Manuscript

Title

Alcohol and Alzheimer’s Disease - does alcohol dependence contribute to beta-amyloid deposition, neuroinflammation and neurodegeneration in Alzheimer’s disease?

Running Title

Alcohol and Alzheimer’s Disease

Authors

Dr. Ashwin Venkataraman, MBBS (Corresponding Author)Alzheimer’s Research UK Clinical Fellow,Centre for Neuropsychopharmacology,Division of Brain Sciences,Department of Medicine,Imperial College London,Burlington Danes Building,Hammersmith campus,160 Du Cane Road,London W12 0NNTel 07824 812223 Fax 020 7594 70470a.venkataraman@imperial.ac.uk

Dr. Nicola Kalk, PhDCentre for Neuropsychopharmacology,Division of Brain Sciences,Department of Medicine,Imperial College London,Burlington Danes Building,Hammersmith campus,160 Du Cane Road,London W12 0NN.n.kalk@imperial.ac.uk

Dr. Gavin Sewell, PhDDepartment of Oncology,Charing Cross Hospital,Fulham Palace Rd, London W6 8RFgavin.sewell85@gmail.com

Professor Craig W Ritchie, PhD Professor of the Psychiatry of AgeingCentre for Clinical Brain Sciences,

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University of Edinburgh,The Chancellor's Building,49 Chancellor's Building,Little France,Edinburgh,EH16 4SBcraig.ritchie@ed.ac.uk

Professor Anne Lingford-Hughes, PhDProfessor of Addiction Biology,Centre for Neuropsychopharmacology,Division of Brain Sciences,Department of Medicine,Imperial College London,Burlington Danes Building,Hammersmith campus,160 Du Cane Road,London W12 0NN.Tel: 020 7594 8682Fax: 020 7594 6548anne.lingford-hughes@imperial.ac.uk

Author Contributions

AV proposed the hypothesis, searched the scientific literature, and designed the

figures. NK and GS contributed to hypothesis and writing. CR and AL-H contributed

to design and background knowledge. All authors wrote the article.

Keywords

Alcohol dependence, neuroinflammation, beta amyloid, dementia, Alzheimer’s, toll-

like receptors

Abstract

Aim: To investigate the underlying neurobiology between alcohol use, misuse and

dependence and cognitive impairment, particularly Alzheimer’s disease.

Methods: Review of the literature using searches of Medline, Pubmed, EMBASE,

PsycInfo, and meeting abstracts and presentations.

Results: The role of alcohol as a risk factor and contributor for cognitive decline

associated with Alzheimer’s disease (AD) has received little attention. This is despite

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the high prevalence of alcohol use, the potential reversibility of a degree of cognitive

impairment and the global burden of AD. Until now the focus has largely been on the

toxic effects of alcohol, neuronal loss and the role of thiamine.

Conclusions: We propose alcohol adds to the cognitive burden seen in dementia

through additional mechanisms to neurodegenerative processes or may contribute at

various mechanistic points in the genesis and sustenance of AD pathology via

neuroinflammation. We describe the common underlying neurobiology in alcohol and

AD, and examine ways alcohol likely contributes to neuroinflammation directly via

stimulation of Toll-like receptors and indirectly from small bowel changes, hepatic

changes, withdrawal and traumatic brain injury to the pathogenesis of AD.

Short Summary

Alcohol use, misuse and dependence cause cognitive impairment. We propose alcohol

adds to the cognitive burden seen in dementia through additional mechanisms to

neurodegenerative processes or may contribute at various mechanistic points in the

genesis and sustenance of Alzheimer’s disease (AD) pathology via

neuroinflammation.

Introduction

Alcohol use, misuse and dependence are associated with cognitive impairment. Such

impairment is commonly seen during intoxication however may be enduring for some

in abstinence. Several cohort studies show alcohol consumption beyond moderate

amounts increases the risk of dementia (National Institute for Health and Care

Excellence 2015). Reduced alcohol consumption was shown to lead to longevity

(Khaw et al. 2008) and more quality-adjusted life years (Myint et al. 2011). Those

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who adopted healthy behaviours, of which one was reduced alcohol consumption,

were 3.3 times more likely to age successfully.

A link between alcohol misuse and Alzheimer's disease (AD) has been described.

Alcohol dependence was a significant and independent predictor of AD with odds

ratio ranging from ~3 to 4 depending on length of alcohol dependence, particularly in

those 65-79 years old (Zilkens et al. 2014). The presence of the apolipoprotein E4

allele, a history of heavy drinking or heavy smoking was each associated with an

earlier onset of AD by 2–3 years. Diagnosis of AD occurred nearly 10 years earlier in

those with all three risk factors compared with those with none (Harwood et al. 2010).

We will propose here a potential underlying biological mechanism for these

consistent epidemiological associations.

The principal cause of a dementia syndrome is AD which is characterised by

aggregation of beta-amyloid (Aβ) and tau with contributory neuroinflammation and

cerebrovascular insufficiency. The influence of alcohol on the critical Aβ seen in AD

and subsequent neurodegeneration is largely unknown. Here we review the evidence

and propose mechanisms in which Aβ pathology may be driven or contributed to by

alcohol, leading to microglial activation, neuroinflammation and ultimately neuronal

cell death. This could be directly through chronic alcohol use and withdrawal via

stimulation of Toll-like receptors (TLR) 2 and 4 or indirectly by systemic effects of

alcohol through the action of circulatory cytokines, prostaglandins and nitric oxide on

the brain. By linking Aβ pathology with such direct and indirect effects of alcohol we

propose important novel mechanistic pathways through which AD and Aβ pathology

may be driven or contributed to that needs further exploration for research. This may

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have wide reaching effects when dealing with alcohol dependent patients and patients

at high risk of AD and their use of alcohol.

Importantly alcohol misuse is also associated with a number of other disorders which

increase the risk of AD and vascular dementia. These include heart disease,

cerebrovascular disease, diabetes mellitus, hypertension, smoking, hyperlipidemia,

hypercholesterolemia and traumatic brain injury (TBI) (Zilkens et al. 2014). We know

that TBI has been shown to be associated with increased 11C-PIB (a marker of

amyloid deposition) binding in PET studies (Sharp et al. 2014; Hong et al. 2014). TBI

following alcohol use may also contribute to this in an additive fashion. However

neuroinflammation is also heavily implicated in these other disorders, so we focus

here on how the inflammatory response to alcohol misuse may be linked with Aβ and

AD.

Notably, there are no in vivo studies of Aβ levels (plasma, CSF or PET imaging) in

alcoholism in man yet. The few published studies in human post-mortem samples

from individuals with alcohol abuse did not find increased neuritic plaques or

aggregation of Aβ (Aho et al. 2009). However in these studies the amount and

severity of alcohol consumption does not appear to have been particularly severe. One

study of CSF markers (tau, Aβ) reported that alcoholism could be differentiated from

AD however none were nutritionally deficient (Kapaki et al. 2005). This is important

given how critical thiamine is to brain metabolism, and how deficiency can lead to

neuronal loss in alcoholism. Hence, the epidemiological evidence relating alcohol use

to cognitive impairment and dementia is such that the biological basis of these

associations requires further elaboration.

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Alcohol

Drinking alcohol is a socially accepted behaviour. Many people consume alcohol

without experiencing any problems but a growing number of people drink in a

harmful manner. Global estimates attribute 5.9% of all deaths, and 5.1% of the global

burden of disease to alcohol in one year alone (World Health Organisation 2014).

Harmful use of alcohol ranks among the top five risk factors for disease, disability

and death worldwide, causing more than 200 disease and injury conditions (World

Health Organisation 2014). Worldwide the prevalence of alcohol use disorders is

4.1%. Exposure to alcohol varies considerably by regions. In established market

economies in Western Europe, Eastern Europe and in North America, average volume

of drinking is the highest, with the lowest being in the Eastern Mediterranean regions

and parts of Southeast Asia including India. Patterns were least detrimental in

Western Europe and in the Western Pacific region, such as Japan. The overall

exposure by volume is quite high and patterns are relatively detrimental throughout

the world (Rehm et al. 2003).

The impact of alcohol misuse is widespread encompassing alcohol related illness and

injuries (such as alcohol dependence, liver cirrhosis, cancers, cardiovascular disease,

diabetes, injuries and infectious diseases amongst others) with significant social

impacts to family, friends and wider community. A dose response relationship exists

between alcohol and many acute and chronic disease outcomes (Rehm et al. 2010).

Alcohol use and driving also increases the risk of being involved in a road traffic

accident, and the severity of resulting injuries (World Health Organization 2015).

The alcohol-related burden of disease among older adults is an increasing concern due

to the rapidly ageing population worldwide. Older adults are more vulnerable to

alcohol related harm from a given volume of alcohol (Moos et al. 2009) as are

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adolescents (World Health Organisation 2014). Alcohol has also been shown to be the

leading risk factor for death aged 15-69 (World Health Organisation 2014).

In addition, women are more vulnerable to alcohol related harm for a given amount of

alcohol compared to men. (Wilsnack et al. 2013). Alcoholic women experience

significant deficits in a wide range of neuropsychological domains compared to non-

alcoholic women, and are more sensitive to alcohol related cognitive damage (Nixon

1994), and have a greater risk of alcoholic cardiomyopathy and myopathy compared

with men (Urbano-Márquez et al. 1995).

Alcohol Related Brain Damage

The term ‘alcohol related brain damage’ (ARBD) incorporates a range of

psychoneurological and cognitive conditions that are associated with long-term

alcohol misuse and related vitamin deficiencies. At one extreme is Wernicke-

Korsakoff syndrome (WKS) and at the other, the more frequent milder and often less

obvious subtle dysfunctions of executive and memory processes due to the

vulnerability of the frontal lobe and hippocampus to adverse effects of alcohol.

Notably these regions are especially vulnerable to Aβ pathology.

ARBD tends to affect people in their 40s and 50s, with females presenting a decade

younger than males (Royal College of Psychiatrists 2014). Women may be more

sensitive to alcohol related brain damage (Nixon 1994). It has been shown that

alcoholic women have greater reductions in the left hippocampal formation and

corpus callosum after shorter duration of alcohol consumption compared to men,

however there is limited data to support this (Hommer et al. 1996; Agartz et al. 1999).

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It appears that alcohol has a differential effect on the central GABA-benzodiazepine

receptors in men and women (Lingford-Hughes et al. 2000). Although ARBD may

substantially recover within 2 to 3 months, cognitive impairment continues to improve

for 2-3 years when abstinent. There is much evidence to suggest that abstinence leads

to a substantial recovery of cognitive deficits in the overwhelming majority of

individuals. However, in some, impairments may persist and are usually related to

concurrent thiamine deficiency or other complications such as vascular dementia in

~25% (Wilson et al. 2012). Older alcoholics show greater volume deficits compared

to age–matched control subjects than younger alcoholics, suggesting that the older

brain is more vulnerable to the effects of alcohol (Pfefferbaum et al. 1992).

Alzheimer’s Disease

The number of people living with dementia worldwide today is estimated at 44

million, set to almost double by 2030 and triple by 2050. The global per annum cost

of dementia was estimated in 2010 at US $604 billion, and this is rising (Prince et al.

2014). Small reductions in potentially modifiable risk factors (such as alcohol

consumption) are therefore likely to have a large impact on the global burden of the

disease.

The most common cause of dementia is AD. This fatal neurodegenerative disorder is

characterised by progressive cognitive impairment, social, functional impairment and

memory loss. There are currently no available treatments to cure or stop progression.

Treatments such as acetylcholinesterase inhibitors seem to show a modest benefit at

best towards the disease (National Institute for Health and Care Excellence 2006).

Pathological evidence gives rise to the amyloid cascade hypothesis where the

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accumulation of Aβ plaques and neurofibrillary tau tangles lead to neurodegeneration

in AD. Current research suggests Aβ peptide is cleaved from the transmembrane

amyloid precursor protein (APP) by β and γ secretases. These peptides aggregate and

form the main component of the diffuse senile plaques found in the brain tissue of AD

patients. This initiates the inflammatory processes, which cause neuronal death

leading to the progression of AD (Heneka et al. 2015).

Alcohol and Neuroinflammation

There is an emerging body of evidence that there is a link between alcohol and

neuroinflammation. Alcohol intoxication, withdrawal and chronic use impacts on the

immune system, causing microglial activation, releasing pro-inflammatory cytokines,

and this is mediated via Toll-like receptors. Alcohol also affects the peripheral

immune system, causing both thiamine deficiency, and increasing pre-cursors of beta-

amyloid in the brain. The effects of short-term, long-term, and binge drinking in both

pre-clinical and clinical studies are examined below.

Alcohol and Microglia

Pre-clinical models show how short-term ethanol impacts on the immune system

(Pascual et al. 2015). Most evidence for a role of neuroinflammation in ARBD comes

from preclinical studies. Activated microglia have been demonstrated in a mouse

model of short-term alcohol dependence (Syapin & Alkana 1988). In a rat model of

long-term alcohol dependence sufficient to produce physical dependence and mild

cognitive deficits, activated microglia have been shown to be present for 14 days after

the last alcohol dose (Obernier et al. 2002).

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The hyperglutamatergic state during alcohol withdrawal has also been suggested to be

a neuroinflammatory stimulus related to microglial activation (Ward et al. 2009). Pre-

clinical binge models suggest that microglia are partly rather than fully activated

during alcohol withdrawal – a non-inflammatory phenotype (Marshall et al. 2013).

Also a single binge ethanol exposure results in low-level of microglial activation,

however, a second binge potentiated the microglial response in pre-clinical models

(Marshall et al. 2016).

In clinical studies, although increased numbers of microglia in the brains of alcohol-

dependent patients at post-mortem have been seen, it is not clear what kind of

inflammatory phenotype has been described (He & Crews 2008). Clusters of activated

microglia and astrocytes have been described in the thalamus and around the third

ventricle in post-mortem specimens from patients with Wernicke–Korsakoff

syndrome (Kril & Harper 2012). The classical areas of Wernicke-Korsakoff syndrome

may not be commonly associated with AD, suggesting a need to think beyond its

classical conceptualisations. Microglial activation has been demonstrated in vivo in

alcohol dependent patients with hepatic encephalopathy (Cagnin et al. 2006) and in

Wernicke’s encephalopathy (Tsukahara et al. 2005). Alcoholic patients with a history

of chronic alcohol dependence uncomplicated by liver cirrhosis and/or nutritional

deficiencies showed microgliosis and astrogliosis in post-mortem samples (Rubio-

Araiz et al. 2016).

Alcohol, cytokines and the peripheral immune system

Alcohol affects both cytokines and the peripheral immune system. Alcohol induced

neuroinflammation includes increased brain levels of pro-inflammatory cytokines

interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF-α). Reduced anti-

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inflammatory cytokine IL-10 have been observed in response to chronic high alcohol

administration in short-term pre-clinical models. Notably these persisted for several

days while peripheral circulating plasma cytokines were undetectable, favouring a

pro-inflammatory milieu in the brain (Qin et al. 2008).

In preclinical models the impact of ethanol may have dichotomous effects on the

peripheral immune system depending on the concentration – having a suppressive

effect at high and stimulatory effect at low concentration (Fernandez-Lizarbe et al.

2008).

Clinical studies show serum proinflammatory cytokines have been increased in

patients with chronic alcoholism (McClain & Cohen 1989; McClain et al. 1999).

Increased pro-inflammatory and anti-inflammatory cytokines early in abstinence have

been described (González-Quintela et al. 2000), as has increased pro-inflammatory

cytokine production by dendritic cells from alcohol-dependent participants. In

addition to the pathways above, peripheral inflammation is highly likely due to

alcohol related increase in gut bacteria translocation. These peripheral cytokines are

known to affect microglial phenotypes (Wang & Hazell 2010).

The effects of alcohol in humans on the peripheral immune system is pertinent given

the discovery of functional meningeal lymphatic vessels that line the dura of the CNS

connected to the deep cervical lymph nodes, changing a fundamental assumption of

neuroimmunology that the brain was an immune privileged site (Louveau et al. 2015).

This communication is particularly important given the wide ranging effects of

alcohol peripherally. Peripheral cytokines are also known to communicate with the

central nervous system via vagal afferents, periventricular macrophages and dedicated

transporter systems (Laso et al. 2007).

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Alcohol and Toll-like receptors

There is evidence for alcohol related induction of neuroinflammation via direct and

indirect mechanisms. Alcohol has been reported to stimulate Toll-like receptors

(TLRs). TLRs are pattern recognition receptors in the innate immune system,

predominantly expressed in glial cells and critical in the generation of an immune

reaction in response to pathogens. Binding of specific ligands to TLRs results in

activation of downstream signalling cascades within immune cells. This ultimately

results in a state of ‘cellular activation’ releasing chemokines and cytokines. TLRs

also express the co-stimulatory molecules for pattern recognition needed for

protective immune responses alongside efficient clearance of damaged tissues (Tahara

et al. 2006). Downstream signalling may proceed via MyD88-dependent and MyD88-

independent pathway, the former is considered more important in microglia. This

culminates in the activation of transcription factors, and thereby generation of

proinflammatory cytokines or free radicals (Keogh & Parker 2011; Pascual et al.

2015). CD14 acts as a coreceptor and interacts with TLR4 and TLR2 containing

dimeric complexes to transduce activation signals in response to bacterial pathogens.

It is shown that exogenous and endogenous TLR ligands activate microglia (Tahara et

al. 2006). This is important given alcohol’s effect on TLRs. Alcohol may have a

direct effect on TLRs as an upstream driver of inflammation as well as a direct effect

on downstream inflammatory process. This may ultimately lead to the aggregation of

Aβ and tau especially in vulnerable areas like the hippocampus.

In pre-clinical models short-term alcohol models, alcohol has also been shown to

activate TLR4 directly in astrocytes and microglia in vitro (Fernandez-Lizarbe et al.

2009). This activation leads to expression of the transcription factor TNF-α and

expression of enzymes which regulate the production of prostaglandins and nitric

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oxide, cyclooxygenase 2 (COX-2) and inducible nitric oxide synthetase (iNOS)

(Vallés et al. 2004). Inflammatory microglial activation and increase in inflammatory

cytokines, nitric oxide and prostaglandins in the brain induced in a rodent model of

long-term alcohol dependence were reduced to control levels when TLR4 was

eliminated by knock-out (Alfonso-Loeches et al. 2010), as were associated cognitive

deficits in pre-clinical short-term models (Pascual et al. 2007). Mice lacking TLR4

and TLR2 receptors were protected against ethanol induced cytokine and chemokine

release, and showed ethanol consumption increases cytokine and chemokine levels in

the striatum and serum of wild type mice, but not in TLR4/TLR2 knockout mice, but

the effects on the peripheral immune system were not considered.

Alcohol and Thiamine Deficiency, APP & BACE

Lastly, preclinical models of thiamine depletion show early microglial activation with

blood–brain barrier disruption in equivalent areas to those affected in human

populations (Todd & Butterworth 1999; Wang & Hazell 2010).

In addition to neuroinflammation, alcohol abuse is also commonly associated with

thiamine deficiency and notably in transgenic AD mouse models, thiamine deficiency

has been shown to exacerbate amyloid plaque pathology (Karuppagounder et al.

2009). Given how critical thiamine is to brain metabolism, it is not surprising

therefore that changes in brain metabolism in AD have been related to changes in

thiamine-dependent enzymatic activity. Thiamine deficiency induces chronic mild

impairment of oxidative metabolism and promotes selective changes in oxidative

stress and inflammation that lead to neuronal loss in specific brain regions. In addition

to thiamine deficiency we also know that mice given alcohol show an increase in APP

and BACE-1 levels compared to controls (Kim et al. 2011). There is little literature

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linking alcohol and tau. One study attempted to evaluate CSF markers tau and beta-

amyloid to differentiate ARBD and AD however the implications remain uncertain

(Kapaki et al. 2005).

Given the importance of neuroinflammation in alcohol it is necessary to elaborate on

where these are in common with those pathways of neuroinflammation in AD to

provide focus for further investigation.

Neuroinflammation in Alzheimer’s Disease

There is emerging evidence of the potential role of neuroinflammation not just in the

progression of AD but as a potential trigger or accelerating event, with a link between

inflammation and amyloid plaque formation. It is known that immune system

mediated actions contribute to and drive the pathogenesis of AD (Heppner et al.

2015). Microglial priming seems to be key to this (Heneka et al. 2014; Perry &

Holmes 2014).

Microglial priming is seen to lead to a self-perpetuating downstream expression of

inflammatory cytokines and chemokines resulting in neurodegeneration and neuronal

loss. It is hypothesised that a combination of Aβ driven priming of microglial cells,

Aβ peptide accumulation within microglia, systemic and local inflammation, together

cause chronic activation and ‘burn out’ of microglia (Heppner et al. 2015). Mice

models show transient systemic inflammation superimposed on neurodegenerative

disease acutely exacerbates cognitive and motor symptoms of disease and accelerates

progression (Cunningham et al. 2009).

In early AD it is believed that activated microglia and reactive astrocytes are

neuroprotective, however in the later stages they fail to clear Aβ leading to

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neuroinflammation and neurodegeneration (Tahara et al. 2006; Gambuzza et al.

2014). Activated microglial cells in AD are therefore seen as a “double-edged sword”

(Song et al. 2011). On one hand they protect neurons by taking up and degrading

aggregated Aβ but on the other they degrade them and cause release of

proinflammatory cytokines, chemokines, and reactive oxygen and nitrogen species,

which harm synapses and neurons (Wyss-Coray 2006; Rivest 2009).

Neuropathological evidence confirms these being found in brains and CSF of AD

patients (Akiyama et al. 2000; Landreth, G and Reed-Geaghan 2009).

TLRs are linked to this process. This is important, given the activation of TLRs by

alcohol and an association between TLRs and AD (Fassbender et al. 2004; Udan et

al. 2008; Richard et al. 2008; Jana et al. 2008). Depending on the disease stage TLRs

and co-receptors are activated to induce Aβ uptake or downstream inflammatory

processes. Fibrillar Aβ (fAβ) is believed to interact directly with TLR2, TLR4 and

CD14 and induce microglial Aβ phagocytosis in the beginning stages, and

neuroinflammatory responses in the late stages (Heneka et al. 2015). Apolipoprotein

E, a protein linked to AD susceptibility by genetic studies, may modulate microglial

function downstream of TLRs resulting in a pro-inflammatory state (Li et al. 2015).

TREM2, another genetic risk factor for AD, has also been linked with macrophage

and microglial function and proinflammatory cytokine release in GWAS (Guerreiro et

al. 2013; Lambert et al. 2013; Yaghmoor 2014). We are not aware anyone has looked

at this in alcohol.

In vitro mouse models support this process. Co-administration of Aβ with TLR4 or

TLR2 ligand in microglial cultures led to additive release of nitric oxide and TNF-α

from microglia (Lotz et al. 2005). Daily intraventricular injection of

lipopolysaccharide (LPS) for 14 days induced premature fAβ deposits in young

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mouse models (Sheng et al. 2003). CD14 and TLR2 and 4 are required for fAβ

stimulated microglial activation which together act to bind fAβ and to activate

intracellular signalling, with both stimulating NADPH oxidase and forming reactive

oxygen species (ROS) (Reed-Geaghan et al. 2009). Hence, activation of TLR

signalling can exacerbate AD by induction of oxidative stress, inflammation and Aβ

deposition.

There is evidence that TLRs are involved in the microglial response to Aβ as

inhibition of TLR4 or TLR2 also prevented fAβ induced nitrite, IL-6, and TNF-

production (Walter et al. 2007; Udan et al. 2008; Jana et al. 2008; Reed-Geaghan et

al. 2009). Furthermore a polymorphism in the TLR4 gene results in a blunted

signalling response and is correlated with a 2·7-fold reduction in risk for late onset

AD (Minoretti et al. 2006).

Conversely, some studies conclude enhanced TLR4 signalling promotes clearance of

Aβ. Mice models homozygous for destructive mutation of TLR4 had increases in Aβ

on immunocytochemistry compared to wild-type models TLR4 (Tahara et al. 2006).

Deficiency in an AD mouse model increased Aβ42 in the brain and exacerbated

cognitive impairments in one model (Richard et al. 2008). Inhibition of TLR2 in

microglia reduced the detrimental effect of inflammatory activation while enhancing

the beneficial effect of Aβ clearance (Liu et al. 2011). This supports the hypothesis

that TLR4 and TLR2 signalling in the early stages of AD pathogenesis may be

neuroprotective, but in the late stages neurotoxic (Song et al. 2011).

Neuronal loss is a prominent element of AD pathology. In one study neuronal

apoptosis was induced by the addition of conditioned media from Aβ-stimulated

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monocytes (Combs et al. 2001). Hippocampal neurons exposed to conditioned media

from microglia treated with aggregated Aβ42 resulted in neuronal death, however,

media from CD14 or TLR4 knockout microglia treated with Aβ42 was unable to kill

neurons in the brain parenchyma (Fassbender et al. 2004; Walter et al. 2007;

Landreth, G and Reed-Geaghan 2009). TLR4 dependent upregulation of cytokines in

a transgenic mouse model of Alzheimer's disease, namely IL-1β, IL-10, IL-17 and

TNF-α in TLR4w AD mice compared to non-AD mice and suggest TLR4 signalling

as a new therapeutic target for AD (Jin et al. 2008).

The role of tau in neuroinflammation is currently uncertain. Alzheimer’s

neuroinflammation can manifest without neurofibrillary tangles.

Linking neuroinflammation in Alzheimer’s disease and alcohol misuse.

Given the above evidence of a role for neuroinflammation in both AD and that

alcohol increases neuroinflammation, it appears highly likely that Aβ pathology is

influenced by alcohol both directly and indirectly via common pathways (See Figures

1 & 2).

There is an emerging consensus on the role of the innate immune system in both AD

and in the effects of alcohol. As described, both AD and alcohol cause

neuroinflammation via the action of TLRs, in particular TLR4 and TLR2. The action

of microglial cells are suggested to be key to this process. Downstream inflammatory

cascades are activated, and release of certain cytokines and chemokines are thought to

lead to neuronal death. Modulation of the effects of Aβ via TLR appears central to

this. Alcohol in stimulating TLR4 may modulate this process and a positive feedback

loop may be established via TNFα (Kuno et al. 2005).

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Indirect systemic effects of alcohol may lead to microglial activation directly, and

contribute to the self-perpetuating cycle of inflammatory processes in the Aβ cascade.

This is due to the small bowel changes, hepatic changes and withdrawal effects and

the resultant circulatory cytokine release and increased endothelial expression of

prostaglandins, and iNOS. Through the action of circulatory cytokines, prostaglandins

and nitric oxide on the brain, this may lead to microglial activation and microglial

burnout further driving Aβ pathology and causing subsequent neuroinflammation and

ultimately neuronal cell death. Given this evidence alcohol might be responsible for

the induction and perpetuation of Alzheimer’s disease.

Conclusion

The global burden of both alcohol and AD is increasing. Although epidemiological

studies reveal alcohol as an important risk factor for AD, the underlying mechanism

has not received focus. We have reviewed here how neuroinflammation may be

involved and provide a link between Aβ pathology and alcohol misuse. This may be

through binge or chronic alcohol use and withdrawal directly stimulating TLR 2 and 4

or indirectly by systemic effects of alcohol through the action of circulatory

cytokines, prostaglandins and nitric oxide on the brain. Both these routes lead to

microglial activation, neuroinflammation and ultimately neuronal cell death, seen in

AD.

The mechanisms described herein require appropriate testing. This area of research

has received limited attention so far and redressing this would be a valuable

contribution to dealing with AD. This may also have wide reaching effects when

considering the adverse effects of alcohol misuse in the general population as well as

when treating alcohol dependent patients. Traditionally the cognitive deficits and

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brain damage associated with alcohol use has shown some improvement and a degree

of reversibility on abstinence from alcohol use. If alcohol dependence and

Alzheimer’s disease share a common mechanistic pathology, then treatment of any

alcohol misuse or dependence to support cessation would be an important clinical

consideration for those at risk of AD and in the treatment of patients with AD to

provide a pivotal disease modifying strategy.

Figure Legends

Figure 1 – Proposed links between alcohol and Aβ cascade in AD through

inflammatory processes. Aβ peptide is cleaved from the transmembrane amyloid

precursor protein (APP) by β and γ secretases. There is an increase in production or

reduced clearance of Aβ with the likelihood of conversion shown by solid arrows, and

less conversion illustrated by dotted arrows. Different forms of Aβ affect microglial

cell activation differently, and Aβ fibrils are shown to activate microglial cells,

leading to inflammatory cytokines and chemokines release, further both re-activating

microglial cells, and also causing increased aggregation of Aβ towards plaque forms.

This self-perpetuating cycle leads to neurodegeneration and cell death. It is suggested

that alcohol acts both directly and indirectly on this system. Directly alcohol causes

TLR2/4 stimulation and together with fAβ it leads to increased microglial activation,

and greater inflammatory cytokines and chemokine release. Indirectly alcohol via

small bowel, hepatic and withdrawal effects causes increased circulatory cytokines, in

particular TNFα, IL-1β, IL-6 and endothelial expression of prostaglandins and iNOS

which cause direct microglial cell activation, and contribute to neuroinflammation by

cytokines and chemokines release. The summation of effects results in more

neurodegeneration and cell death, greater microglial burnout, and increased

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aggregation of Aβ due to a combination of increased inflammatory mediators, and

decreased or saturated microglial cells.

Figure 2 – Proposed Toll-like receptor (TLR) 4 signalling in microglia. Both alcohol

and fibrillar Aβ bind to TLR4, resulting in neuroinflammation via the MyD88-

dependent and MyD88 independent response.

List of Abbreviations

Aβ beta amyloid

AD Alzheimer’s Disease

APP amyloid precursor protein

ARBD alcohol related brain damage

BACE-1 beta-secretase 1

CCL CC chemokine ligands

CD cluster of differentiation

COX cyclooxygenase

fAβ fibrillar beta amyloid

GWAS genome wide association study

IL interleukin

iNOS inducible nitric oxide synthase

LPS lipopolysaccharide

MCP monocyte chemoattractant protein

MyD88 myeloid differentiation primary response gene 88

NADPH nicotinamide adenine dinucleotide phosphate

NFB nuclear factor kappa-light-chain-enhancer of activated B cells

NICE National Institute of Clinical Excellence

PET positron emission tomography

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ROS reactive oxygen species

TBI traumatic brain injury

TLR Toll-like receptors

TNF tumour necrosis factor

TREM2 triggering receptor expressed on myeloid cells 2

WKS Wernicke-Korsakoff syndrome

Conflicts of interest

Dr Venkataraman reports grants from Alzheimer’s Research UK, NIHR and BRC, outside the submitted work. He is working on a GSK funded study and receives no financial or other support from them. He receives non-financial support from Eli-Lilly.

Dr Kalk reports previous support from Wellcome Trust, from GSK, other from the NIHR, during the past 36 months.

Dr Sewell has nothing to declare.

Prof. Ritchie has nothing to declare.

Prof. Lingford-Hughes reports other from Wellcome Trust, other from GSK, non-financial support from Imanova, during the conduct of the study; grants, personal fees and non-financial support from Lundbeck, non-financial support from GSK, grants from NIHR RfPB & HTA, grants from MRC, grants from Imanova , outside the submitted work.

Acknowledgments

The authors wish to thank Dr Magdalena Sastre for her comments on the manuscript.

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