The case for a viral role in Alzheimer's disease

Most of us harbour in our body several types of herpes virus—perhaps as many as five—and we provide them with a safe and secluded haven for life, as there are no methods for eliminating or expelling them. Much of the time they are latent, i.e., dormant, and in that state they are probably relatively harmless in most people. However, they can reactivate if the host is stressed or immunosuppressed, and they then replicate, causing damage and death in the host cells via direct viral action and viral-induced inflammation. In some cases though the overall damage is minimal, so that the person appears asymptomatic even though infected. Herpes simplex virus type 1 (HSV1) displays this behaviour in that it infects most people by the age of about 60, and reactivates periodically in the peripheral nervous system (PNS), but causes visible damage (cold sores) in only some 25% of infected people.

We decided to investigate a possible role for HSV1 in Alzheimer's disease (AD) for three main reasons: firstly, the fact that as HSV1 remains in the body lifelong and can reactivate, it has the potential to cause damage later in life, consistent with the late development of AD; secondly, a number of viruses (measles, HIV, JC virus) are known to affect the brain and can cause fatal dementing illness, sometimes years after the initial infection; thirdly, the rare and very serious brain disease herpes simplex encephalitis (HSE), which is caused usually by HSV1, affects the same brain regions as those mainly affected in AD [1].

It has been known for decades that HSV1 lurks in the PNS of "normal" people. However, whether it could reach the central nervous system too, and reside there was totally unknown. In the 1970s and 1980s, several groups sought viral DNA in brain using solution or in situ hybridisation. The results were inconclusive, with some positive and some negative findings. Using PCR, under very stringent conditions, my group detected HSV1 DNA in brain [2] (a discovery which elicited cries of shock and outrage from some reviewers), and we later found that about 70% of postmortem brains from elderly people and from AD patients were HSV1 DNA-positive [3]. Subsequently, five other laboratories detected HSV1 DNA in human brains. We then found a strong causal association in AD patients between HSV1 presence in brain and carriage of an APOE-ε4 allele. Our data showed that the association was not artefactual in that AD patients, and those carrying an APOE-ε4, were not more susceptible to HSV1 infection than were controls or carriers of the other APOE alleles. In other words, the virus and APOE-ε4 were a cause, not an effect, of the disease, accounting for about 60% of cases (I stress a cause, as the disease is clearly multifactorial). APOE-ε4 carriage also proved to explain the relatively low proportion of infected people who suffer cold sores, as it is a risk for that disorder as well [3]. In fact, we later discovered that APOE genotype determined susceptibility to infection, or severity of infection damage, in several other diseases of microbial cause [4].

These findings reflect two key features of herpes and other microbes, of which many people seem unaware, judging by the comments often raised after my talks and by reviewers of my group's grant applications and papers: "How can they think that a virus is involved in AD when they find that a high proportion of elderly people as well as AD patients have HSV1 in brain?" Firstly, being infected with a microbe yet unaffected by it is a common phenomenon, strikingly illustrated by tuberculosis, as the bacterium Mycobacterium tuberculosis infects millions of people yet causes tuberculosis only in some 10%; similarly with HSV1 in the periphery, with cold sores occurring in only some 25% of those infected. Secondly, microbes can cause chronic effects, but are often regarded as causes of acute damage only, after which they fly away—or kill their host; lingering on in the body is not even contemplated.

There is now good though indirect evidence from a number of groups that HSV1 not only resides in most elderly brains but also reactivates there [5], probably repeatedly, when the immune system is weakened (as in aging), so that damage accumulates gradually, eventually leading to AD. The damage is presumably mild or limited, as otherwise, it would be apparent—the patient would display symptoms of HSE. What then is the connection, if any, between reactivation and AD? One answer is that in HSE, which (in adults) is always caused by HSV1, many who survive show symptoms similar to those of AD: memory loss, personality and behavioural changes, and aphasia, and as mentioned above, HSE damage occurs in the very regions of brain that suffer most damage in AD. Also, HSE occurs occasionally in a "mild" form (probably unrecognised and under-diagnosed), and can be recurrent [6], just as we suggest takes place during the development of AD. There are several precedents for viruses reactivating in brain when the immune system is weakened, causing vulnerability to an endogenous virus: for example, overt JC virus infection can occur in brain after iatrogenic immunosuppression, giving rise to the very serious disease, progressive multifocal leukoencephalopathy.

From cell culture studies, another HSV1-AD link becomes apparent: infection by the virus causes accumulation of both amyloid-beta and of AD-like tau, the main components respectively of the characteristic amyloid plaques and neurofibrillary tangles of AD brain [7]. Further, in AD brains, the viral DNA is located very specifically within plaques (arguments against an artefactual co-localisation are given in detail in [8] ). Another major connection is that HSV1 causes synaptic damage, one of the main features of AD [9].

These data and concepts linking HSV1 to AD are often termed "controversial"—a singularly inappropriate term as it implies that there are arguments pro and con. However, opponents never divulge any scientific cons. A frequent bleat is that there is no evidence for the concept, an odd statement, as there are now over 100 publications directly or indirectly supporting it (with only three that conflict, all published over 12 years ago), and the topic is amply discussed in over 20 recent reviews. One can infer only that opponents either have not accessed the relevant information or have not absorbed it. The opponents are mainly devotees of amyloid-beta or AD-like tau, implicating one or other as the cause of AD, despite the absence of any data revealing what causes their appearance, and despite the fact that the virus-AD concept actually accommodates both proteins. And even the current boost for a major role of amyloid-beta in AD, provided by data indicating that it (or an oligomer) is an anti-microbial peptide [10-14], seems only to tiptoe around the concept that infection is involved. Another comment is "How do we know if HSV1 in brain is a cause or an effect of the disease". The answer was stated almost 20 years ago when the frequency of viral DNA presence in brain was found to be similar in AD patients and elderly normal people [3]. Yet this and the "controversial" mantra are still chanted in response to the word "microbe". It is surprising though, as many people are aware of parallel effects caused by well known viruses such as HIV and measles, specifically dementia, as well as amyloid plaque and/or neurofibrillary tangle formation.

Bacteria have also been implicated strongly in AD, in particular, Spirochaetes [15] and Chlamydia pneumoniae [16], so a major question in respect to HSV1 and AD is: does the virus act together with either of these bacteria or is one of them responsible for the disease in those patients who do not have HSV1 in brain and APOE-ε4 carriage? A related question is whether HSV1 acts together with another non-microbial factor, though so far, no such factor has been identified.

As for treatment of the disease, surely the evidence presented warrants usage of an antiviral agent. In fact, studies on cell cultures have shown that various antivirals, which target HSV1 by different mechanisms, not only reduce virus replication greatly, as expected, but also reduce HSV1-induced P-tau almost to zero and HSV1-induced amyloid-beta very substantially [17,18]. Probably the most effective treatment would be an antiviral that inhibits viral DNA replication, together with one that blocks viral entry. However, initial trials would necessarily use only the former type—the standard agent for combating HSE, valacyclovir (VCV), which is very effective and has few side-effects. VCV, though, targets viral replication, and so could not deal with latent virus, but presumably, it would act against new virions produced after viral reactivation.

In summary, the three main problems appear to be: 1) Is there significant damage during latency and if so, how could it be stopped? 2) Could an anti-inflammatory substance be used with VCV to combat virus-induced inflammation? 3) Could methods be devised which monitor viral load and activity in brain during treatment? One can only hope that those of us in the microbe-AD field will not have to wait much longer for acceptance of the relevant concepts, and subsequent provision of resources for answering these questions.

References
[1] Ball MJ (1982) Limbic predilection in Alzheimer dementia: is reactivated herpesvirus involved?. Can J Neurol Sci 9, 303–306.
[2] Jamieson GA, Maitland NJ, Wilcock GK, Craske J, Itzhaki RF (1991) Latent herpes simplex virus type 1 in normal and Alzheimer’s disease brains. J Med Virol 33, 224–227.
[3] Itzhaki RF, Lin W-R, Shang D, Wilcock GK, Faragher B, Jamieson GA (1997) Herpes simplex virus type 1 in brain and risk of Alzheimer’s disease. Lancet 349, 241–244.
[4] Itzhaki MS, Wozniak M (2009) Apolipoprotein E: microbial friend or foe? In Apoprotein Research, Penfield LR, Nelson RT, eds. Nova Science Publishers, New York, pp. 99–112.
[5] Itzhaki RF (2014) Herpes simplex virus type 1 and Alzheimer’s disease: Increasing evidence for a major role of the virus. Front Aging Neurosci 6, 1–9.
[6] Klapper PE, Cleator GM, Longson M (1984) Mild forms of herpes encephalitis. J Neurol Neurosurg Psychiatry 47, 1247–1250.
[7] Itzhaki RF, Wozniak MA (2012) Could antivirals be used to treat Alzheimer’s disease? Future Microbiol 7, 307–309.
[8] Wozniak MA, Itzhaki RF (2010) Antiviral agents in Alzheimer’s disease: hope for the future? Ther Adv Neurol Disord 3, 141–152.
[9] Piacentini R, Li Puma DD, Ripoli C, Marcocci ME, De Chiara G, Garaci E, Palamara AT, Grassi C (2015) Herpes Simplex Virus type-1 infection induces synaptic dysfunction in cultured cortical neurons via GSK-3 activation and intraneuronal amyloid-β protein accumulation. Sci Rep 5, 15444.
[10] Soscia SJ, Kirby JE, Washicosky KJ, Tucker SM, Ingelsson M, Hyman B, Burton MA, Goldstein LE, Duong S, Tanzi RE, Moir RD (2010) The Alzheimer’s disease-associated amyloid beta-protein is an antimicrobial peptide. PLoS One 5, e9505.
[11] White MR, Kandel R, Tripathi S, Condon D, Qi L, Taubenberger J, Hartshorn KL (2014) Alzheimer’s associated β-amyloid protein inhibits influenza A virus and modulates viral interactions with phagocytes. PLoS One 9, e101364.
[12] Bourgade K, Garneau H, Giroux G, Le Page AY, Bocti C, Dupuis G, Frost EH, Fülöp T (2015) β-Amyloid peptides display protective activity against the human Alzheimer’s disease-associated herpes simplex virus-1. Biogerontology 16, 85–98.
[13] Bourgade K, Le Page A, Bocti C, Witkowski JM, Dupuis G, Frost EH, Fülöp T (2016) Protective effect of amyloid-β peptides against herpes simplex virus-1 infection in a neuronal cell culture model. J Alzheimers Dis 50, 1227–1241.
[14] Kumar DK V., Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD (2016) Amyloid- peptide protects against microbial infection in mouse and worm models of Alzheimers disease. Sci Transl Med 8, 340ra72–340ra72.
[15] Miklossy J (2015) Historic evidence to support a causal relationship between spirochetal infections and Alzheimer’s disease. Front Aging Neurosci 7, 46.
[16] Little CS, Joyce TA, Hammond CJ, Matta H, Cahn D, Appelt DM, Balin BJ (2014) Detection of bacterial antigens and Alzheimer’s disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae. Front Aging Neurosci 6, 304.
[17] Wozniak MA, Frost AL, Preston CM, Itzhaki RF (2011) Antivirals reduce the formation of key Alzheimer’s disease molecules in cell cultures acutely infected with herpes simplex virus type 1. PLoS One 6, e25152.
[18] Wozniak M, Bell T, Dénes Á, Falshaw R, Itzhaki R (2015) Anti-HSV1 activity of brown algal polysaccharides and possible relevance to the treatment of Alzheimer’s disease. Int J Biol Macromol 74, 530–540.

Last comment on 19 August 2016 by Brian Balin, PhD

Comments

First, a geriatrician’s personal remarks on Prof. Itzhaki’s blog. I first met Ruth Itzhaki at a gerontological congress in 2000 in Bologna, Italy. During the speakers’ dinner we discussed her ideas about herpesviridae and Alzheimer's disease. The topic was new to me, but at the Helsinki University Hospital I had been trained for internal medicine in a clinic with special interest in gastroenterology, and I clearly remembered the enthusiasm at the end of 1980s when the connection between Helicobacter pylori and duodenal ulcer was revealed and how it transformed clinical practice. In 2000, the increasing dementia burden was well recognized, but cholinergic drugs were emerging as only routine medications (as they are today) and practically the only advice for prevention was to wear a helmet while cycling.

So, I was fascinated by Prof. Itzhaki’s ideas about Alzheimer’s disease etiology, and after returning from Bologna, promptly tested our 75+ DEBATE study sera for some viral and bacterial antibodies. It turned out that viral (herpes simplex virus type 1 [HSV-1], herpes simplex virus type 2 [HSV-2], and cytomegalovirus [CMV]) seropositivity was clearly associated with worse cognition [1]. Of note, similar association was not observed between bacterial burden (Chlamydia pneumoniae and Mycoplasma pneumoniae) and cognition in our cohort. In a further analysis, we also noted that when viral burden was associated with low education and presence of APOE epsilon4, the association with worse cognition was ever stronger (risk ratio 6.1, 95% CI 2.4-15.2) pointing to the multifactorial nature of late-onset cognitive dysfunction [2].

Seropositivity is naturally indirect evidence, and with ever increasing interest I have followed the sophisticated analyses performed by Prof. Itzhaki’s group and convincingly summarized in her present blog. In our email correspondence during the years, I have also shared her sadness about disbelief among reviewers of her grant applications. We both agreed that proving or disproving the herpes-Alzheimer’s disease connection would need a randomized treatment trial with an antiviral. This kind of trial was actually designed and approved by authorities in Helsinki, but unfortunately intervening duties have so far prevented our group from realizing it.

The year 2016 will hopefully mark a turning point for the microbe-Alzheimer’s disease theory; the Journal has published the Editorial about microbes and Alzheimer’s disease [3], and supporting experimental evidence was reported by a Harvard group [4]. But for sure, treatment of herpesviridae does not alone solve the dementia problem. Most of late-onset cognitive dysfunction is a multifactorial disease, at one end, “pure” neurodegenerative disease of the Alzheimer type, the other end a “pure” vascular disease. My educated guess, based on our unpublished longitudinal data among others, is that neurodegenerative dysfunction without clear vascular involvement or relation to vascular risk factors could be responsible for 25% of all progressing dementias in old age. That is a lot of patients and if antiviral treatment could specifically relieve that burden, it would be a great leap forward.

References
[1] Strandberg TE, Pitkala KH, Linnavuori KH, Tilvis RS (2003) Impact of viral and bacterial burden on cognitive impairment in elderly persons with cardiovascular diseases. Stroke 34, 2126-2131.
[2] Strandberg TE, Pitkala K, Eerola J, Tilvis R, Tienari PJ (2005) Interaction of herpesviridae, APOE gene, and education in cognitive impairment. Neurobiol Aging 26, 1001-1004.
[3] Itzhaki RF, Lathe R, Balin BJ, et al. (2016) Microbes and Alzheimer's Disease. J Alzheimers Dis 51, 979-984.
[4] Kumar DK, Choi SH, Washicosky KJ, Eimer WA, Tucker S, Ghofrani J, Lefkowitz A, McColl G, Goldstein LE, Tanzi RE, Moir RD (2016) Amyloid-β peptide protects against microbial infection in mouse and worm models of Alzheimer's disease. Sci Transl Med 8, 340ra72.

The blog by Dr. Itzhaki on “The case for a viral role in Alzheimer’s disease” highlights the ongoing struggle faced by many in the Alzheimer’s disease (AD) research arena to gain acceptance for the EVIDENCE that INFECTION FOUND in AD BRAIN TISSUES could actually have causative effects leading to this horrible disease. I can reiterate what Dr. Itzhaki has stated in this blog, but up to now this approach has fallen on deaf ears and blind eyes. The public, researchers and medical personnel who are truly concerned with seeing that a PARADIGM shift take place need to see the alternative reality. In this regard and amazingly, many of us have been told that there is not ENOUGH EVIDENCE to move forward with the consideration that infections are involved with this disease and until there is (by what mechanism, I don’t know), that the EVIDENCE will not be regarded as something significant in our understanding of what REALLY causes the vast majority of late-onset AD. What is even more amazing is that the very same people who do not believe that there is enough evidence to move forward with this PARADIGM shift, have not studied directly or refuse to actually examine the evidence that infection is involved with this disease. In this regard, IGNORANCE may be bliss, BUT what is being overlooked is the actual scourge on society and the affects and effects on the millions who suffer with or through (caregivers) this process. For this researcher, and I am sure others, this is OUTRAGEOUS and UNACCEPTABLE.

How many trial failures with amyloid and tau modifying approaches are NEEDED before the pharmaceutical, medical and research communities decide to look elsewhere in the search for the REAL triggers and drivers of this disease? As there are currently over 400 trial failures [1] over numerous years, what number do we have to reach before the LIGHTS go on? Ironically, as we consider this dilemma, the current (August 15, 2016) AlzForum website has released information stating that “In the wake of a decade of clinical trial setbacks, researchers all around the world are building integrated systems to run cheaper, faster, and hopefully more successful trials. Large registries, partnerships with health-care providers, and other outreach efforts are supposed to reach hundreds of thousands of people, and entice them to join cohorts where those at high risk get phenotyped and invited to join therapeutic trials. Europe, North America, Australia, and Japan are all on this path. Will it work?” Cheaper and faster will not help if we are looking under an INCORRECT lamp-post!

Now, turning back to infectious processes in AD and how to combat these issues; what clinical trials can be designed currently to address this issue? As Dr. Itzhaki has proposed, anti-viral trials for HSV1 infections seem appropriate and would have a high benefit to risk ratio. Trials to address other types of infection possibly with anti-bacterials and anti-inflammatories in conjunction may be warranted as well. Older populations at risk can be assessed for serotiters against many of these infectious agents, and if positive, could be enrolled for study. Individuals demonstrating early symptoms such as change in sense of smell and early cognitive change could be enrolled especially if positive by serotitering for infectious agents already found associated with AD. These evidenced based approaches should be considered just as important as those of imaging and biomarker based approaches for which amyloid and tau accumulations are noted in AD brains and used as a basis for AD determination and trial enrollment.

In summary, we cannot afford to delay initiating trials addressing infection in AD. The ongoing physical and emotional suffering as well as the economic burden for so many warrants trying to make a difference based on CURRENT EVIDENCE. I fully support Dr. Itzhaki’s position and challenge all of us to be bold and courageous in our efforts to change the course of AD.

Reference
[1] Cummings JL, Morstorf T, Zhong K (2014) Alzheimer's disease drug-development pipeline: few candidates, frequent failures. Alzheimers Res Ther 6, 37.

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