Alzforum Ruth Itzhaki

8/7/2019 Alzforum Ruth Itzhaki

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HERPES SIMPLEX VIRUS TYPE 1, APOE-4 AND ALZHEIMERS DISEASE: THE

CASE FOR ANTIVIRAL TREATMENT

Ruth Itzhaki, Matthew Wozniak, Alison Frost

Molecular Neurobiology Lab

Faculty of Life Sciences

University of Manchester


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MICROBES & DAMAGE

Some microbes can remain in the body lifelong after acuteinfection they dont just hit and run.

Infection can be latent, but reactivation may occur.

Infect doesnt necessarily mean affect, so controls may be

infected. Severity of damage probably depends on host genes.

Susceptibility to microbial infection, like susceptibility to

microbial damage, can depend on host genes.

Effects of microbe may depend on tissue/cell type involved & on

microbe strain.


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HSV1 & THE NERVOUS SYSTEM

HSV1 infects most humans in infancy. Remains latent in the PNS.

Latency: viral DNA is present, only one abundant set oftranscripts, no viral proteins.

Stress, immunosuppression, etc, can reactivate HSV1: wholeviruses form, i.e., acute infection occurs.

On reactivation, 20+% of people develop herpes labialis (coldsores); the remainder are unaffected.

In acute infection, HSV1 subverts the cell machinery, & stimulatescells into cycle (to G2/M). Occurs in AD too


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QUESTIONS

1. Is HSV1 present in normal brains?

2. Is the virus active in brain?

3. Is HSV1 in brain associated with AD?

4. Is there a causal link with AD neuropathology?

5. If a link is found, might antivirals work against AD?


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HSV1 & APOE-4 IN AD (& COLD SORES)

Answers to questions 1-3

HSV1 DNA is present (latently) in many elderly normal & AD brains

(shown by solution & in situ PCR)(J Med Virol. 1991. et seq.). Subsequently, 5

other groups found HSV1 DNA in human brains.

It has reactivated, then replicated there, perhaps recurrently (shown

by intrathecal antibodies to HSV1) (J Med Virol. 2005).

HSV1 in brain of APOE-4 carriers confers a strong risk of AD,

accounting for ~ 60% of patients(Lancet, 1997; Alz Repts. 1998).

APOE-4 is a risk factor for herpes labialis (Lancet, 1997; Alz Repts. 1998).(Confirmed by Koelle et al., 2010)

Conclusion: in CNS & PNS, HSV1 damage is greater in

APOE-4 carriers.


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HSV1 LINKS TO AD NEUROPATHOLOGY

Previous relevant work:

HSV1 glycoprotein B peptides form fibrils ultra-structurallyidentical to A; HSV1 might seed plaques (Cribbs et al., 2000).

APP is associated with HSV1 during virus anterogradetransport; might affect APP degradation & synaptic function(Satpute-Krishnan et al., 2003).


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HSV1 INCREASES LEVELS OF A & AD-LIKETAU

HSV1 infection of human neural cells increases:

intracellular A 1-42 & A 1-40 & also BACE & nicastrin(Neurosci.Lett., 2007).

tau phosphorylation at AD-relevant sites, & also PKA &GSK3(J. Alz. Dis., 2009).

Also, HSV1-infected mice show A accumulation in brain

NB:

BACE increase is caused by phosphorylation of eIF2, due to activation of PKR (Ill-Raga et al.,submitted)

HSV1 encodes a protein kinase, US3; this activates & functionally overlaps cell kinase PKA.

GSK3 regulates A production


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IN SITUPCR & THIOFLAVIN S OR IHCWozniak et al., J.Pathol 2009

HSV1 DNA (in situ PCR)

20X

Plaques (Thioflavin S)

20X

HSV1 DNA (in situ PCR, brown) &

A (IHC, purple) 20X

>90% plaques

contain viral DNA


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PERCENT AMYLOID PLAQUES CONTAINING

HSV1 DNA, AND HSV1 DNA CLUSTERS

ASSOCIATED WITH PLAQUES

* p


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CO-LOCALISATION OF HSV1 DNA &

PLAQUES(J Pathol. 2009).

In AD & aged normal brains, 80-90% of amyloid plaquescontain HSV1 DNA.

In AD brains, over 70% of the HSV1 DNA is located in plaques;

only 24% in aged normals, possibly because less produced&/or more cleared.

Artefactual co-localisation can be refuted.

Of course association does not prove causality, but asHSV1 infection causes A accumulation the viral DNA-

amyloid co-localisation indicates that HSV1 is a major

cause of toxic A products & plaques.


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ACYCLOVIR

ACV is a nucleoside analogue that stops viral replication. Its action requires HSV1 thymidine kinase (TK).

TK phosphorylates ACV to its mono-phosphate form.

Cell enzymes further phosphorylate to di- and tri-phosphate.

The tri-phosphate competes with dGTP as a DNA polymerase

substrate; in new DNA it prevents elongation.

Thus ACV finds HSV1-infected cells & stops HSV1

replication.

NB. In practice, valacyclovir, the biodrug of ACV, is

used orally as its absorption is very much greater.


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P-tau

(pS214)

A

(A1-42)

HSV1

Uninfected

50uM ACV

Uninfected

No ACV

HSV1-infected

no ACV

HSV1-infected

50uM ACV

EFFECTS OF ACYCLOVIR ON THE AD-LIKE CHANGES IN HSV1-

INFECTED CELLS


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HSV1-INDUCED CHANGES IN A & P-tau,

& EFFECT OF ACV

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

0uM ACV 100uM ACV 0uM ACV 100uM ACV

HSV1-infected (10pfu/cell) HSV1-infected (10pfu/cell) Uninfected Uninfected

p


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ANSWERS

1. Is HSV1 present in normal brains?Yes, in the elderly.

2. Is the virus active in brain?

Yes, perhaps recurrently.

3. Is HSV1 in brain associated with AD?

Yes, in APOE-4 carriers.

4. Is there a causal link with AD neuropathology?

Yes, with plaques and AD-like tau.

5. If a link is found, might antivirals work against AD?

Based on our cell culture work, they should reduce not

only a major cause but also A and P-tau.


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FUTURE POSSIBILITIES FOR

PREVENTION OR TREATMENT OF AD

Immunisation against HSV1 in infancy (more

feasible now, with rising age of primary infection).

Use of antiviral agents to retard diseaseprogression.

N.B. Antivirals (e.g., VCV/ACV) would target only

HSV1, i.e., a cause of AD, rather than thesymptoms/features, & not host cells. In fact they

cause very few side-effects.


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PROOF OF CONCEPT TRIAL

We hope to recruit 100 HSV-seropositive patients with

mild to moderate AD, in four centres.

Treat half with VCV (2x500mg/day), half with placebo.

Follow for 12 months, investigating cognitive function

(ADAS-Cog & MMSE), activities of daily living (IDDD),

behavioural and psychological symptoms (NPI), Quality

of life (DEMQOL), & global impression of change (GIC),assessing pre-randomisation and at 3, 6, 9 & 12 months.

Check renal function.


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PAST LAB MEMBERS

Curtis Dobson

Ann CooksonSuzanne Shipley

Tom Lloyd

Woan-Ru Lin

Gordon Jamieson

Dazhuang Shang

Acknowledgements include

Alzheimers Society; Henry

Smith Charity.

COLLABORATORS

Andrew Mee

Gordon Wilcock

Stacey Efstathiou

Margaret Esiri

Seth Love

Brian Faragher

Roy Jennings

Marc CombrinckBob Cooper

Paul Klapper

Chris Preston

Raj Kalaria

David MannNigel Hooper


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RELEVANT WORK BY OTHERS (1) Measles virus causes NFT in brain, & HIV causes amyloid plaques

and NFT. *

Systemic infection causes cognitive decline in the elderly (Strandberg

et al., 2003; Holmes et al., 2003) - consistent with a viral role in AD: peripheral

infection brain inflammation latent HSV1 reactivation in brain

both inflammatory and direct damage

Serum IgM evidence of recent HSV reactivation (Letenneur et al.2008).

Genetic support for a viral role (Porcellini et al., 2010)

*HSV1 is the only relevant virus found so far in elderlybrains and so is uniquely placed to cause AD-like

damage


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RELEVANT WORK BY OTHERS (2)

In HSV1-infected APOE-transgenic mice, viral load in brain is greaterin APOE-4 than APOE-3 animals (Burgos et al., 2003, 2006;

Bhattacharjee et al, 2008), & APOE alleles differentially affect HSV1

expression (Miller & Federoff, 2007).

Repeated stress reactivates latent HSV1 in brains of IL-/-mice,

causing widespread lesions. HSV1 damage is greater in brains of

APOE-4 than of APOE-3 mice, & in older than in young mice (Sawtell,IHW abstracts, 2010, & http://www.cincinnatichildrens.org/health/subscribe/ped-

insights/November2010/herpes.htm).

HSV1 infection of primary cultures of embryonic neurons causesfunctional changes, AD-like tau phosphorylation, increased A, &

triggers AD-like caspase-3 activation & tau cleavage (Piacentini et al, 2010;Santana et al., 2011; Zambrano et al., 2008; Lerchundi et al., 2010).


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SUMMARY (1)

HSV1 DNA resides, and the virus has replicated,in brain of many elderly people.

HSV1 DNA in brain and APOE-4 confer a strongrisk of AD (cf. APOE-4 and herpes labialis).

HSV1 causes A accumulation & AD-like tau, &

increases levels of relevant enzymes.


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SUMMARY (2)

HSV1 DNA co-localises with amyloid plaquesin AD brains.

ACV reduces HSV1-induced A formation

& AD-like tau phosphorylation in cell cultures.

Vaccination of mice prevents HSV1 latency in

brain.

APOE determines severity of damage (or

susceptibility to infection) by diverse

pathogens.


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Questions (2)Holtzman:

Q: In humans, is there evidence in living humans that those

developing AD pathology and neurodegeneration (pre and post-

symptomatically) have differences (or not) in prior or current CNS

infections with different herpes or other viruses?

A:Its not known if they are more or less prone, though many elderly

normals as well as AD patients harbour HSV1 in brain. As to overt

CNS infections, luckily these are very rare and so the sample numbers

would be very small. HSV1 causes herpes simplex encephalitis, a

very severe acute event affecting 1 to 3 people per million. Previously

most would have died but now ACV/VCV treatment prevents death -complicating this issue.


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Questions (3)

Anonymous (!):Is active HSV1 seen more commonly before and during the onset of

typical AD symptoms than in age-matched elderly who did not develop

AD or its precursor, mild cognitive impairment (MCI)? This could be done

by demonstrating links between HSV1 titers and AD biomarkers (e.g. low

cerebrospinal fluid A42/40 ratio, high PIB signal in brain amyloid

imaging)

A:it is unknown whether active HSV1 infection is more common before

and during AD onset. HSV antibody titres (IgG) in serum are not

indicative as they differ little between reactivation and latency episodes

(probably because reactivation frequency is high). In brain, HSV1 cant be

detected ante mortem, let alone latent or reactivated virus except

via CSF taken weekly (hardly feasible)! However, intrathecal antibodies

(IgG) are present in many elderly controls and AD patients, so HSV1 must

have reactivated in brain, perhaps recurrently. Also, see Letenneur et al

(2008) re detection of IgM in serum of those who will develop AD

indicating viral reactivation in PNS and possibly also in the CNS.

Questions (4)


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Questions (4)

Schlossmacher:

Q. Is there any study in the neuropathology literature that has examined brains

of survivors of a monophasic HSV encephalitis to determine whether A andtau are dysregulated versus survivors of other encephalitides?

A. Nothing relevant has been published, but with so much damage it wouldprobably be hard to detect.

Q. Do patients who are on chronic antiviral treatment with Zovirax (e.g., for

prevention of recurrence of genital herpes) have a lesser risk of ADdevelopment or AD progression?

A. We have been unable to find any data on this (or on HSE survivors) butwould expect little effect in brain as absorption of ACV is poor (the biodrugVCV is much better absorbed).

Q. Do patients with classical herpes simplex type 1 infections carry tau-positive tangles in their ganglion Gasseri of cranial nerve 5 at postmortemexamination?

A. Nothing published. NB, It is difficult to obtain human trigeminal ganglia.


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HSV1 DNA PRESENCE IN

PLAQUES IS NOT ARTEFACTUAL

Might pre-existing plaques attract & engulf viral DNA from other sites? No: viral DNA is large(so movement is unlikely), & does not persist extra-cellularly.

Might microglia engulf HSV1 DNA or be infected & then carry the viral DNA to plaques (towhich they are attracted)? No: infection of microglia in brain is rare (Esiri et al. 1995).

Might pre-existing plaques reactivate HSV1 via inflammatory effects? No: that would requireall the 80-90% of plaques with viral DNA to have been located next to latently infected cells.

Might viral DNA be stuck to plaques? No: they contain no cell DNA (Ginsberg et al. 1997) though itsamount is vastly greater - & 10-20% contain no viral DNA at all.

Also, average plaque size is ~50 um diameter but sections are only 7 um thick; thus, most

sections display plaque interiors - & all reveal viral DNA throughout.


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0

7

41

0

5

10

15

20

25

30

35

40

45

HSV1 infection + PBS HSV1 infection + ISCOM vaccine ISCOM Vaccine

(N=39)

(N=41)

(N=9)%

%

%

PROTECTION OF MICE FROM HSV1 LATENCY IN BRAIN

BY VACCINATION WITH HSV1 GLYCOPROTEINS (Neurobiol.Aging., 2001)


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PROPORTION OF BRAINS HARBOURING

HERPESVIRUSES (J. Pathol. 2002)

* OR = 3.9, 95%CI 1.54-9.64

0

10

20

30

40

50

60

70

80

HSV1 HSV2 CMV HHV6Virus

AD

Normal

N = 53

N = 35

N = 45N = 29

N = 50

N = 35

N = 48

N = 61

EFFECT OF APOE ON OUTCOME OF


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EFFECT OF APOE ON OUTCOME OF

INFECTION

a. whole group & females, b. females only, c. whole group only, d. whole group & males.

Conclusion: APOE is a major factor in determining outcome of infection. Alleleinvolved probably depends on cell type as well as pathogen type.

Disease Agent Risk Protective

Herpes labialis HSV1 APOE-4 (p

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