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Wozniak 1

Apolipoprotein E-ε4 deficiency and cognitive function in hepatitis C virus -infected patients

M. A. Wozniak, PhD,1 L. M. Lugo Iparraguirre,2 M. Dirks, MD,2 M. Deb-Chatterji,MD,2 H.

Pflugrad,MD,2 A. Goldbecker,MD,2 A. B.Tryc, MD,2 H.Worthmann,MD,2 M. Gess, MD, 3 M. M.E.

Crossey,4 D. M. Forton,MD,3 S. D. Taylor-Robinson, Professor,4 R. Itzhaki, Professor,1* K.

Weissenborn,Professor.2

1Faculty of Life Sciences, University of Manchester, Manchester M60 1QD, United Kingdom

2Dept. of Neurology, Hannover Medical School, 30623 Hannover, Germany

3Dept. of Gastroenterology and Hepatology, St George’s Hospital and Medical School, London

SW17 0QT, United Kingdom

4 Dept. of Medicine, St Mary´s Hospital Campus, Imperial College London, London W2 1NY,

United Kingdom

*Present address: Nuffield Dept. Clinical Neurosciences, University of Oxford, Level 6, West

Wing, John Radcliffe Hospital, Oxford, OX3 9DU

Corresponding Author:

Professor Karin Weissenborn

Department of Neurology

Hannover Medical School,

30623 Hannover, Germany

Phone: 0049-511-532-2339; Fax: 0049-511-532-3115;

e-mail: [email protected]

running title: ApoE and cognition in HCV patients

mailto:[email protected]

Wozniak 2

e-mail addresses of all authors:

Matthew A Wozniak: [email protected]

Lea Margrit Lugo Iparraguirre: [email protected]

Meike Dirks: [email protected]

Milani Deb-Chatterji: [email protected]

Henning Pflugrad: [email protected]

Annemarie Goldbecker: [email protected]

Anita Blanka Tryc: [email protected]

Hans Worthmann: [email protected]

Markus Gess: [email protected]

Mary ME Crossey: [email protected]

Daniel M Forton: [email protected]

Simon D Taylor-Robinson: [email protected]

Ruth Itzhaki: [email protected]

Karin Weissenborn: [email protected]

The statistical analysis of the data was conducted by M.A. Wozniak; Faculty of Life Sciences,

University of Manchester, Manchester M60 1QD, United Kingdom

Supplemental references and tables are available electronically (SR-1 to SR-10, table e-1, table e-2)

Study funding: The genotyping was partially financed by a grant from Pfizer Inc. to SDT-R. SDT-R

and MMEC are grateful to the United Kingdom National Institute for Health Research (NIHR)

Biomedical Facility at Imperial College London for infrastructure support. MMEC is supported by

a Fellowship from the Sir Halley Stewart Trust (Cambridge, UK).



Wozniak 3

Search terms: All Clinical Neurology [14], All Cognitive Disorders [25], Viral Infections [142

Word count:

Abstract: 227 words

Main Text: 2878 words

Number of tables: 2

Number of figures: 0

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Authors’ Contributions

Matthew A Wozniak: study concept and design, APO E genotyping, analysis and interpretation of

data, statistical analysis, drafting of the manuscript; critical revision of the manuscript for important

intellectual content

Lea Margret Lugo Iparraguirre: recruitment of participants, psychometric testing, analysis and

interpretation of the data, critical revision of the manuscript for important intellectual content

Meike Dirks: recruitment of participants, psychometric testing, analysis and interpretation of the

data, critical revision of the manuscript for important intellectual content

Milani Deb-Chatterji: recruitment of participants, critical revision of the manuscript for important


Henning Pflugrad: recruitment of participants, psychometric testing, analysis and interpretation of

the data, critical revision of the manuscript for important intellectual content

Annemarie Goldbecker: recruitment of participants, analysis and interpretation of the data, critical

revision of the manuscript for important intellectual content

Anita Blanka Tryc: recruitment of participants, critical revision of the manuscript for important


Hans Worthmann: recruitment of participants, critical revision of the manuscript for important


Markus Gess: critical revision of the manuscript for important intellectual content

Mary ME Crossey: critical revision of the manuscript for important intellectual content

Daniel M Forton: drafting of the manuscript, critical revision of the manuscript for important


Simon D Taylor-Robinson: drafting of the manuscript, critical revision of the manuscript for

important intellectual content, obtained funding

Ruth Itzhaki: study concept, interpretation of data; drafting of the manuscript; critical revision of

the manuscript for important intellectual content

Wozniak 5

Karin Weissenborn: study concept and design, recruitment of participants, analysis and

interpretation of data, drafting of the manuscript; critical revision of the manuscript for important


Study funding: The genotyping was partially financed by a grant from Pfizer Inc. to SDT-R. SDT-R

and MMEC are grateful to the United Kingdom National Institute for Health Research (NIHR)

Biomedical Facility at Imperial College London for infrastructure support. MMEC is supported by

a Fellowship from the Sir Halley Stewart Trust (Cambridge, UK).

Disclosures

Matthew A Wozniak reports no disclosures.

Lea Margrit Lugo Iparraguirre reports no disclosures

Meike Dirks reports no disclosures

Milani Deb-Chatterji reports no disclosures

Henning Pflugrad reports no disclosures

Annemarie Goldbecker reports no disclosures

Anita Blanka Tryc reports no disclosures

Hans Worthmann reports no disclosures

Markus Gess reports no disclosures

Mary ME Crossey reports no disclosures

Daniel M Forton reports no disclosures

Simon D Taylor-Robinson reports a grant from Pfizer Inc. that helped financing the genotyping

Ruth Itzhaki reports no disclosures

Karin Weissenborn reports no disclosures

Wozniak 6

List of abbreviations

HCV – hepatitis C virus

APOE - apolipoprotein E gene

apo E – apolipoprotein E protein

HIV – human immunodeficiency virus

DNA - deoxyribonucleic acid

PCR - Polymerase-Chain-Reaction

TAP- test battery for the assessment of attention

HADS - Hospital Anxiety and Depression Scale

BDI - Beck Depression Inventory

FIS - Fatigue Impact Scale

WFMT - word-figure memory test

RFT - Recurring Figures Test

BBB – blood brain barrier

MRS magnetic resonance spectroscopy

PET - positron emission tomography

Wozniak 7

Abstract

Objective: Hepatitis C virus causes not only liver damage in certain patients but can also lead to

neuropsychiatric symptoms. Previous studies have shown that the type 4 allele of the gene for

apolipoprotein E is strongly protective against hepatitis C virus-induced damage in liver. In the

present study, we have investigated the possibility that APOE genotype is involved in the action of

hepatitis C virus in brain.

Methods: 100 hepatitis C virus-infected patients with mild liver disease underwent a neurological

examination and a comprehensive psychometric testing of attention and memory function. In

addition, patients completed questionnaires for the assessment of fatigue, health-related quality of

life and mood disturbances. APOE-genotyping was done on saliva using buccal swabs.

Results: The APOE-ε4 allele frequency was significantly lower in patients with an impairment of

working memory, compared to those with a normal working memory test result (p=0.003). A lower

APOE-ε4 allele frequency was also observed in patients with definitely altered attention ability

(p=0.008), but here the p-value missed the level of significance after application of the Bonferroni

correction.

Conclusions: Our data suggest that the APOE-ε4 allele is protective against attention deficit and

especially against poor working memory in HCV-infected subjects with mild liver disease.

Considering the role of apolipoprotein E in the life cycle of the virus the findings shed interesting

new light upon possible pathomechanisms behind the development of neuropsychiatric symptoms

in hepatitis C infection.

Keywords: virus replication, neurodegeneration, cerebral lipoprotein metabolism, attention, HCV

encephalopathy

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Introduction

Current World Health Organisation estimates suggest that, globally, 170 million people are infected

with hepatitis C virus (HCV), with 3-4 million new infections each year. Like many infectious

agents, HCV causes a variety of outcomes: patients can experience no symptoms, but liver

involvement may encompass a spectrum from mild liver disease, cirrhosis to liver cancer. A

considerable amount number of patients also exhibit neuropsychiatric symptoms, such as

depression, fatigue, and deficits in attention, concentration and memory.1 The reason why there are

different clinical syndromes probably reflects both underlying host and viral factors. In the case of

liver disease caused by HCV, one factor that is involved is apolipoprotein E (gene: APOE; protein:

apoE). This polymorphic gene has three common alleles (APOE-ε2, -ε3 and -ε4) that give rise to

three common protein isoforms (apoE2, apoE3 and apoE4).2 The main functions of the apoE protein

are the transport of lipids around the body and the repair of damaged tissue.2 APOE is also a key

determinant of the outcome of infection, with possession of certain alleles conferring risk of, or

protection against, damage caused by several infectious agents, including HIV3, Herpes simplex

virus types 14 and 25 ,Mycobacterium tuberculosis6 and Plasmodium falciparum7 For HCV, the

APOE-ε4 allele confers protection against severe liver disease.8 Subsequent studies have confirmed

the beneficial role of APOE-ε4 in HCV disease: Richardson and colleagues9 demonstrated that

HCV-induced liver damage progression was more rapid in those who lacked an APOE-ε4 allele;

Price and colleagues10 found that the APOE-ε2 and APOE-ε4 alleles were both associated with

increased clearance of the virus and that the APOE-ε3 allele was a risk for HCV persistence; and

Toniutto and colleagues11,12 showed that HCV-infected individuals who received a liver transplant

and had an APOE-ε4 allele had a slower liver disease progression rate than recipients carrying the

other alleles.

We hypothesised that HCV-induced development of neuropsychiatric symptoms might also depend

on possession of a particular APOE allele.. We therefore investigated the role of APOE in the

occurrence of neuropsychiatric complications in HCV-infected patients.

Ruth Itzhaki, 19/02/15,

Why is APOE italicised - just in this section?


not ilalicised

Wozniak 9

Materials and Methods

Patients

100 HCV-infected patients with mild liver disease (32 male) aged 25-75 years were recruited at

Hannover Medical School, Hannover, Germany. 84 patients were infected with HCV genotype 1.

Eighty-one patients were HCV-RNA positive. Forty-six patients had undergone interferon treatment

several years previously. The time period since infection was more than 25 years in most of those in

whom the route of transmission was clear. The majority of patients had been infected via blood

transfusion (n=37), or by treatment with immunoglobulins (n=34). Seven patients had a history of

intravenous drug abuse more than 25 years ago. In four patients, occupational transmission was

considered likely, and in one, maternal transmission. In 17 patients, the route of transmission

remained unclear. Clinical findings, liver biopsy results and/or APRI scores showed that none of the

patients had cirrhosis. Patients with concomitant diseases, or with medication which might impair

brain function, were excluded from the study, as were patients with ongoing alcohol or drug abuse.

Clinical assessment

Besides neurological examination the patients underwent comprehensive psychometric testing. In

addition, they completed questionnaires for the assessment of health-related quality of life and

mood disturbances. The psychometric test battery applied comprised the PSE-Syndrome-TestSR-1 the

cancelling “d”-testSR-2 and several subtests of the TAP-battery, a battery of attention testsSR-3.

Furthermore, the Hospital Anxiety and Depression Scale (HADS)SR-4, Beck Depression Inventory

(BDI)SR-5, the Fatigue Impact Scale (FIS)SR-6, and the SF-36 questionnaireSR-7 were performed.

Seventy-four patients performed Luria’s list of wordsSR-8, the word-figure memory test (WFMT)SR-9

and the Recurring Figures Test (RFT)SR-10 for the assessment of learning ability and memory.

The individual results of the cancelling “d”-test, the TAP-battery subtests and the RFT were

compared to the available norm data and expressed as percentiles. Since in practice percentiles less

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than 10 are considered clinically relevant, the 10th percentile (accordingly z-scores ≤ -1.3) was used

as cut-off between normal and abnormal results. To achieve a sum score for each patient’s

individual attention ability, we calculated the ratio between of the maximal achievable number of

abnormal results and to the patient’s number of abnormal results (attention test sum score).

Attention tests results were summarized in a score ranging from 0-1. Scores above 0.35 are were

considered abnormal.

Test results of the WFMT are were expressed as z-scores after comparison with normal data,

adjusted for age and education. In accordance with the evaluation of the attention tests, z-scores ≤ -

1.3 were scored as abnormal. PSE-test results of less than -4 points were regarded as abnormal, as

recommended in the test manual.

APOE genotyping

Buccal swabs were used for APOE genotyping. DNA was prepared from the swabs using the

Nucleospin tissue preparation kit (ABgene, Epsom, UK) according to manufacturer’s instructions.

Swabs were left in PBS for 1 hour and then proteinase K and lysis buffer were added. After a 10-

min incubation at 70ºC, the DNA was ethanol-precipitated, bound to a column, washed and then

eluted. The extracted DNA was checked for quality and quantity using the Nanodrop 1000

spectrophotometer (Thermo Scientific, Wilmington, USA) and stored at -20C. The APOE genotype

was then determined using the method of Wenham et al.13, but using Hotstartaq® (Qiagen, Crawley,

UK) and agarose gel electrophoresis.

Standard Protocol Approvals, Registrations, and Patient Consents

The study was approved by the institutional ethics committee and complied with the precepts set

out in the 1975 Helsinki Declaration on human rights. Patients were included after giving their

written, informed consent.


not italicised


Italics intentional?


Changed because you've used past tense elsewhere

Wozniak 11

Statistics

To determine the influence of APOE on the psychometric tests conducted, subjects were divided

into those with and those without a specific allele. For example, subjects were divided into APOE-

ε2 allele possessors (i.e., those subjects with genotypes APOE-ε2ε2, ε2ε3 and ε2ε4) and those who

did not possess an APOE-ε2 allele (i.e., APOE-ε3ε3, ε3ε4 and ε4ε4 subjects). The means of the

different tests were compared by ANOVA using SPSS version 15.0 for Windows. Similarly, to

determine the influence of the APOE-ε4 allele, subjects were divided into APOE-ε4 allele

possessors and non-possessors. The subjects were not divided into those with and those without an

APOE-ε3 as there were far too few subjects without an APOE-ε3 allele. For some categories,

subjects were divided into abnormal and normal scores and the APOE allele frequencies were

determined for each group. Statistical significance in these cases was determined using 2 analysis.

A Bonferroni correction was applied and results were considered significant if p≤0.003.

Results

There were no differences in any of the psychometric test results between APOE-ε2 allele

possessors and non-possessors, and the same was true for APOE-ε4 (Tables e-1 and e-2). There

were also no differences in terms of allele frequency between the groups when divided into those

individuals with abnormal and normal fatigue, depression and anxiety scores (data not shown).

However, there were statistically significant differences in the working memory test of the TAP

battery (table 1). In this test, the APOE- ε4 allele frequency was higher (25.9 %) in those patients

who performed normally on this task compared to patients who performed expressing an

abnormally high number of errors (4.5%, p=0.003).

The results were also analysed for males and females separately. In women, the APOE-ε4 allele

frequency was higher in those with correct responses (25%) compared to those with a high number

of errors (2.8%, p=0.004). A difference was also seen in the men (27.5 % v 12.5%) but this did not

reach statistical significance.

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There were further trends for a beneficial effect of the APOE-ε4 allele in performance in attention

tasks (Intermodal comparison test p=0.01, Incompatibility test p=0.05, Alertness test p=0.008) but

these did not meet our criterion for statistical significance (table 1). Finally, when we analysed the

attention tests sum score, the APOE-ε4 allele frequency was significantly higher in those with

preserved cognitive function compared to those with a high number of pathological scores (24.6 %

v 9.5 %, p=0.008). There were no significant results for the APOE-ε2 and APOE-ε3 alleles.

Memory function was not found to be related to the frequency of any of the APOE alleles (table 2).

Discussion

Patients with neuropsychiatric sequelae of HCV frequently complain of cognitive problems or

“brain fog”.14 A large number of studies have reported objectively measured impairments in

cognitive function, most frequently characterised by deficits in attention and working memory .14-18

The precise aetiology remains to be elucidated but imaging studies, including the use of cerebral

magnetic resonance spectroscopy (MRS) and positron emission tomography (PET), have reported

abnormalities consistent with immune activation within the CNS.14, 17-19 The two main explanations

offered for these findings are 1) the penetration of HCV through the blood brain barrier into the

CNS and 2) a cerebral effect of the peripheral immune response 16, 20-24

In relation to this, our main finding was that in HCV-infected subjects with mild liver disease the

APOE-ε4 allele frequency was significantly lower in those with an abnormal number of errors in the

working memory test, compared to those with a normal result. Other psychometric tests that assess

attention deficit also displayed a tendency to lower APOE-ε4 allele frequency, although the level of

significance did not hold up after Bonferroni correction. This suggests that the APOE-ε4 allele is

protective against attention deficit and especially against poor working memory in HCV-infected

subjects with mild liver disease. Given that impaired working memory is the commonest measured

Wozniak 13

impairment in these patients, it follows that the APOE-ε4 allele may in some way be protective

against the effect of HCV infection on the brain.

HCV infection is intimately linked to the metabolism of lipids within infected hepatocytes and

dysregulates the metabolism of circulating lipoproteins as well. HCV uses the very-low density

lipoprotein (VLDL) secretion pathway for the production of infectious particles, known as lipoviral

particles (LVP). There is evidence that in vivo, there is significant exchange of virions between

different lipoproteins. It has been suggested that the association of virions with lipoproteins enables

the virus to bind to lipoprotein receptors that are needed for entering the host cells, as well as to

hide from the host’s immune surveillance.25,26 The protein apoE has been found to be required for

HCV virion infectivity and production. Its level correlates with HCV infectivity.27 ApoE mediates

the binding of the virus to heparin sulphate proteoglycan (HSPG) receptors.28 HSPGs are

structurally heterogeneous and thereby serve as receptors for a multitude of viruses and cell types. It

is hypothesized that apoE-mediated binding of HCV to the HPSG receptor or LDL receptor (LDLr)

facilitates the interaction of the virus with other cell surface receptors for cell entry, such as CD81,

claudin, occludin, and SRBI.29

In one study, the level of apoE was found to correlate strongly with the LVP ratio, i.e. the ratio of

HCV RNA load in the highly infectious LVP to the total HCV RNA load in all plasma fractions.30

This and previous studies31 also show that apoE levels are lowest in those with APOE-ε4 alleles.

Thus, the APOE-ε4 genotype may potentially impair incorporation of HCV into LVP and reduce

the binding of HCV to cell surface receptors. In considering possible HCV infection of the CNS,

Fletcher and colleagues have shown that, in in vitro experiments, endothelial cells of the blood

brain barrier (BBB) express all the receptors necessary for viral entry. Furthermore, in vitro

infection of endothelial cells resulted in apoptosis and conformational change that might lead to a

leaky BBB.20 This raises the possibility that the APOE-ε4 genotype might protect against HCV-

Wozniak 14

mediated BBB permeability and viral entry. If HCV penetration into the CNS underlies the

observed cognitive impairment, this might explain the protective effect of APOE-ε4 in our study.

Another possible explanation is an interaction between HCV and lipid metabolism within the brain.

The human apoE protein has its highest expression in the liver and importantly, in the brain. In the

brain it is predominantly expressed by astrocytes and microglia in high-density lipoprotein (HDL)-

like particles. As in the periphery, apoE functions as a ligand in receptor-mediated endocytosis of

lipoprotein particles in the CNS. Experimental data hint at a possible effect of apoE in neuronal

sprouting, synaptogenesis and maintenance of synaptic connections after brain injury. A protective

role of APOE-ε4 in patients with HCV infection is somehow unexpected, in view of the role of

apoE4 in the development of dementia and in severe cognitive dysfunction in patients with HIV

encephalopathy.32,33 However, the difference may be explained by the interactions between HCV

and lipoproteins. It may be assumed that similar to the periphery, the apoE4 isoform hampers HCV

binding to the HPSG and LDL receptors present on the membrane of their target cells in the CNS,

namely astrocytes and microglial cells, and thereby impedes infection of the glial cells. To this end,

several groups have shown evidence for HCV replication within the CNS.21-23 Wilkinson et al.24

provided evidence for HCV replication in microglia and astrocytes in autopsy brain tissue. One

autopsy study on the brain of HIV/HCV infected subjects found the highest density of HCV

infected cells in the frontal white matter and basal ganglia, and a predominance of HCV

immunoreactive microglial cells around blood vessels.23 This finding suggests that the virus, similar

to the human immunodeficiency virus (HIV), enters the brain as a “blind passenger”, using

circulating monocytes, which cross the blood-brain barrier. Within the brain, lipoviral particles

released from these monocytes are probably channelled into the CNS lipoprotein pathways with the

first step being the attachment to microglia and astrocytes via the HPSG and LDL receptors,

followed by the use of the VLDL secretion pathway for the production of infectious particles, as in

the periphery. Owing to the different binding capacities of the apoE isoforms, this process ought to

be less effective in APOE-ε4 allele carriers, and thus virus replication would be reduced and

Wozniak 15

microglial function preserved. Microglia maintain a regular net-like pattern within the CNS, with

each of the cells covering a constant area of brain tissue ready to react to any alteration of brain

homeostasis.34 Recently Grover et al.19 showed microglia activation in the caudate nucleus and

thalamus of HCV-infected patients with mild liver disease, using PK11195-PET. PK11195 is a

selective marker of activated microglia in vivo. In accordance with their PET findings, they also

showed an increase of the myoinositol/creatine (mI/Cr) and choline/creatine (Cho/Cr) ratio in the

basal ganglia using MRS. Both brain metabolites are increased in case of glial proliferation and

neuroinflammation. Their findings also confirm a quantitative MRS study that showed an increase

of choline and creatine in the white matter and basal ganglia, and an increase of myo-inositol within

the basal ganglia in HCV patients without significant neuropsychiatric impairment, compared to

neurocognitively impaired HCV patients.18 The data suggest that reduced impairment of microglia

function by HCV in APOE-ε4 allele carriers results in better cognitive function.

Furthermore, several studies indicate that APOE-ε4 predisposes to glial activation and

neuroinflammation.35 It has been shown, that APOE-ε4 allele carriers have decreased expression of

apoC-I which is immunosuppressive, resulting in increased innate immune activity – an attribute

that might be beneficial in the presence of HCV within the brain .36

An alternative hypothesis for the neuropsychological effects of HCV infection is a CNS effect of

chronic systemic immune activation. ApoE levels correlate with blood levels of IP-10, a marker of

interferon-stimulated genes (ISGs) in patients with chronic HCV infection.37 Furthermore, pro-

inflammatory cytokines, such as γ-interferon induce the enzyme indoleamine 2 3-dioxygenase,

resulting in downstream alterations of serotonin and tryptophan metabolites, which may affect

cerebral function. Thus, if the finding of an association between ApoE and ISG induction can be

substantiated, the APOE genotype can be expected to affect this interaction.

In conflict with the above suggestions some data may put a protective role of APOE-ε4 into

question: apoE protein has a direct role in maintaining the integrity of the BBB and APOE-ε4

appears to offer least protection. Nishitsuji et al. showed that the integrity of the BBB in APOE-ε4-


not italicised

Wozniak 16

knock-in mice is poorer than in APOE-ε3-knock-in animals.38 Further conflicting data include the

harmful effect of the ε4 allele in Alzheimer’s disease, and the recent findings that suggest it may

have an adverse effect on working memory, in cognitively normal middle-aged adults.39 In a smaller

study of patients with hepatitis C, low apoE levels were associated with more depression, although

cognitive function was not tested.37 Finally, HCV-infected patients with a ε4 allele were more likely

to be referred to a psychiatrist and had more neuropsychiatric symptoms during antiviral treatment

with interferon than those without an ε4 allele.40 Thus, the complex interactions between APO-E

genotypes, apoE levels, HCV, LVP, cell receptor binding, a potential effect on BBB permeability,

and neuropsychological outcomes requires further research.

Although our findings potentially fit with the current knowledge about the interaction between

HCV and apoE isoforms, some limitations of the study must be taken into account. Owing to the

limited number of patients included in the study, a significant association between the APOE-ε4

genotype and cognitive function in HCV patients could only be proven for working

memory/attention deficit. Probably, further associations would have been detected in a far larger

group of patients. Some of our data hint at a sex effect upon the APOE/HCV interaction. An

interpretation of these data, however, is hampered by the imbalance of the sexes in the study group

and should be addressed in further work.


should be APOE


In fact there are data suggesting that APOE-e4 carriers have better cognition up till middle age than those with the other alleles (a pleiotropic effect?)


I think the harmful role of e4 in Alzheimer's shouldn't be described as conflicting with HCV data in brain. We found that the dependence of degree of damage on APOE allele can differ between different microbes, different diseases and perhaps different tissues also. Perhaps say elsewhere that it "contrasts with" - and very briefly say it can vary with type of microbe, disease, etc.

Wozniak 17

Acknowledgements: We thank the following for their excellent technical assistance at different

periods in the study: Ms. Ann Cookson, Miss Alison Frost, Ms. Nicola A. Cook, and Ms. Ruth

Noone.

Wozniak 18

References

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apolipoprotein E binding to cell surface heparan sulfate. J Virol 2012; 86: 7256–7267.

29. Jiang J, Wu X, Tang H, Luo G. Apolipoprotein e mediates attachment of clinical hepatitis C

virus to hepatocytes by binding to cell surface heparan sulfate proteoglycan receptors. PLoS One.

2013 Jul 2;8(7):e67982. doi: 10.1371/journal.pone.0067982. Print 2013

30. Sheridan DA, Bridge SH, Felmlee DJ et al. Apolipoprotein-E and hepatitis C lipoviral particles

in genotype 1 infection: evidence for an association with interferon sensitivity. J Hepatol 2012;

57(1):32-38.

31. Vincent-Viry M, Schiele F, Gueguen R, Bohnet K, Visvikis S, Siest G. Biological variations

and genetic reference values for apolipoprotein E serum concentrations: results from the

STANISLAS cohort study. Clin Chem 1998;44(5):957-965

32. Holtzman DM, Herz J, Bu G. Apolipoprotein E and Apolipoprotein E Receptors: Normal

Biology and Roles in Alzheimer Disease. Cold Spring Harb Perspect Med 2012; 2:a006312. doi:

10.1101/cshperspect.a006312.

33. Letendre S, Paulino AD, Rockenstein E et al. Pathogenesis of hepatitis C virus coinfection in

the brains of patients infected with HIV. J Infect Dis 2007; 196(3): 361–370

34. Cronk JC, Kipnis J. Microglia - the brain's busy bees. F1000Prime Rep. 2013 ;3;5:53.

eCollection 2013; doi: 10.12703/P5-53

35. Zhu Y, Nwabuisi-Heath E, Dumanis SB et al. APOE genotype alters glial activation and loss of

synaptic markers in mice. Glia 2012;60(4):559-569

36. Cudaback E, Li X, Yang Y et al. Apolipoprotein C-I is an APOE genotype-dependent

suppressor of glial activation. J Neuroinflammation 2012; 9:192. doi: 10.1186/1742-2094-9-192

37. Sheridan DA, Bridge SH, Crossey MM et al. Depressive symptoms in chronic hepatitis C are

associated with plasma apolipoprotein E deficiency.Metab Brain Dis 2014;29(3):625-634.

Wozniak 19

38. Nishitsuji K, Hosono T, Nakamura T, Bu G, Michikawa M. Apolipoprotein E regulates the

integrity of tight junctions in an isoform-dependent manner in an in vitro blood-brain barrier model.

J Biol Chem 2011; 286(20):17536-17542.

39. Chen CJ, Chen CC, Wu D et al. Effects of the apolipoprotein E ε4 allele on functional MRI

during n-back working memory tasks in healthy middle-aged adults.AJNR Am J Neuroradiol

2013;34(6):1197-1202

40. Gochee PA, Powell EE, Purdie DM et al. Association between apolipoprotein E epsilon4 and

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Psychosomatics 2004; 45(1):49–57

Wozniak 20

Table 1 - APOE allele frequencies of subjects and attention test results

Total

N % N % N %

Whole group 14 7,6 148 75,8 38 16,7 200

Test for Attention Performance (TAP) batteryAttention Switch Test

Median reaction timePathological 6 12,0 37 74,0 7 14,0 50Non-pathological 8 5,6 106 73,6 30 20,8 144p-value

Number of errorsPathological 3 7,1 33 78,6 6 14,3 42Non-pathological 11 7,2 110 72,4 31 20,4 152p-value

Intermodal comparison testMedian reaction time

Pathological 9 9,4 75 78,1 12 12,5 96Non-pathological 4 4,2 68 70,8 24 25,0 96p-value


Incompatibility test Median reaction time



Go/No Go TestMedian reaction time



Divided attention testMedian reaction time



Number of missesPathological 6 9,7 47 75,8 9 14,5 62Non-pathological 8 6,1 97 73,5 27 5,3 132p-value

Alertness testMedian reaction time


Working memory testMedian reaction time



Number of missesPathological 4 9,1 35 79,5 5 11,4 44Non-pathological 8 7,1 78 69,6 26 23,2 112

. p-value

0.36 0.73 0.32

0.28 0.20 0.003

0.68 0.21 0.10

0.23 0.86 0.51

0.27 0.09 0.008

0.67 0.59 0.88

0.84 0.92 1.0

0.93

0.37

0.15

0.09

0.42 0.46 0.17

0.33 0.35

0.05 0.06

0.12

0.26

0,129 0,96 0,29

1.0 0.42

0.11

APOE- 2 APOE- 3 APOE- 4

0.25 0.03

0.100.95

0.42

Wozniak 21

Table 2 - APOE allele frequencies of subjects and memory test results

Total

N % N % N %

Whole group 14 7,6 148 75,8 38 16,7 200

Word-Figure-Memory testWords


PicturesPathological 3 10,0 19 63,3 8 26,7 30Non-pathological 9 7,6 87 73,7 22 18,6 118p-value

Recurring figures testGeometric


NonsensePathological 1 12,5 6 75,0 1 12,5 8Non-pathological 11 7,9 100 71,4 29 20,7 140p-value

APOE-2 APOE-3 APOE-4

0,18 0,54 0,83

0,67 0,26 0,33

0,21 0,88 0,31

0,63 0,83 0,57

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