ND, n = 20) compared to patients without neurodegenerative disorders (non-ND, n = 11): 2.80 versus 2.30 (p < 0.005) and 2.83 versus 2.30 ng/mL (p = NS), respectively. CSF PCSK9 were positively correlated with AD biomarkers (Aβ_1-42, T-tau, and P-tau). PCSK9 concentrations in CSF are increased in neurodegenerative disorders rather than specifically in AD.

Keywords

cerebrospinal fluid frontotemporal lobar degeneration LDL-cholesterol P-tau PCSK9 T-tau

INTRODUCTION

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a critical regulator of LDL-cholesterol (LDL-C) metabolism. Indeed, the canonical role of PCSK9 is to act as an endogenous inhibitor of the LDL receptor (LDLR) pathway [1]. Genetic studies demonstrated that Pcsk9 gain-of-function mutations are associated with autosomal dominant hypercholesterolemia and premature atherosclerosis [2], while Pcsk9 loss-of-function variants conversely lead to low levels of LDL-C and cardiovascular protection [3]. Briefly, after intracellular autocleavage, PCSK9 is secreted by the liver in the circulation, binds to the extracellular domain of the LDLR at the cell surface of the hepatocytes, and targets it to lysosomes for degradation [1]. Pharmacological inhibition of PCSK9 by PCSK9 inhibitors (i.e., human monoclonal antibodies directed against PCSK9) leads to a ≈ 60% reduction of LDL-C and a significant decrease of major cardiovascular events [4 –6].

Beyond its role in the liver, an increasing body of evidence suggests that PCSK9 can also exert some extra-hepatic actions [7]. PCSK9 has been cloned as the ninth member of proprotein convertases in primary cerebellar neurons under apoptosis and was initially called NARC-1 (neural apoptosis-regulated convertase 1) [8]. PCSK9 seems to play a role in neurogenesis since its overexpression in a primary culture of embryonic neural progenitor cells stimulates the differentiation of cortical neurons [8]. While PCSK9 downregulates LDLR levels during brain development in mice [9], total PCSK9 knockout mice did not show signs of altered central nervous system (CNS) development [10]. Beside LDLR, PCSK9 also promotes the degradation of the apolipoprotein E receptor E2 (ApoER2) [11], which is involved in the regulation of neuronal apoptosis [12].

A more controversial issue is the role of PCSK9 on amyloid deposition and Alzheimer’s disease (AD). PCSK9 has been shown to modulate the disposal of BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1), the enzyme involved in the generation of the amyloid β-peptides 1–40 and 1–42 (Aβ), with increased levels of BACE1 and total Aβ in the brain of PCSK9 knockout mice [13]. These results were, however, not confirmed in a subsequent study [14]. Recently, Zimetti et al. reported that PCSK9 levels in cerebrospinal fluid (CSF) were higher in AD than in non-AD subjects [15].

Here, we compared CSF PCSK9 in AD and non-AD patients (including subgroups of patients with (non-AD ND) and without (non-ND) neurodegenerative disorders) and investigated the correlations between PCSK9 and biomarkers of AD [Aβ_1-42, total tau (T-tau), and phosphorylated tau (P-tau)] in CSF.

PATIENTS AND METHODS

Patients

CSF samples from patients with the three biomarkers in favor of biological AD (Aβ_1-42 <700 ng/L, T-tau≥350 ng/L and P-tau≥60 ng/L) (n = 36) and age- and gender-matched non-biological AD subjects, with the three biomarkers normal (Aβ_1-42 >800 ng/L, T-tau <350 ng/L, and P-tau <50 ng/L) (n = 31) were collected at the Neurological Memory Centre, Nantes University Hospital from April 2013 to May 2014. CSF studies were performed for routine biological diagnosis for patients presenting with cognitive impairment suspected to be AD or related disorders. All procedures followed were in accordance with the Helsinki Declaration of 1964, as revised in 2013. The biocollection was approved by the local ethical committee and the patients given their written informed consent.

Clinical diagnosis

The diagnoses were retained from the medical records. They were made according to clinical criteria, before the results of CSF biomarkers. Patients fulfilled the diagnosis criteria for probable AD dementia with evidence of the AD pathophysiological process [16], frontotemporal lobar degeneration (FTLD) [17], primary progressive aphasia [18], progressive supranuclear palsy [19], corticobasal syndrome [20], Lewy body disease [21], or multiple sclerosis [22].

Biochemical analyses

CSF were obtained by lumbar puncture. The transport to the laboratory was performed within 1 hour and CSF samples were centrifuged at 2,100 g for 10 min at 4°C within 2 h. A part of CSF was used for routine analysis, including measurement of total cells, total protein, and glucose level. CSF and serum IgG and albumin levels were measured by immunochemical nephelometry method using Immage^® 800 Analyzer (Beckman Coulter, Villepinte, France). In all patients, blood-CSF-barrier (B-CSF-B) dysfunction was determined by the CSF/serum albumin quotient (QAlb).

In the same time, CSF samples were aliquoted in polypropylene tubes of 2 mL and stored at –80°C until measurement of AD biomarkers were done. CSF Aβ_1-42, T-tau, and P-tau were measured in duplicate using commercially available sandwich ELISAs Innotest, (Fujirebio, Les Ulis, France) following the manufacturer’s instruction. PCSK9 concentrations were assayed in duplicate using a commercially available quantitative sandwich ELISA assay (Circulex CY-8079; CycLex Co, Nagano, Japan) and following the manufacturer’s instructions, as previously described [23].

Statistical analysis

Results were presented as median (interquartile range), minimum, and maximum. Categorical variables were presented as counts (proportions). The Mann–Whitney-U test and Kruskal Wallis test were performed to test for statistical differences in continuous parameters between two groups or three groups. The χ² or the Fisher exact test (based on expected frequency) were used to compare categorical variables between groups. A Bonferroni correction was used for multiple comparisons. Spearman’s correlations and linear regression were used to determine the association between PCSK9 levels and the biomarkers of AD in CSF. Statistical significance was set at p < 0.05 or p < 0.02 (with Bonferroni correction). Statistical analysis was performed using SAS, version 9.4 (SAS Institute, Cary, North Carolina).

RESULTS

Baseline characteristics of the patients

The baseline clinical and biological characteristics of patients are listed in Table 1. There was no significant difference between AD and non-AD patients for gender (≈60% female) and age (≈68 years). Non-AD patients were classified into the following two subgroups according to their final clinical diagnosis: non-AD neurodegenerative disorders (non-AD ND, n = 20) and non-AD non-neurodegenerative diseases (non-ND, n = 11). FTLD was the most frequent diagnosis in the non-AD ND group, while alcohol abuse and normal pressure hydrocephalus were the most common causes in non-ND group. Mean Mini-Mental State Examination (MMSE) score was <25 in all three groups. As stated in the methods section, AD patients had a typical AD neurobiomarker pattern, with a significant decrease in Aβ_1-42 and a concomitant increase in T-tau and P-tau, whereas neurobiomarkers were normal in the non-AD group. There was no difference in the CSF neurobiomarker profile between the non-AD ND and non-ND subgroups. Markers of brain-blood barrier permeability, such as QAlb and IgG index all were comparable between all three groups.

Table 1

Demographic data, clinical diagnosis, and biological parameters

		non-AD (n = 31)
	AD (n = 36)	non-AD ND (n = 20)	Non-ND (n = 11)	p-value
Demographics
Female sex, n (%)	22 (61%)	12 (60%)	7 (64%)	0.98
Age (years)	68 [55–82]	64.5 [53–84]	72 [46–78]	0.51
MMSE	21.5 [6–29]	24.5 [11–29]	23 [10–29]	0.14
Clinical diagnosis
Alzheimer’s disease	36	–	–
Amnestic presentation	26
Mixed dementia (vascular cognitive impairment and AD)	2
Aphasic presentation	2
Posterior cortical atrophy	2
Parietal presentation	2
Executive presentation	1
Amyloid precursor protein duplication	1
Frontotemporal lobar degeneration	–	15	–
Progressive supranuclear palsy	–	3	–
Behavioral variant	–	7	–
Non fluent primary progressive aphasia	–	2	–
Semantic variant primary progressive aphasia	–	3	–
Lewy body dementia	–	2	–
Multiple sclerosis	–	2	–
Atypical parkinsonian syndrome, without criteria for FTLD or LBD	–	1	–
Alcohol abuse	–	–	3
Normal pressure hydrocephalus	–	–	3
Temporal epilepsy	–	–	1
Endocarditis	–	–	1
Memory complaints, without sign of neurodegeneration	–	–	1
Depression	–	–	1
Psychogenic	–	–	1
Biological parameters (CSF)
Aβ_1-42 (ng/L)	522 [367–695]	1249 [812–1727]*	1095 [<891–1339]^α	<0.001
T-Tau (ng/L)	715 [388–1874]	251 [157–319117–284]^α	<0.001
p-Tau (ng/L)	91 [62–189]	43 [24–56]*	31 [27–49]^α	<0.001
QAlb (CSF albumin/serum albumin) (normal value <8.10³)	8.3 [3.1–15.8]	7.3 [3.5–10.1]	5.6 [2.8–9.5]	0.16
Index IgG (QIgG/QAlb) (normal value <0.065)	0.46 [0.38–0.58]	0.49 [0.40–1.01]	0.48 [0.42–0.53]	0.25

AD, Alzheimer’s disease; MMSE, Mini-Mental State Examination; NS, not significant; CSF, cerebrospinal fluid; FTLD, Frontotemporal lobar degeneration; LBD, Lewy body dementia. Data are expressed as Mediane [Min-Max]; Nonparametric two-sided Kruskal Wallis test was applied to compare the three groups. *Nonparametric two-sided Mann Whitney test to compare AD versus other neurodegenerative process group (p < 0.001). ^αNonparametric two-sided Mann Whitney test to compare AD versus Non-degenerative group (p < 0.001).

CSF PCSK9 levels

There were no significant differences in CSF PCSK9 concentration between AD and non-AD patients (median [IQ25-IQ75]: 2.80 [2.34–3.63] versus 2.62 [1.73–2.98] ng/mL, p = 0.14) and between AD and the non-AD ND subgroup (2.80 [2.34–3.63] versus 2.83 [2.02–4.59] ng/mL, p = 0.90). A significant difference was only observed when AD group was compared to the non-ND group (2.80 [2.34–3.63] versus 2.30 [1.60–2.62] ng/mL, p = 0.005). In non-AD patients, CSF PCSK9 levels were increased in non-AD ND group compared to non-ND group but this difference did not reach statistical significance (2.83 [2.02–4.59] versus 2.30 [1.60–2.62] ng/mL, p = 0.04) (Fig. 1).

Fig.1

CSF PCSK9 levels in Alzheimer’s disease (AD), non-AD non neurodegenerative (non-AD ND), and non-neurodegenerative (non-ND) groups. Data are expressed as median (IQ25; IQ75) in ng/mL; Non-parametric two-sided Kruskal Wallis test was applied to compare the three groups (p = 0.03). Nonparametric two-sided Mann Whitney test with a Bonferroni correction (statistical significance: p < 0.017) was applied to compare AD versus non-AD ND (p = 0.90), AD versus non ND (p = 0.005), and non-AD ND versus ND (p = 0.04, NS). NS, not significant.

Correlation between CSF PCSK9 and AD biomarkers

As a next step, we investigated the correlations between PCSK9 and the neurobiomarkers of AD in the CSF (Fig. 2). PCSK9 was positively associated with both Aβ_1-42 (p = 0.02) and P-tau (p = 0.0004), regardless of patients subgroups. In addition, PCSK9 was also positively associated with T-tau, but only in AD (p = 0.004) and non-AD ND (p = 0.009) groups. Of note, PCSK9 was neither significantly associated with markers of B-CSF-B permeability nor MMSE score.

Fig.2

Tau, P-Tau, and Aβ_1-42 levels (ng/L) in CSF as a function of PCSK9 levels (ng/mL) in Alzheimer’s disease (AD, triangles), non-AD non neurodegenerative (non-AD ND, circles), and non-neurodegenerative (non-ND, squares) groups. PCSK9 was positively associated with both Aβ_1-42 (p = 0.02) and P-tau (p = 0.0004), regardless of patients subgroups, and with T-tau, but only in AD (p = 0.004) and non-AD neurodegenerative (p = 0.009) groups (Spearman’s correlations and linear regression).

DISCUSSION

It has been recently shown that PCSK9 levels in CSF are increased in AD versus non-AD subjects [15]. In the present study, we demonstrated that CSF PCSK9 concentrations are not specifically increased in AD since there was no significant difference when compared to patients with neurodegenerative processes (mainly FTLD). In addition, we showed for the first time that CSF PCSK9 concentrations are positively associated with neurobiomarkers of AD, such as Aβ_1-42, T-tau, and P-tau. Finally, the variations of CSF PCSK9 levels were not associated with changes in B-CSF-B permeability markers.

Despite some apparent discrepancy, our results are in accordance with those previously published by Zimetti et al. [15]. Indeed, the majority of the patients in their control non-AD group have non-neurodegenerative diseases (mainly psychiatric disorders and hydrocephalus) [15]. Accordingly, in our study CSF PCSK9 levels in AD patients were also significantly increased when compared to non-AD patients without neurodegenerative disease (non-ND group). We also find a clear trend for an increase of CSF PCSK9 levels in other neurodegenerative patients (non-AD ND), suggesting that the PCSK9 CSF level is linked to the neurodegenerative process but not specific for AD. Since the CSF biomarkers are strictly normal in this non-AD ND group, we exclude the participation of an AD process to the cognitive decline and to the increase of PCSK9. Importantly, CSF PCSK9 levels are not correlated with biomarkers of B-CSF-B permeability. Thus, increased CSF PCSK9 level seems to be a marker of neurodegeneration, not explained by an unspecific aggression of the CNS. However, it should be underlined that our sample size is small and does not allow to detect some small statistically significant difference, if it exists, between the AD and non-AD groups. Further studies in larger cohorts are required to clarify this point.

Interestingly, a significant correlation between CSF PCSK9 and T-tau, a marker that reflects neurodegeneration, was observed in AD and neurodegenerative groups only [24]. Several authors have shown that high T-tau level predict a rapid progression in AD [25 –27]. In a future study, it would be interesting to evaluate if higher PCSK9 level in CSF is associated with a worse cognitive evolution, arguing for a more aggressive neurodegenerative process.

A significant and positive correlation was observed between CSF PCSK9 and neurobiomarkers of AD: Aβ_1-42 and P-tau in all the three groups. This finding may suggest that PCSK9 may interfere with Aβ production, as suggested by one previous study in mouse [13]. However, a Japanese study found no link between genetic Pcsk9 polymorphisms and AD risk [30]. Finally, the results of the prospective randomized controlled trial EBBINGHAUS with evolocumab, a PCSK9 inhibitor, did not find any changes in neurocognitive tests assessed using the Cambridge Neuropsychological Test Automated Battery toll and in patient-reported changes in memory and executive function, compared to placebo after a short median follow-up of 2.2 years [31], even in patients achieving a very low LDL-C levels (i.e., <0.5 mmol/L) [32]. It should be underlined, however, that PCSK9 monoclonal antibodies only block the extracellular form of PCSK9 in the circulation and do not alter intracellular PCSK9 in the CNS.

The mechanism by which PCSK9 could be involved in neurodegenerative processes is unknown. One can speculate on a proapoptotic effect of PCSK9 on neurons [12, 28], or alterations of lipid homeostasis in CNS, following a downregulation of LDLR, ApoER2, VLDLR, or LRP1 [11, 29], generating neuronal injuring. In addition, it has been recently demonstrated that brain endothelial-specific LRP1 deletion increases Aβ brain accumulation in mouse [33]. Beside AD pathogenesis, there are some pre-clinical data suggesting that PCSK9 overexpression stimulates the differentiation of cortical neurons [8]. Conversely, the silencing of PCSK9 expression in zebrafish eggs impaired the early CNS development, leading to an embryonic lethality between 2 to 4 days after fertilization [34]. However, PCSK9 KO mice did not show any signs of altered CNS development, suggesting a species-specific dependent action of PCSK9 on neurogenesis [10]. In accordance with a potential role for PCSK9 in CNS development, it has been shown recently that reduced PCSK9 plasma concentrations could serve as a biomarker for neural tube defects (spina bifida) in human [35]. These observations during CNS development could let us hypothesize an implication of PCSK9 in neuronal plasticity also later in life, after apparition of a neurodegenerative process, explaining its elevation in these conditions. However, a link between PCSK9 and neuroplasticity has never been demonstrated: although an elevation of PCSK9 in brain after ischemic stroke was found in mice, no elevation of markers of neurogenesis was associated, and the mice’s behavior as well as the extend of lesions were the same in PCSK9 KO and wild-type mice [9].

Our study has several limitations that should be underlined. Due to the retrospective design and the small sample, we cannot exclude that we have missed some correlations due to selection bias or lack of statistical power. We have only collected CSF samples and we were unable to perform some correlation with plasma PCSK9 and neurodegenerative diseases, although CSF PCSK9 concentrations are not correlated with plasma PCSK9 levels in a previous study performed in healthy volunteers [36]. The observational design of the study allows us to draw some associations but not causation, regarding PCSK9 role in neurodegenerative diseases.

In conclusion, we demonstrated that CSF PCSK9 levels are increased in neurodegenerative diseases rather than specifically in AD and are positively correlated with neurobiomarkers of AD. Additional studies are warranted to unravel the exact role of PCSK9 in neurodegenerative processes.

Footnotes

ACKNOWLEDGMENTS

This work by supported by the Fondation LEDUCQ (13CVD03) (for BC) and from the Fundazione CARIPLO (for BC).

Authors’ disclosures available online ().

References

Seidah

, Awan

, Chrétien

, Mbikay

(2014) PCSK9: A key modulator of cardiovascular health. Circ Res 114, 1022–1036.

Abifadel

, Varret

, Rabes

, Allard

, Ouguerram

, Devillers

, Cruaud

, Benjannet

, Wickham

, Erlich

, Derré

, Villéger

, Farnier

, Beucler

, Bruckert

, Chambaz

, Chanu

, Lecerf

, Luc

, Moulin

, Weissenbach

, Prat

, Krempf

, Junien

, Seidah

, Boileau

(2003) Mutations in PCSK9 cause autosomal dominant hypercholesterolemia. Nat Genet 34, 154–156.

Cohen

, Boerwinkle

, Mosley

Jr , Hobbs

(2006) Sequence variations in PCSK9, low LDL, and protection against coronary heart disease. N Engl J Med 354, 1264–1272.

Farnier

, Gaudet

, Valcheva

, Minini

, Miller

, Cariou

(2016) Efficacy of alirocumab in high cardiovascular risk populations with or without heterozygous familial hypercholesterolemia: Pooled anlysis of eight ODYSSEY phase 3 clinical program trials. Int J Cardiol 223, 750–757.

Koren

, Sabatine

, Giugliano

, Langslet

, Wiviott

, Kassahun

, Ruzza

, Ma

, Somaratne

, Raal

(2017) Long-term low-density lipoprotein cholesterol-lowering efficacy, persistence, and safety of evolocumab in treatment of hypercholesterolemia: Results up to 4 years from the open-label OSLER-1 extension study. JAMA Cardiol 2, 598–607.

Sabatine

, Giugliano

, Keech

, Honarpour

, Wiviott

, Murphy

, Kuder

, Wang

, Liu

, Wasserman

, Sever

, Pedersen

, FOURIER Steering Committee and Investigators (2017) Evolocumab and clinical outcomes in patients with cardiovascular disease. N Eng J Med 376, 1713–1722.

Cariou

, Si-Tayeb

, Le May

(2015) Role of PCSK9 beyond liver involvement. Curr Opin Lipidol 26, 155–161.

Seidah

, Benjannet

, Wickham

, Marcinkiewicz

, Jasmin

, Stifani

, Basak

, Prat

, Chretien

(2003) The secretory proprotein convertase neural apoptosis-regulated convertase 1 (NARC-1): Liver regeneration and neuronal differentiation. Proc Natl Acad Sci U S A 100, 928–933.

Rousselet

, Marcinkiewicz

, Kriz

, Zhou

, Hatten

, Prat

, Seidah

(2011) PCSK9 reduces the protein levels of the LDL receptor in mouse brain during development and after ischemic stroke. J Lipid Res 52, 1383–1391.

10.

Rashid

, Curtis

, Garuti

, Anderson

, Bashmakov

, Ho

, Hammer

, Moon

, Horton

(2005) Decreased plasma cholesterol and hypersensitivity to statins in mice lacking Pcsk9. Proc Natl Acad Sci U S A 102, 5374–5379.

11.

Poirier

, Mayer

, Benjannet

, Bergeron

, Marcinkiewicz

, Nassoury

, Mayer

, Nimpf

, Prat

, Seidah

(2008) The proprotein convertase PCSK9 induces the degradation of low density lipoprotein receptor (LDLR) and its closest family members VLDLR and ApoER2. J Biol Chem 283, 2363–2372.

12.

Kysenius

, Muggalla

, Mätlik

, Arumäe

, Huttunen

(2012) PCSK9 regulates neuronal apoptosis by adjusting ApoER2 levels and signalling. Cell Mol Life Sci 69, 1903–1916.

13.

Jonas

, Costantini

, Puglielli

(2008) PCSK9 is required for the disposal of non-acetylated intermediates of the nascent membrane protein BACE1. EMBO Rep 9, 916–922.

14.

Liu

, Wu

, Baysarowich

, Kavana

, Addona

, Bierilo

, Mudgett

, Pavlovic

, Sitlani

, Renger

, Hubbard

, Fisher

, Zerbinatti

(2010) PCSK9 is not involved in the degradation of LDL receptors and BACE1 in the adult mouse brain. J Lipid Res 51, 2611–2618.

15.

Zimetti

, Caffarra

, Ronda

, Favari

, Adorni

, Zanotti

, Bernini

, Barocco

, Spallazzi

, Galimberti

, Ricci

, Ruscica

, Corsini

, Ferri

(2017) Increased PCSK9 cerebrospinal fluid concentrations in Alzheimer’s disease. J Alzheimers Dis 55, 315–320.

16.

McKhann

, Knopman

, Chertkow

, Hyman

, Jack

, Kawas

, Klunk

, Koroshetz

, Manly

, Mayeux

, Mohs

, Morris

, Rossor

, Scheltens

, Carrillo

, Thies

, Weintraub

, Phelps

(2011) The diagnosis ofdementia due to Alzheimer’s disease: Recommendations from the National Institute on Aging-Alzheimer’s Associationworkgroups on diagnostic guidelines for Alzheimer’s disease. Alzheimers Dement 7, 263–269.

17.

Rascovsky

, Hodges

, Knopman

, Mendez

, Kramer

, Neuhaus

, van Swieten

, Seelaar

, Dopper

, Onyike

, Hillis

, Josephs

, Boeve

, Kertesz

, Seeley

, Rankin

, Johnson

, Gorno-Tempini

, Rosen

, Prioleau-Latham

, Lee

, Kipps

, Lillo

, Piguet

, Rohrer

, Rossor

, Warren

, Fox

, Galasko

, Salmon

, Black

, Mesulam

, Weintraub

, Dickerson

, Diehl-Schmid

, Pasquier

, Deramecourt

, Lebert

, Pijnenburg

, Chow

, Manes

, Grafman

, Cappa

, Freedman

, Grossman

, Miller

(2011) Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia. Brain 134, 2456–2477.

18.

Gorno-Tempini

, Hillis

, Weintraub

, Kertesz

, Mendez

, Cappa

, Ogar

, Rohrer

, Black

, Boeve

, Manes

, Dronkers

, Vandenberghe

, Rascovsky

, Patterson

, Miller

, Knopman

, Hodges

, Mesulam

, Grossman

(2011) Classification of primary progressive aphasia and its variants. Neurology 76, 1006–1014.

19.

Litvan

, Agid

, Calne

, Campbell

, Dubois

, Duvoisin

, Goetz

, Golbe

, Grafman

, Growdon

, Hallett

, Jankovic

, Quinn

, Tolosa

, Zee

(1996) Clinical research criteria for the diagnosis of progressive supranuclear palsy (Steele-Richardson-Olszewski syndrome): Report of the NINDS-SPSP international workshop. Neurology 47, 1–9.

20.

Boeve

(2011) The multiple phenotypes of corticobasal syndrome and corticobasal degeneration: Implications for further study. J Mol Neurosci 45, 350–353.

21.

McKeith

, Boeve

, Dickson

, Halliday

, Taylor

J-P

, Weintraub

, Aarsland

, Galvin

, Attems

, Ballard

, Bayston

, Beach

, Blanc

, Bohnen

, Bonanni

, Bras

, Brundin

, Burn

, Chen-Plotkin

, Duda

, El-Agnaf

, Feldman

, Ferman

, Ffytche

, Fujishiro

, Galasko

, Goldman

, Gomperts

, Graff-Radford

, Honig

, Iranzo

, Kantarci

, Kaufer

, Kukull

, Lee

VMY

, Leverenz

, Lewis

, Lippa

, Lunde

, Masellis

, Masliah

, McLean

, Mollenhauer

, Montine

, Moreno

, Mori

, Murray

, O’Brien

, Orimo

, Postuma

, Ramaswamy

, Ross

, Salmon

, Singleton

, Taylor

, Thomas

, Tiraboschi

, Toledo

, Trojanowski

, Tsuang

, Walker

, Yamada

, Kosaka

(2017) Diagnosis and management of dementia with Lewy bodies: Fourth consensus report of the DLB Consortium. Neurology 89, 88–100.

22.

Polman

, Reingold

, Banwell

, Clanet

, Cohen

, Filippi

, Fujihara

, Havrdova

, Hutchinson

, Kappos

, Lublin

, Montalban

, O’Connor

, Sandberg-Wollheim

, Thompson

, Waubant

, Weinshenker

, Wolinsky

(2011) Diagnostic criteria for multiple sclerosis: 2010 revisions to the McDonald criteria. Ann Neurol 69, 292–302.

23.

Cariou

, Langhi

, Le Bras

, Bortolotti

, Lê

, Theytaz

, Le May

, Guyomarc’h-Delasalle

, Zaïr

, Kreis

, Boesch

, Krempf

, Tappy

, Costet

(2013) Plasma PCSK9 concentrations during an oral fat load and after short term high-fat, high-fat high-protein and high fructose diets. Nutr Metab (Lond) 10, 4.

24.

Blennow

, Hampel

, Weiner

, Zetterberg

(2010) Cerebrospinal fluid and plasma biomarkers in Alzheimer disease. Nat Rev Neurol 6, 131–144.

25.

Degerman Gunnarsson

, Ingelsson

, Blennow

, Basun

, Lannfelt

, Kilander

(2016) High tau levels in cerebrospinal fluid predict nursing home placement and rapid progression in Alzheimer’s disease. Alzheimers Res Ther 8, 22.

26.

Kester

, van der Vlies

, Blankenstein

, Pijnenburg

, van Elk

, Scheltens

, van der Flier

(2009) CSF biomarkers predict rate of cognitive decline in Alzheimer disease. Neurology 73, 1353–1358.

27.

Wallin

, Blennow

, Zetterberg

, Londos

, Minthon

, Hansson

(2010) CSF biomarkers predict a more malignant outcome in Alzheimer disease. Neurology 74, 1531–1537.

28.

Bingham

, Shen

, Kotnis

, Lo

, Ozenberger

, Ghosh

, Kennedy

, Jacobsen

, Grenier

, DiStefano

, Chiang

, Wood

(2006) Proapoptotic effects of NARC1 (=PCSK9), the gene encoding a novel serine proteinase. Cytometry A 69, 1123–1131.

29.

Canuel

, Sun

, Asselin

, Paramithiotis

, Prat

, Seidah

(2013) Proprotein convertase subtilsin/kexin type 9 (PCSK9) can mediate degradation of the low density lipoprotein receptor-related protein 1 (LRP-1). Plos One 8, e64145.

30.

Shibata

, Ohnuma

, Higashi

, Usui

, Ohkubo

, Watanabe

, Kawashima

, Kitajima

, Ueki

, Nagao

, Arai

(2005) No genetic association between PCSK9 polymorphisms and Alzheimer’s disease and plasmacholesterol levels in Japanese patients. Psychiatr Genet 15, 239.

31.

Giugliano

, Mach

, Zavitz

, Kurtz

, Im

, Kanevsky

, Schneider

, Wang

, Keech

, Pedersen

, Sabatine

, Sever

, Robinson

, Honarpour

, Wasserman

, Ott

, EBBINGHAUS Investigators (2017) Cognitive function in a randomized trial of evolocumab. N Engl J Med 377, 633–643.

32.

Giugliano

, Pedersen

, Park

, De Ferrari

, Gaciong

, Ceska

, Toth

, Gouni-Berthold

, Lopez-Miranda

, Schiele

, Mach

, Ott

, Kanevsky

, Lira Pineda

, Somaratne

, Wasserman

, Keech

, Sever

, Sabatine

, FOURIER Investigators (2017) Clinical efficacy and safety of achieving very low LDL-cholesterol concentrations with the PCSK9 inhibitor evolocumab: A prespecified secondary analysis of the FOURIER trial. Lancet 390, 1962–1971.

33.

Storck

, Meister

, Nahrath

, Meißner

, Schubert

, Di Spiezio

, Baches

, Vandenbroucke

, Bouter

, Prikulis

, Korth

, Weggen

, Heimann

, Schwaninger

, Bayer

, Pietrzik

(2016) Endothelial LRP1 transport amyloid-β(1-42) across the blood-brain barrier. J Clin Invest 126, 123–136.

34.

Poirier

, Prat

, Marcinkiewicz

, Paquin

, Chitramuthu

, Baranowski

, Cadieux

, Bennett

, Seidah

(2006) Implication of the proprotein convertase NARC-1/PCSK9 in the development of the nervous system. J Neurochem 98, 838–850.

35.

, Wei

, Li

, Huang

, Zhao

, Liu

, Wang

, Chen

, Ma

, Zhang

, Cao

, Yuan

(2015) Identification of PCSK9 as a novel serum biomarker for the prenatal diagnosis of neural tube defects using iTRAQ quantitative proteomics. Sci Rep 5, 17559.

36.

Chen

, Troutt

, Konrad

(2014) PCSK9 is present in human cerebrospinal fluid and is maintained at remarkably constant concentrations throughout the course of the day. Lipids 49, 445–455.