Alzheimer’s disease plaques and tangles: Cemeteries of a Pyrrhic victory of the immune defence network against herpes simplex infection at the expense of complement and inflammation–mediated neuronal destruction.

 C.J.Carter Neurochem Int,2010

 Running title: HSV-1 binding proteins in Alzheimer’s disease plaques or tangles

Summary

 Plaques and tangles are highly and significantly enriched in herpes simplex (HSV-1) binding proteins (by 11 and 15-fold respectively (P < 4.47E-39) and 132/341 (39%) of the known HSV-1 binding partners or associates are present in these structures. The classes involved include the majority (63-100%) of the known HSV-1 host protein carriers and receptors, 85-91% of the viral associated proteins involved in endocytosis, intracellular transport and exocytosis and 71% of the host proteins associated with the HSV-1 virion. The viral associated proteins found in plaques or tangles trace out a complete itinerary of the virus from entry to exocytosis and the virus also binds to plaque or tangle components involved in apoptosis, DNA transcription, translation initiation, protein chaperoning, the ubiquitin/proteasome system and the immune network. Along this route, the virus deletes mitochondrial DNA, as seen in Alzheimer’s disease, sequesters the neuroprotective peptide, ADNP, and interferes with key proteins related to amyloid precursor protein processing and signalling as well as beta-amyloid processing, microtubule stability and tau phosphorylation, the core pathologies of Alzheimer’s disease. Amyloid-containing plaques or neurofibrillary tangles also contain a large number of complement, acute phase and immune-related proteins, and the presence of these pathogen defence related classes along with HSV-1 binding proteins suggests that amyloid plaques and tangles represent cemeteries for a battle between the virus and the host’s defence network. The presence of the complement membrane attack complex in Alzheimer’s disease neurones suggests that complement mediated neuronal lysis may be a consequence of this struggle.  HSV-1 infection is known to increase beta-amyloid deposition and tau phosphorylation and also results in cortical and hippocampal neuronal loss, cerebral shrinkage and memory deficits in mice. This survey supports the contention that herpes simplex viral infection contributes to Alzheimer’s disease, in genetically predisposed individuals. Genetic conditioning effects are likely to be important, as all of the major risk promoting genes in Alzheimer’s disease, (Apolipoprotein E, clusterin, complement receptor 1 and the phosphatidylinositol binding clathrin assembly protein PICALM), and many lesser susceptibility genes, are related to the herpes simplex life cycle Database . 33 susceptibility genes are related to the immune system. Vaccination or antiviral agents and immune suppressants should therefore perhaps be considered as viable therapeutic options, prior to, or in the early stages of Alzheimer’s disease.

Introduction

 

Alzheimer’s disease is a devastating degenerative disorder characterised by extensive neuronal loss particularly of the cholinergic system (Sims, Bowen et al. 1980) , severance of the afferent and efferent hippocampal connections (Van Hoesen and Hyman ,1990) , loss of corticocortical glutamatergic association fibres (Young ,1987) and massive cerebral shrinkage (Thompson, Hayashi et al. 2003) . It is characterised pathologically by the presence of amyloid containing plaques (Nikaido, Austin et al. 1970) and neurofibrillary tangles (Ishino and Otsuki ,1975) containing the hyperphosphorylated microtubule protein, tau (Kosik ,1990) . Plaques and tangles are thought by many to be the factors leading to the neuronal devastation in this disease (Perl ,2010). An understanding of their genesis is a key goal in Alzheimer’s disease research. These features, including beta-amyloid deposition,  tau phosphorylation (Itzhaki, Cosby et al. 2008) (Piacentini, Civitelli et al. 2010;Wozniak, Frost et al. 2009) , entorhinal and hippocampal cell loss and cerebral shrinkage (Armien, Hu et al. 2009) can all be induced by herpes simplex viral infection in mice.

Herpes simplex can enter the brain via neural routes, typically via the trigeminal nerve, from whence the virus targets the limbic system (Becker ,1995;Damasio and Van Hoesen ,1985) , but can also enter via the bloodstream. Infection via this route is favoured by the possession of the APOE4 variant in mice and also by gender, with female mice showing a greater degree of cerebral infection (Burgos, Ramirez et al. 2005;Burgos, Ramirez et al. 2006;Burgos, Ramirez et al. 2003;Burgos, Ramirez et al. 2002)  . The virus binds to all classes of lipoprotein (VLDL, LDL and HDL) and the viral glycoprotein B binds to APOA1 and APOE containing lipoproteins (Huemer, Menzel et al. 1988) . The virus also binds to complement receptor 1 (CR1) on erythrocytes (Powers, Buster et al. 1995) . As CR1 is expressed in phagocytic Kolmer cells in the choroid plexus (Singhrao, Neal et al. 1999) , this receptor, a key player in Alzheimer’s disease genetics (Lambert, Heath et al. 2009) ,  could provide a means of viral cerebral entry.

There is evidence for herpes simplex infection (HSV-1) as a risk factor in Alzheimer’s disease, acting in synergy with possession of the apolipoprotein E APOE4 allele (Itzhaki, Lin et al. 1997) . A recent study has also shown that anti-HSV-1 immunoglobulin M seropositivity, a marker of primary viral infection or reactivation, in a cohort of healthy patients, was significantly associated with the subsequent development of Alzheimer’s disease. Anti-HSV-1 IgG, a marker of lifelong infection showed no association with subsequent Alzheimer’s disease development (Letenneur, Peres et al. 2008) .  Herpes simplex viral DNA is also found in the beta-amyloid containing plaques that characterise Alzheimer’s disease (Wozniak, Mee et al. 2009) ,

 During the course of viral infection, the herpes virus engages with a number of human proteins which it uses to gain cellular entry, and to traffic from the cell membrane to the nucleus and back, via endosomal and other compartments. It also uses the host’s transcriptional machinery to replicate, and binds to proteins that control immune surveillance or apoptosis. Numerous evasion strategies related to these interactions regulate the survival and propagation of the virus. Concomitantly, host proteins attempt to destroy the virus, and the eventual outcome of this host/pathogen war determines the success of the virus, and the degree of damage it may cause. Paradoxically, successful viral elimination by the host’s immune system could well lead to greater cellular damage, mediated via immune and inflammatory targeting of the cells containing the virus.

A literature survey of herpes simplex binding proteins and of the proteins identified in Alzheimer’s disease plaques or neurofibrillary tangles (NFT’s) identified a large number of HSV-1 related proteins in these structures. These proteins relate to all stages of the viral life cycle .Both structures also contain a number of immune related proteins, suggesting that they may represent the record of a host/viral battle, whose outcome is viral elimination at the expense of massive neuronal destruction.

 

 

Methods

 

HSV-1 viral binding chemicals and proteins were identified by literature survey and are stocked and referenced in a database at http://www.polygenicpathways.co.uk/herpeshost.html

 which serves as the reference source for the viral/host interactions described below. At the time of writing, the database contains 341 direct interactions between the virus and host proteins or chemicals and a number of other effects such as mitochondrial gene deletion. Indirect effects, such as those on gene or protein expression, are not included. The constituents of amyloid containing plaques or neurofibrillary tangles (NFT’s) in Alzheimer’s disease were trawled by literature survey and a large number were identified from a proteomics study of laser-dissected plaques, which identified 488 proteins within these structures (Liao, Cheng et al. 2004) and from a similar study of  NFT’s which reported 79 tangle related proteins (Wang, Woltjer et al. 2005). These datasets were used to cross reference the protein accession numbers of HSV-1 interacting proteins.  Genes implicated in Alzheimer’s disease are referenced at http://www.polygenicpathways.co.uk/alzpolys.html. Gene symbols recognised by the Human Gene Nomenclature committee (HUGO) are used throughout the text and figures, and full names are given in Tables 3 -8.

 

 

 

Results

 

Statistical analysis and the types of viral interacting protein found in plaques and tangles

 

For the two proteomics studies, HSV-1 binding proteins were highly and significantly enriched in both the plaque (10.7 fold: p=2.5229E-125) and tangle (14.4 fold:  p=4.47466E-39) datasets. Conversely, plaque and tangle components were highly and significantly enriched in the HSV-1 binding dataset (Plaque proteins = 7.4 fold: p=5.19975E-77: Tangle proteins = 61.6 fold: P= 9.5142E-39) (Table 1). Viral associated proteins were also second of the functional classes observed in plaques and first if mitochondrial proteins are included (mitochondria cluster around the virus at intracellular infection sites (Table 1, see below).

64 viral associated proteins were present in the plaque proteome and 14 were present in the tangle proteome (19% and 4% respectively of the known HSV-1 binding proteins). Further proteins in plaques and tangles were identified by literature survey .111 viral associated proteins were identified as amyloid plaque components, and 68 as tangle components. In total 132 exact protein/chemical matches (39% of the known viral associated proteins) were identified in either compartment.

A summary of the types of protein found in plaques or tangles is provided in Table 2 and references for their association with either of these structures in Tables 3 to 8 which are organised by functional category. These categories are illustrated in Figs 1-4.

There was considerable overlap between the two compartments as shown in Table 2, although the profiling of the proteins in relation to function was distinct in the two compartments (Figs 3-4). However a number of proteins were plaque (67) or tangle (23) specific. These are highlighted in Table 2, and illustrated in Figs 1-4. Moreover, the viral interacting proteins found in plaques and tangles are clustered in distinct functional classes sequentially related to different phases of the viral life cycle (Table 2, Figs 1-4). For example, almost all of the known carriers and primary receptors for the herpes simplex virus (100 and 63% respectively) are present in plaques or tangles (Table 2) and a high proportion of host viral associated proteins related to herpes simplex endocytosis (91%), intervesicular transport (88%), anterograde and retrograde transport (85 and 94%) and host virion components (71%) are also present in these structures. Host nuclear proteins, or those binding to the viral genome, or latency transcript, are much less represented (Fig 3, 4) (see below).

 

Viral associated proteins in amyloid plaques (Fig 1, Fig 3)

 

Amyloid plaques contain numerous herpes simplex carriers, (defined as soluble molecules occupying the extracellular space), including APOE, as well as a large number of known viral receptors (Table 2,3, Fig 1). Two of these, syndecans 1 and 2, are substrates for the APP protease complex, gamma-secretase (Hemming, Elias et al. 2008).

Herpes simplex binds to heparan sulphate and chondroitin sulphate proteoglycans; although within this family only syndecans 1 and 2 have been specifically tested as entry receptors (Cheshenko, Liu et al. 2007) . Agrin, Aggrecan, Decorin, Glypican 1, Perlecan, syndecans 3 and 4, neurocan and Versican belong to these families and are all present in plaques or tangles. These might all be considered as potential HSV-1 receptors (Table 2, 3).as could a chondroitin sulphate proteoglycan form of APP, appican, which is expressed in astrocytes (Pangalos, Shioi et al. 1996) . Calculations with or without these receptors are provided in Tables 1 and 2.

The cellular uptake of the herpes virus protein VP22 is mediated via lipid raft-dependent endocytosis that depends on dynamins and ADP ribosylation factors. Dynamin 1 (DNM1) is expressed in both plaques and tangles and numerous ARF’s (virion components), which play a general role in membrane traffic (D'Souza-Schorey and Chavrier ,2006) are predominantly expressed in amyloid containing plaques (Table 4, Fig 1). The routing of viral glycoprotein D to the endosomal compartment is also mediated by mannose-6-phosphate receptors (IGF2R and M6PR) both of which also play a role in viral entry (Table 4, Fig 1).  (Brunetti, Dingwell et al. 1998) Clusterin is a ligand for M6PR (Lemansky, Brix et al. 1999) while PICALM (Phosphatidyl inositol binding clathrin assembly protein) overexpression reduces its endosomal localisation, suggesting blockade of its transport from the plasma membrane or the trans-Golgi network to endosomes (Tebar, Bohlander et al. 1999)(Fig 1) .Thus plaques contain many elements of the machinery delivering the virus to intracellular vesicular compartments. Two major Alzheimer’s disease susceptibility genes, clusterin and PICALM) are implicated in this machinery, while APOE is involved in serum viral transport.

Retrograde viral transport towards the nucleus (Table 4, Fig1) is mediated by dynein motors which carry the virus and walk it along the microtubule network. The dynein motor is composed of numerous dynein light, intermediate and heavy chains and three dynactins. The herpes virus binds to dynactin1 (p150/glued) and to dyneins DYNC1I1, DYNLL1, DYNLT1 and DYNLT3, although other components of the motor are likely to be involved in viral transport (Lyman and Enquist ,2009). The amyloid plaque contains the viral binding dynein DYNC1I1 and DYNLL1 (as well as DYNC1H1, and DYNLL2, which have not been shown to bind to the virus but which are generally involved in retrograde transport) (Fig 1).

The herpes simplex virion associates with host actins (ACTB, ACTG1), cofilin 1, which disassembles actin filaments,  profilin1, which regulates actin polymerisation (Bugyi and Carlier ,2010) and cysteine rich protein , CSRP1,  which binds to the actin cross-linking protein, actinin (Harper, Beckerle et al. 2000).  Actins also play a role in HSV-1 intracellular transport including within, and to and from the nucleus (Lyman and Enquist ,2009). Nuclear export of the virus is mediated by crm1 (exportin XPO1)  a protein that binds to the product of a major Alzheimer’s disease susceptibility gene, PICALM. This is discussed below (Fig 1).

The anterograde transport  (nucleus to cell surface) of the virus along microtubules involves APP (Satpute-Krishnan, DeGiorgis et al. 2003) (see below) and kinesin motors (Lyman and Enquist ,2009) (Table 4, Fig 1). The kinesin-1 (KIF1A or KIF5B) viral associated proteins were not found the proteomics study of amyloid plaques, although kinesin light chain KLC3 was present. Kinesins also play a role in APP transport, which is discussed below. Multiple forms (45) of kinesin exist, and their individual role in herpes viral or APP transport, or in plaque deposition remains to be assessed.

Numerous virion-incorporated RAB proteins (members of the Ras superfamily of monomeric G proteins) are amyloid plaque components (Table 5). These play multiple roles in intracellular traffic (endoplasmic reticulum, Golgi, endosome, lysosome and autophagosome).  Their distribution in these different compartments, based on recent reviews (Stenmark ,2009;Cardoso, Jordao et al. 2010), is illustrated in Fig 1. The trans-Golgi network protein TGOLN2 is also involved in protein transport within the trans-Golgi network, endosomes, and the cell membrane (McNamara, Grigston et al. 2004). Many viral associated proteins including RABS and the autophagy protein beclin 1 are present in these intervesicular compartments.

            Several annexins are both virion and plaque components (Table 5) and ANXA2 has also been isolated as a virion associate from HSV-1 infected cells (Padula, Sydnor et al. 2009). Annexins are involved in endo- and exocytosis while the growth cone associated protein, GAP43 and the synaptosomal protein, SNAP25, are involved in viral exocytosis (Fatimathas and Moss ,2010).

A recent proteomics study of 14-3-3 proteins delivered many binding partners. Prominent among these were a number of kinesins (KIF1B, KIF1C, KIF23, KLC2, KLC3, KLC4 and the viral binding KIF5B) and tubulins (TUBB, TUBB4 and TUBB4A), shared between YWHAE, YWHAG and YWHAZ. YWHAG also bound to dynein (DYNC1H1) (Jin, Smith et al. 2004). 14-3-3 proteins have many diverse roles but their relationship to transport systems is highlighted in Figs 2 and 3. Their positioning relates to the binding partners in the proteomics publication.  

In addition to these transport related proteins, amyloid plaques contain several viral binding protein chaperones (calnexin and heat shock proteins), as well as ubiquitin and the proteasomal unit PSMA2 and the translation initiation factor, EIF4A2. (Table 7, 8) .The chaperone/proteasome/ubiquitin machinery is hijacked by herpes simplex which moves these complexes into discrete nuclear foci probably involved in the quality control of viral proteins (Burch and Weller ,2004). The ubiquitin proteasome system, necessary for the elimination of abnormal proteins such as beta-amyloid and tau, is dysfunctional in Alzheimer’s disease (Paul ,2008), a situation that might well be induced by the effects of the virus on the chaperone/proteasome/ubiquitin machinery. A number of viral binding nuclear proteins (BRCA1, HNRNPK, APEX1, PARP1, XRCC6, and numerous Histones) are likely to be involved in viral DNA replication. Clusterin appears in this compartment as it binds to the Ku antigen and DNA repair protein (XRCC6) in nuclei, an interaction promoting apoptosis (Leskov, Klokov et al. 2003).

Plaques contain a number of viral binding coagulation factors. These are part of a cascade eventually leading to thrombin activation. Thrombin is involved in the proteolysis of APP, generating a c-terminal derived peptide that may be a precursor for beta-amyloid (Igarashi, Murai et al. 1992) (Fig 5).     

Other classes of viral associated proteins included metabolic proteins (Glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase and nucleoside diphosphate kinase A). Several viral binding free radical related proteins, peroxiredoxin and thioredoxin are also plaque components. Immune and defence related viral associated proteins are well-represented plaque components ( Table 6) including complement C3, cyclophilin (PPIA) the viral DNA activated kinase EIF2AK2 , also known as protein kinase R, and its activator PRKRA,  immunoglobulin G , Interleukin 6, macrophage inhibitory factor and  HLA-antigens. The proteasome unit PSMA2 is also involved in the immune system as part of the immunoproteasome that processes MHC peptides (Wang and Maldonado ,2006). XRCC6 is included in this list as it doubles as a protein involved in somatic recombination, a process that generates multiple T cell receptors and immunoglobulins that recognise viruses and other pathogens (Gu, Jin et al. 1997).

The proteomics study of amyloid containing plaques reported a high proportion (53/488: 11%) of mitochondrial elements related to the Krebs cycle and oxidative phosphorylation (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) a result that might reflect the clustering of  mitochondria around the herpes simplex virus following infection (Bello-Morales, Fedetz et al. 2005).  In addition many enzymes related to glycolysis (18) and pyruvate metabolism (8) were observed in amyloid plaques. HSV-1 infection is known to increase cerebral glucose consumption in infected brain areas (Saito and Price ,1984).

 The viral associated proteins found in amyloid plaques effectively, and sequentially, trace out the whole process of the viral life cycle, from carriage and entry, through transport to nuclear or endosomal compartments, nuclear DNA replication, nuclear egress, protein synthesis and packaging, anterograde transport and release. Along this route, the sequestration of many proteins by the virus is likely to promulgate its traffic through diverse neuronal compartments. In addition, such sequestration is likely to influence such diverse effects as APP traffic, neurotransmitter release, neurite outgrowth, neuronal survival, neuroprotection, inflammation, and astrogliosis – all key processes disrupted in the Alzheimer’s disease brain. 

 

 

Viral associated proteins in neurofibrillary tangles (Fig 2, Fig 4)

 

The proteomics laser dissection study of NFT’s reported much fewer proteins (79) than the plaque study. 14 of which (18%) exactly match known viral associated proteins. HSV-1 also binds to tubulins, although only TUBA1B has been specifically reported. Several members of this family were NFT components in this study (TUBA4A, TUBB2A, TUBB3, TUBB4 and TUBB4Q). The virus also binds to Histones, 11 of which were present in this NFT proteomics study (Table 6). These are involved in DNA packaging. As in plaques, the chondroitin sulphate proteoglycan, Versican was present in NFT’s. If these proteins are included, 30/79 (38%) tangle components can be related to the virus (Table 1).

Other studies have shown that the viral binding proteins APOE and fibroblast growth factor ,FGF2, and the viral receptors Heparan sulphate, Chondroitin sulphate , FGFR1, IGF2R, syndecans SDC1, SDC2 as well as heparan sulphate and chondroitin sulphate proteoglycans are present in NFT’s,  as in plaques (Table 2, 3, Fig 2). Viral associated proteins related to endocytosis, retrograde, nuclear and anterograde transport, including the microtubule associated protein tau (MAPT), are also well represented in tangles (Table 2, 4, Fig 2, 4).

The viral binding kinases CDC2 and casein kinase CSNK2B  both phosphorylate tau (Singh, Grundke-Iqbal et al. 1994) while the viral binding protein, SET, is a phosphatase inhibitor that regulates tau dephosphorylation (Chohan, Khatoon et al. 2006). HSV-1 infection also increases glycogen synthase kinase GSK3B and protein kinase a (PRKRCA) expression and tau phosphorylation (Wozniak, Frost, and Itzhaki ,2009).

As with plaques, NFT’s contain nuclear proteins, heat shock proteins, translation initiation factors, 14-3-3 proteins, GAPDH , and peroxiredoxins. The binding of 14-3-3 proteins to tubulins and kinesins has been covered above. In addition YWHAZ, the predominant form in tangles, binds to tau (MAPT) and enhances its phosphorylation by cAMP dependent kinase (Hashiguchi, Sobue et al. 2000). Tangles also contain ubiquitin and proteasome components. As with plaques, tangles contain a number of immune/defence related proteins including complement C3, the membrane attack complex inhibitor CD59, cyclophilin, HLA antigens and the viral activated kinase EIF2AK2.

The virus uses the microtubule network for both anterograde and retrograde transport, and its association with a number of elements related to tau phosphorylation are likely to impact upon microtubule function.

 

Differences in the functional profiles of viral-binding proteins in plaques and tangles (Fig 3, Fig 4).

 

There is an interesting gradient in relation to the types of viral proteins that bind to the host proteins, which is similar in both amyloid plaques and tangles; glycoprotein > virion > capsid > tegument > viral genome for plaques, and glycoprotein > virion > tegument > viral genome > capsid for tangles,  roughly corresponding to the layering and sequential processing of the virus. Plaques contain a higher proportion of viral receptors and carriers, and of the viral associated proteins involved in endocytosis, intervesicular transport and exocytosis. They also specifically contain host proteins binding to the viral latency transcript (Fig 3, 4), suggesting that plaque components may be involved in viral reactivation.

Amyloid plaques are also enriched in the viral associated proteins involved in APP and beta-amyloid processing (Fig 3, 4), and contain 53 mitochondrial related proteins (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) which may reflect mitochondrial clustering around the virus after infection (Bello-Morales, Fedetz, Alcina, Tabares, and Lopez-Guerrero ,2005). Tangles are relatively enriched in viral associated proteins related to tau phosphorylation, and anterograde and retrograde transport (Fig 4), as might be expected. Tangles also contain relatively higher proportions of viral-binding proteins involved in signalling, although these are also mostly related to tau phosphorylation.

In both cases, nuclear related viral binding host proteins (replication, repair, transcription, chromatin, or proteins binding to the viral single stranded DNA binding protein ICP8, (involved in viral genome replication and recombination) are relatively poorly represented. A speculative interpretation of the sequential ordering of the viral compartments is that viral replication, entailing host nuclear compartments, has not been very successful and that the structural components of the virus (glycoprotein, virion, tegument, and capsid) have been eliminated. In this respect, both plaques and tangles contain relatively high proportions of autophagy and immune related viral associated proteins (Table 7 , Fig 3, 4) (see below). Autophagy is the process of cellular self-digestion mediated by lysosomes, that also plays a more controlled role in viral destruction and in the destruction of aberrant proteins such as beta-amyloid. This system is disrupted in the Alzheimer’s disease brain(Kim, Lee et al. 2010) (Itzhaki, Cosby, and Wozniak ,2008).

 

Immune related proteins in amyloid plaques and neurofibrillary tangles (Figs 1- 5).

 

As well as the immune or defence-related herpes simplex binding proteins covered in this survey, a number of other immune-system related proteins are found in amyloid plaques or NFT’s. The cytokines, Interleukin 1 alpha, IL6 and tumour necrosis factor TNF have also all been localised within amyloid containing plaques (Veerhuis, Janssen et al. 1999). Acute phase proteins involved in inflammation, such as amyloid P, alpha-1 antichymotrypsin and C-reactive protein are also plaque components (Eikelenboom, van Exel et al. 2010) while Immunoglobulin G is located in the plaque corona (Eikelenboom and Stam ,1982).  

As well as The herpes simplex binding protein Complement C3, a number of other complement related proteins are found in amyloid plaques or NFT’s. Complement components Clq, C3d, and C4d are present in plaques, dystrophic neurites and NFT’s (McGeer, Akiyama et al. 1989).  The membrane attack complex (MAC: complements complex C5b, C6, C7 C8 C9) is not present in amyloid plaques, but is observed in dystrophic neurites and tangle containing neurones (McGeer, Akiyama, Itagaki, and McGeer ,1989) . The MAC complex has also been detected in the neuronal cytoplasm in AD brains, associated with NFT’s and lysosomes, a distribution that suggests endocytosis of membrane bound MAC and its retrograde transport to the lysosome (Itagaki, Akiyama et al. 1994). The MAC complex is a channel that is inserted into pathogen membranes, or into host cell membranes, causing death by osmotic-related lysis. The complement inhibitors, complement receptor 1 (CR1), CD59, decay accelerating factor (CD55) or CD46 are not present in plaques (Zanjani, Finch et al. 2005) although clusterin which inhibits formation of the MAC attack complex  (Fig 3)  is present in these structures (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004): CD59, which inhibits formation of the MAC attack complex (Fig 3) is also present in tangled neurones and dystrophic neurites (McGeer, Walker et al. 1991).  In the normal brain complement receptor 1 is specifically localised to phagocytic Kolmer cells of the choroid plexus suggesting an important first line of defence (Singhrao, Neal, Rushmere, Morgan, and Gasque ,1999) . However, as the herpes simplex virus binds to this receptor (Powers, Buster, Reist, Martin, Bridges, Sutherland, Taylor, and Scheld ,1995), it may also provide an entry portal for cerebral viral infection.

Cytokines, immunoglobulins, HLA-antigens, acute phase proteins and the complement pathways are all involved in pathogen defence and the localisation of components of these systems in amyloid plaques is consistent with a pathogenic relationship to plaque formation.  The presence of the MAC complex in neurones might also suggest that neuronal lysis by the MAC complex could contribute to neuronal cell death.

 

The immune system, herpes simplex and other pathogens.

 

HSV-1 DNA is found in many healthy brains (Jamieson, Maitland et al. 1992) , and the virus is obviously able to live in harmony with the host in cerebral tissue, without provoking cell death. Indeed the viral latency transcript may even have beneficial effects as it inhibits apoptosis and promotes neurite sprouting in neuroblastoma cells (Li, Carpenter et al. 2010) . Upon reactivation, the ability of HSV-1 to inhibit the complement pathways (see above) is likely to protect it, and the neuronal cells in which it is harboured, from destruction. HSV-1 infection can per se produce entorhinal cortex and hippocampal cell loss along with memory deficits , in mice (Armien et al,2009) . In man, it may be necessary to  disrupt the harmony between the virus and the host and it is possible that activation of the immune system by other pathogens implicated in Alzheimer’s disease (e.g. Helicobacter Pylori, Chlamydia Pneumoniae and others (Honjo, van Reekum et al. 2009) ) might disturb this fragile balance, allowing viral destruction at the expense of neuronal loss. Chlamydia Pneumoniae antibodies have recently been identified in the Alzheimer’s disease brain, colocalising in apposition to plaques and tangles in vulnerable brain regions (Hammond, Hallock et al. 2010) .   Immune activation, while promoting latent rather than active infection can also produce collateral neuronal damage via inflammatory mediators (Conrady, Drevets et al. 2010) . Paradoxically, while the immune system is suppressed, the virus and the host cells could peacefully coexist, while immune activation and viral destruction might be at the terrible cost of neuronal death. 

  

 

APP processing and herpes Simplex. (Fig 5)

 

Gamma-secretase plays a key role in Alzheimer’s disease, cleaving the amyloid precursor protein APP to form the toxic beta amyloid peptide or a non-toxic intracellular signalling peptide, the APP intracellular domain (AICD). A component of this enzyme complex, Nicastrin, is upregulated by HSV-1 infection in cell culture, and beta amyloid deposition is also observed in this model, and in the brains of HSV-1 infected mice (Wozniak, Itzhaki et al. 2007). Gamma secretase also cleaves the herpes virus receptors syndecans 1 and 2 (Hemming, Elias, Gygi, and Selkoe ,2008).

The intracellular processing of full length APP follows the same route as that of the virus.  The anterograde transport of APP and HSV-1 (towards the cell surface) requires Heavy chain Kinesin-1 which binds to both APP (Szodorai, Kuan et al. 2009) and to HSV-1. Furthermore APPBP2 (Pat1), an APP binding protein, is a kinesin light chain binding protein that also binds to HSV-1 and is involved in the anterograde transport of both APP (Zheng, Eastman et al. 1998) and the virus.  Anterograde APP transport requires the viral binding protein RAB3A GTPase and the APP transport vesicle contains the viral binding synaptosomal protein SNAP25 (Szodorai, Kuan, Hunzelmann, Engel, Sakane, Sasaki, Takai, Kirsch, Muller, Beyreuther, Brady, Morfini, and Kins ,2009). APP is directly involved in the anterograde transport of the virus (in squid axons) and is a major component of the viral particles (Satpute-Krishnan, DeGiorgis, and Bearer ,2003).

APP endocytosis, like that of HSV-1 is mediated by dynamins, including the viral binding DNM2, and is regulated by the viral binding protein RAB5A (Marquez-Sterling, Lo et al. 1997). Decreasing DNM2 levels increases beta-amyloid secretion in neuroblastoma cells (Kamagata, Kudo et al. 2009). Such an effect could theoretically be duplicated by viral binding to DNM2. The retrograde transport of APP, as for HSV-1, involves dyneins and dynactins, and dynein intermediate chain knockdown increases intracellular APP and tau levels in neuroblastoma cells (Kimura, Imamura et al. 2007)

The gamma secretase mediated cleavage of APP results in the formation of  beta-amyloid and also of an intracellular signalling peptide, APP intracellular domain (AICD), which complexes with Fe65 (APPB1) and Tip60 (KAT5) or with APPB1 and TFCP2, creating active transcription complexes that increases the expression of various target genes, including APP, the APP beta-secretase BACE1 and GSK3B (Tip 60 complex) or of GSK3B (TFCP2 complex),  effects accompanied by increased  phosphorylation of the microtubule associated protein tau   (Muller, Meyer et al. 2008)  The nuclear viral binding protein Lamin A/C binds to Tip60 (Stelzl, Worm et al. 2005)and TFCP2 bind to the viral origin of replication site (Dabrowski, Carmillo et al. 1994). AICD is degraded by caspase 3 (Chow, Mattson et al. 2009).

Following its entry into the nucleus the HSV-1 viral protein ICP0 localises in nuclear dot 10 (ND10) bodies. Its ubiquitin E3 ligase activity, in concert with the host ubiquitin E2 conjugating enzyme UBE2D1, degrades the PML and SP100 components of the ND10 body (Gu and Roizman ,2009). PML binds to TIP60, recruiting it to PML nuclear bodies (Wu, Hu et al. 2009). The major susceptibility gene PICALM, binds to an exportin involved in nuclear viral egress (see below) and is thus in a position to regulate the viral effects on PML3 and tip60, and APP signalling.

Thus, as shown in Fig 5, the virus is clearly in a position to regulate APP processing, intracellular signalling and tau phosphorylation which in turn are potentially able to influence viral DNA production and the transport of the virus along the microtubule network.

 

Beta-amyloid processing and herpes simplex (Fig 5)

 

Fibrillar beta-amyloid, the toxic form of the peptide, enhances the infectivity of several viruses including herpes simplex in various cell lines. It was suggested that this might be due to enhanced fusion of the lipid envelope of these viruses with the cell membrane as the amyloid peptides and other artificial fibril producing peptides also promoted the association of lipid vesicles with cells (Wojtowicz, Farzan et al. 2002). In  addition amino acids 1-42 of HSV-1 glycoprotein B show 33% amino acid identity with beta-amyloid (Pyles ,2001), raising the possibility of viral-induced autoimmunity to beta-amyloid, a process that could continue after elimination of the virus.   Indeed, beta-amyloid autoantibodies have been found in elderly patients and AD patients , although viral cross-reactivity has not been assayed (Sohn, So et al. 2009). A peptide from the homologous region of glycoprotein B, like beta-amyloid, forms fibrillar aggregates, accelerated beta-amyloid fibril formation  and displays cytotoxic properties superior to those of beta-amyloid (Pyles ,2001).

Lipoprotein receptors play a key role in the clearance of potentially toxic beta-amyloid. The principal routes are mediated by the lipoprotein receptor LRP1, which binds to A2M- or APOE-bound beta-amyloid  and by LRP2 and LRP8 which bind to clusterin-bound beta-amyloid. Beta amyloid is also degraded by the viral binding insulin degrading enzyme (IDE) (Bates, Verdile et al. 2009). The APOA1 lipoprotein transporter, ABCA1 is also involved in beta-amyloid clearance (Koldamova, Staufenbiel et al. 2005).

The HSV-1 binding protein, complement C3 is also a ligand for LRP1 and LRP8, both of which play a role in C3 cellular uptake (Meilinger, Gschwentner et al. 1999). Beta amyloid in the bloodstream is processed by its binding to C3, which subsequently binds to complement receptor 1 on erythrocytes(Rogers, Li et al. 2006). Viral binding to A2M, C3, APOA1, or APOE as well as to IDE suggest multiple means by which the virus might influence beta-amyloid clearance as shown in Fig 5. The effects of clusterin, which inhibits the formation of the MAC attack complex by binding to several of its components are also shown. These compartments may well  be linked as Megalin (LRP2) antibodies induce a marked activation of the MAC complex in kidney subepithelial cells, suggesting that impaired clusterin import via LRP2  reduces its inhibitory effects on MAC complex formation  (Ronco and Debiec ,2007).

 

Thus, many components the APP physiological and toxic network are also involved in the herpes simplex life-cycle and the virus binds to several key components involved in full length APP processing, APP signalling and beta-amyloid processing. These networks are also related to GSK3B expression and tau phosphorylation, and these relationships perhaps explain why HSV-1 infection leads to increased beta-amyloid deposition and tau phosphorylation. Similarly these APP processing networks are likely to impinge upon viral function.

 

 

Mitochondrial gene deletion  (Fig 1, Fig 2)

 

Mitochondrial gene deletion is a feature in the Alzheimer’s disease brain although this can also be observed in normal subjects (Corral-Debrinski, Horton et al. 1994). However, increased levels of mitochondrial DNA deletion, relative to controls, have been observed in the cerebral vasculature in Alzheimer’s disease (Aliyev, Chen et al. 2005) . Mitochondrial gene deletion can be produced by the herpes viral protein UL12.5, which enters mitochondria, deleting mitochondrial DNA (Saffran, Pare et al. 2007). As mentioned above, mitochondria cluster around the virus during infection and a large number of amyloid plaque related proteins are of mitochondrial origin.

 

Major susceptibility genes and herpes simplex

 

APOE, clusterin, complement receptor 1 and phosphatidylinositol binding clathrin assembly protein, PICALM are the major genetic risk factors in Alzheimer’s disease (see http://www.polygenicpathways.co.uk/alzpolys.html for references).  Each of these is related to herpes simplex. The virus binds to both APOE and complement receptor 1. It also binds to the mannose- 6-phosphate receptor M6PR, a clusterin receptor whose endosomal routing is controlled by PICALM. The herpes simplex virus also uses Crm1 (exportin 1, XPO1) –dependent pathways for nuclear egress, of both viral RNAs and the HSV-1 protein UL47 (Williams, Verhagen et al. 2008). PICALM and other endocytic-regulatory proteins bind to Crm1 and it has been suggested that these might control the nuclear localisation of transcription factors (Vecchi, Polo et al. 2001).

Thus, all of the major Alzheimer’s disease genetic risk factors can be implicated in the viral life-cycle. 28 lesser susceptibility genes are also directly related to the herpes simplex life cycle. 32 minor susceptibility genes are related to immune defence, and 35 susceptibility genes are related to cholesterol and lipoprotein function, an important factor, as herpes simplex viral entry in Vero cells is cholesterol and lipid raft dependent, blocked by the cholesterol synthesis inhibitor , nystatin, and restored by the addition of cholesterol (Bender, Whitbeck et al. 2003) (Table 2).

It is plausible that polymorphisms in CR1, clusterin and PICALM, and many other genes related to herpes simplex, might also influence viral virulence. As HSV-1 seropositivity has been reported in 66% of the American population over the age of 12  (Schillinger, Xu et al. 2004) and in 90% of the population over 70 (Smith and Robinson ,2002) , and viral DNA has also been detected in the brains of patients without Alzheimer’s disease (Jamieson, Maitland, Wilcock, Yates, and Itzhaki ,1992) , clearly  any ability to contribute to Alzheimer’s disease has to be conditioned – perhaps by various Alzheimer’s disease susceptibility genes, or other environmental or infection-related  risk factors. In this context, Helicobacter pylori infection is a major cause of gastritis and also linked to duodenal and gastric ulcers. Its effects on gastritis are both genetically and environmentally conditioned. H.Pylori afflicts a substantial proportion of the world population (~50%), not all of whom develop gastrointestinal problems (Brown ,2000) .

ADNP activity-dependent neuroprotector homeobox .  

ADNP is a neuroprotective peptide active at femtomolar concentrations against a variety of toxic insults including glutamate receptor overactivation and beta-amyloid . ADNP stabilises microtubules, and peptide derivatives of ADNP are in clinical trials and have already shown promise in mild cognitive impairment (Gozes, Stewart et al. 2009). The HSV-1 protein ICP8 binds to ADNP, and ADNP sequestration may be an important hub of the viral toxic effects. Blockade of this interaction might be considered as a therapeutic option.

 

HSV-1 Proteins in the Alzheimer’s disease brain.  

 

Detection of HSV-1 proteins in Alzheimer’s disease has seldom been studied .The limited data are recorded here. HSV-1 immunoreactivity was detected in one Alzheimer’s disease subject, and also in one control in a UK study. In Alzheimer’s disease, labelling was predominantly observed in macrophages in the temporal cortex, hippocampus and cerebellum (Mann, Tinkler et al. 1983) .  In a Japanese study, HSV-1 immunoreactivity was observed in the cortical neuronal cytoplasm of three Alzheimer’s disease patients, with prior identification of HSV-1 glycoprotein D RNA in these patients  (Mori, Kimura et al. 2004). In a CSF study anti HSV-1 IgG antibodies were found in 14/27 Alzheimer’s disease patients but also in 9/13 controls (Wozniak, Shipley et al. 2005) . There has however been no systematic study, for example using panels of antibodies against specific viral proteins, to detect HSV-1 constituents in brain tissue in Alzheimer’s disease.Serum antibodies to HSV-1 (IgM) in elderly patients have however been shown to predict the subsequent development of Alzheimer’s disease(Letenneur, Peres, Fleury, Garrigue, Barberger-Gateau, Helmer, Orgogozo, Gauthier, and Dartigues ,2008).  

During its sojourn in the brain, the virus exists predominantly in a latent state, where few viral proteins are expressed. Indeed, during this state the virus may exert protective effects via inhibition of apoptosis and the promotion of neurite growth (Li, Carpenter, Hsiang, Wechsler, and Jones ,2010) . However, reactivation from time to time may occur, that could reignite the host/pathogen battle. Factors able to reactivate the virus from this dormant state include cytokines and growth factors  and 17-beta estradiol (Kriesel ,1999;Laycock, Brady et al. 1994) (Vicetti Miguel, Sheridan et al. 2010) . It has also been suggested that immune activation by the virus, while driving it to a latent state and inhibiting replication, produces collateral damage via the activation of inflammatory, and toxic, mediators and the production of toxic free radicals (Conrady, Drevets, and Carr ,2010) .   The establishment of latency and the expression of viral immediate early genes is influenced by APOE4 (Miller and Federoff ,2008).

One could also argue that the complement and immune systems have successfully eliminated the antigenic viral constituents, leaving behind the non-antigenic cellular proteins with which it was associated.

Any such successful elimination or suppression of the virus might have been at the cost of destruction of the cells containing the virus, a scenario that may be supported by the presence of numerous immune related proteins in plaques and tangles and the demonstration of the membrane attack complement complex in Alzheimer’s diseases dystrophic neurites and in the neuronal cytoplasm (McGeer, Akiyama, Itagaki, and McGeer ,1989;Itagaki, Akiyama, Saito, and McGeer ,1994).

 

Discussion. 

There is evidence that HSV-1 infection is a risk factor in Alzheimer’s disease and infection has been reported to induce beta-amyloid deposition and tau phosphorylation in animal models (Wozniak, Itzhaki, Shipley, and Dobson ,2007;Wozniak, Frost, and Itzhaki ,2009 Refs) . Herpes simplex infection in mice also causes cortical and hippocampal neuronal loss, cerebral shrinkage and memory deficits, as observed  in Alzheimer’s disease (Armien et al,2009) .  HSV-1 DNA is also found in amyloid containing plaques in Alzheimer’s disease (Wozniak, Mee, and Itzhaki ,2009) , although evidence for viral proteins is clearly lacking. However, this may reflect the possibility that the periods of viral reactivation may be brief, and that destruction of the virus, and its proteins, has been successful. The survey of HSV-1 interacting proteins in relation to their presence in amyloid-containing plaques and tangles in Alzheimer’s disease showed a highly significant enrichment of the known HSV-1 binding proteins in these structures. 40% of 338 known viral associated proteins or chemicals are present in amyloid plaques or neurofibrillary tangles.

These plaque and tangle related proteins are general cellular constituents and the viral relationships could simply reflect the use of many cellular compartments by the virus. However, the statistical analysis suggests a highly significant enrichment of HSV-1 binding proteins in plaques and tangles, and of plaque and tangle related proteins in the HSV-1 dataset. In addition, this analysis was based on exact protein matches between the various datasets. There are, for example, multiple actins, ADP ribosylation factors, annexins, heterogeneous ribonucleoproteins, integrins, RAB’s, heat shock or 14-3-3 proteins, but those found in plaques or tangles are precisely the ones that bind to the virus. Furthermore, other factors such as mitochondrial gene deletion, as well as mitochondrial clustering around the virus and the stimulation of glucose consumption by the virus, reflected by the presence of many glycolytic enzymes and mitochondrial elements in plaques, add weight to a viral implication in the disease process. The HSV-1 binding proteins in plaques and tangles are specifically related to APP processing and tau phosphorylation respectively (Figs 4 and 5) The viral associated proteins are clearly able to affect APP processing and signalling and well as beta-amyloid processing and tau phosphorylation (Fig 3). This is indeed the case in neuroblastoma cells and in the brains of infected mice. The ordering of the human binding proteins with respect to the viral layers (envelope, virion, capsid, tegument, DNA) also suggests physiological relevance, perhaps reflecting destruction of the outer structural viral layers.  Finally, the viral associated proteins in plaques or tangles etch out a very clearly defined pathway, relating to all steps of the viral life cycle, an effect difficult to relate to chance.

The proteins present in these structures trace out the entire life cycle of the virus ranging from viral carriage in extracellular compartments, through receptors, endocytosis, intracellular vesicular traffic, mitochondrial gene deletion, nuclear transport, nuclear DNA synthesis, protein translation and quality control, the bypass of autophagy, apoptosis and protein destruction, inhibition of the complement and other defence systems, retrograde and anterograde transport and finally to exocytosis.  Most viral receptors and carriers, as well as the majority of proteins involved in HSV-1 transport, and most of the host proteins incorporated as HSV-1 virion components are also present in amyloid containing plaques. Viral associated proteins are also components of neurofibrillary tangles, although the class of proteins involved are more restricted to carriers, receptors, anterograde and retrograde transport and kinases/phosphatases involved in tau phosphorylation.

Throughout these networks there are abundant relationships between the virus and APP or tau processing, the core pathological deficits in Alzheimer’s disease. The virus and APP use the same kinesin motors and microtubule network for transport to the cell surface, beta-amyloid facilitates the entry of HSV-1 and other enveloped viruses, and hijack of the ubiquitin proteasome network by the virus is likely to interfere with beta-amyloid processing. The virus also binds to components of the intracellular APP signalling network and to many of the components that are crucial for beta-amyloid clearance.

The use of the microtubule network as a railway track for HSV-1 transport suggests a relationship with microtubule dynamics that is supported by the ability of viral infection to promote tau phosphorylation (Wozniak, Frost, and Itzhaki ,2009;Zambrano, Solis et al. 2008) . Many of the viral associated proteins present in tangles are related to tau phosphorylation.

The implication of many Alzheimer’s disease susceptibility genes in the viral life cycle, or in immunity, suggest that viral involvement might be genetically conditioned.  

The concentration of viral- and immune related proteins in plaques and tangles, and the presence of the complement membrane attack complex in neurones suggests that plaques and tangles represent cemeteries for a battle between the virus and the host’s immune defence mechanisms, which may well have been won, but at the terrible cost of  extensive complement-mediated neuronal loss. 

.          

In summary, the high proportion of viral associated proteins in amyloid containing plaques and/or NFT’s supports accumulating evidence for the involvement of HSV-1 in the pathology of Alzheimer’s disease. Therapies directed towards the immune network or the complement system might therefore be of benefit in this condition. Blockade of the interaction between the neuroprotective peptide ADNP, and the viral protein ICP8, may also be envisaged. In addition, as the virus is essentially cytoprotective in its latent state, blocking apoptosis and promoting neurite extension in neuroblastoma cells (Li, Carpenter, Hsiang, Wechsler, and Jones ,2010), factors that drive the virus to latency, or prevent its reactivation may well be beneficial. As already suggested (Wozniak, Mee, and Itzhaki ,2009;Wozniak, Frost, and Itzhaki ,2009), vaccination against HSV-1 or antiviral therapies in the early stages of Alzheimer’s disease could be considered as viable therapeutic options in Alzheimer’s disease.

 

Acknowledgements: I would like to thank Oliver Chao and Nasire Mahmudi for help in obtaining reprints, the authors who provided information for the HSV-1 database and Bob Blizard for statistical advice.

Table 1: Statistical analysis of the HSV-1 binding proteins present in plaques or tangles (A) and of plaque and tangle components found in the HSV-1 binding protein dataset (B). Chi squared and p values are provided.

A)Enrichment of HSV-1 binding proteins, (VBP’s), in the plaque or tangle proteomics datasets. Expected values are based on a total of 27478 contigs in the human genome http://www.ensembl.org/Homo_sapiens/Info/Index. The plaque proteome also contained tubulins (N=2), H1-H4 histones (N=4) and the chondroitin sulphate proteoglycans, appican (APP), neurocan and Versican which are potential viral associated proteins, while dynamins, dyneins and kinesin (N=4) in these structures are involved in viral transport. Tubulins (N=4), H1 or H2 histones (N=11) and the chondroitin sulphate proteoglycan, Versican, were also components of the tangle proteome and are potential viral binding partners and/or involved in viral transport. Calculations with or without these proteins are included.

 

Enrichment of HSV- binding proteins in plaques or tangles	Number of VBPs	Number of proteins in plaque/tangle  dataset	Expected percentage of VBP’s in any dataset (338/27478)%	Observed percentage of VBP’s	Expected number of HSV-1 interacting proteins in dataset	Observed number, of HSV-1 proteins in  dataset	Fold enrichment (By N)	Chi squared DF=1	P value
Plaques	338	488	1.23	13.1	6	64	10.7	567	2.5229E-125
Tangles	338	79	1.23	17.7	1	14	14.4	171	4.47466E-39
Plaques Including potential partners	338	488	1.23	15.8	6	77	12.8	851	4.4072E-187
Tangles Including potential partners	338	79	1.23	38.0	1	30	30.9	851	4.4072E-187

B) Enrichment of plaque or tangle components in the HSV-1 binding protein dataset. Calculations with or without potential binding partners are included.

Enrichment of plaque or tangle proteins in the HSV-1 binding dataset	Number of proteins in plaques or tangles	Number of VBP’s	Expected percentage of plaque or tangle proteins in any other dataset 488/27478% or 79/27478%	Observed percentage	Expected number of plaque /tangle proteins in  HSV-1 dataset	Observed number of plaque/tangle proteins in HSV1 dataset	Fold enrichment (By N)	Chi squared DF=1	P value
Plaques	488	338	1.78	18.9	8.7	64	7.4	345	5.2E-77
Tangles	79	338	0.29	4.1	0.23	14	61.6	169	9.51E-39
Plaques Including potential partners	488	338	1.78	22.8	8.7	77	8.9	527	1.3E-116
Tangles Including potential partners	79	338	0.29	8.9	0.23	30	132.1	843	2.4E-185

 

Calculation rationale: The human genome contains 27478 contigs. 338 viral associated proteins are known; 488 proteins were found in plaques and 79 in tangles in the two proteomics studies. The expected percentage of each class of protein in any other dataset is thus 338, 488 or 79 /27478 %, from which the expected number of each class of proteins in any particular dataset can be calculated. The observed and expected numbers of proteins absent from each dataset were also calculated, providing the 2*2 table for calculating Chi squared and the p values.

 

E.g.

HSV-1 binding proteins in plaques	Observed	Expected	Chi2	Value
In plaques	64	6
Not in plaques	424	482	567	2.5229E-125

 

Table 2

 

HSV-1 binding proteins (VBP) classified by function and their representation in amyloid plaques and neurofibrillary tangles. Alzheimer’s disease susceptibility genes directly and indirectly related to herpes simplex are also shown. 

 

Carriers are defined as soluble ligands occupying extracellular compartments (e.g. A2M, APOA1, APOE, and Factor XII). *HSV-1 binds to chondroitin sulphate or heparan sulphate proteoglycans, which are present in amyloid plaques, but which have not been individually tested for HSV-1 binding. Viral associated proteins are referenced in an online database at http://www.polygenicpathways.co.uk/herpeshost.html. Genes implicated in Alzheimer’s disease are referenced at http://www.polygenicpathways.co.uk/alzpolys.html

Proteins are identified by Gene symbols (See Tables 3 to 8 for names and references). Proteins in bold are specific to plaques or tangles.

 

Viral Class of protein	Percent in plaques	VBP’s in plaques	Percent in tangles	VBP’s in tangles	% in both
Carrier (soluble ligands)	100% (4/4)	A2M, APOA1, APOE, FGF2	50% (2/4)	APOE FGF2	100% (4/4)
Receptors	68% (15/22) 77% ( 23/30 with CSPG’s and HSPG’s	Heparan sulphate Chondroitin sulphate ANK2, FGFR1, IDE, IGF2R, ITGA5, ITGB2 ITGB3, ITGAM, M6PR, MAG, SDC1, SDC2: Also LTF which blocks viral entry.	27% (6/22) 47% (14/30) with CSPG’s and HSPG’s	Heparan sulphate Chondroitin sulphate FGFR1, NCL, SDC1, SDC2: and LTF	64% (21/33)   70% (28/40) with CSPG’s and HSPG’s
* Chondroitin sulphate proteoglycans	-	APP (Appican) Neurocan, Versican	-	Versican
* Heparan sulphate proteoglycans	-	Aggrecan, Agrin, Glypican 1, Perlecan,  Syndecans SDC3, SDC4	-	Agrin, Glypican , Perlecan Syndecans SDC3, SDC4
Free radical	67% (2/3)	TXN, PRDX1	67% (2/3)	PRDX1, PRDX2	100%
Metabolic	71% (5/7)	ATP5J, GAPDH, NME1, TPI1, SLC25A5	29% (2/7)	GAPDH SLC25A5	71% (5/7)
Endocytosis	82% (9/11)	ALIX, ARF1, ARF3, ARF4, ARF5, IGF2R, M6PR, RAB10 RAB5A	18% (2/11)	GRB2 IGF2R	91% (10/11)
Intervesicular	88% (7/8)	IGF2R, M6PR, RAB2A, RAB2B, RAB33B, RAB4B RAB6A	13% (1/8)	IGF2R	88% (7/8)
Retrograde to nucleus	31% (5/16)	DCTN1, DYNC1L1,  DYNLL1, YWHAG YWHAZ	63% (10/16)	CDK1 CSNK2B SET tau TUBA4A TUBB2A TUBB3 TUBB3 TUBB4 TUBB4Q YWHAZ	94% 15/16
Nuclear transport	40% (6/15)	ACTB, ACTG1, CFL1, CSRP1, LMNA, PFN1	40% (6/15)	ACTB, ACTG1, CFL1, KPNA2, NCL, NUP358	60% (9/15)
Actin related	86% (6/7)	ACTB, ACTG1, CDC42, CFL1, CSRP1, PFN1	43% (3/7)	ACTB ACTG1 CFL1	6/7
Anterograde to plasma membrane	25% (4/16)	APP, YWHAE , YWHAG, YWHAZ	69% (8/16)	CDK1 CSNK2B SET tau TUBA4A TUBB2A TUBB3 TUBB3 TUBB4 TUBB4Q YWHAZ	85% (14/16)
Exocytosis	78% (7/9)	ANXA1, ANXA2, ANXA5, GAP43, RAB35, RAB3A, SNAP25	11% (1/9)	ANXA5	78% (7/9)
APP processing	72% (13/18)	APOA1, APOE, APP, C3, CASP3, DCTN1, DYNC1L1, F12, F8, LMNA, RAB3A, RAB5A, SNAP25	17% (3/18)	APOE, C3, CASP3	72% (13/18)
Tau phosphorylation	29% (3/7)	PRKACA YWHAZ tau	86% (6/7)	CDC2, CSNK2B PRKACA SET, tau, YWHAZ	86% (6/7 )
Coagulation factors	50% (4/8)	F3, F8, F12 Kallikrein	0	-	50% (4/8)
Globins	75% (3/4)	HBA1, HBB, HBE1	0		75% 3 /4)
Autophagy	75% (6/8)	BECN1 DCTN1 RAB10 RAB33B RAB35 RAB5A	38% (3/8)	BECN1 CHMP2B EIF2AK3	100% (8/8)
Heat shock proteins and protein stress	63% (5/8)	CANX, HSPA1A ,HSPA8, HSAP1L HSP90AA1	50% (4/8)	HSP40, HSPA1A ,HSPA8 EIF2AK3 (pretangles)	86% (6/7)
Mitochondrial	75% (3 / 4)	DNA deletion SLC25A5 Impaired respiration	50% (2/4)	DNA deletion SLC25A5	50%
Immune and defence-related	39%(14/36)	BECN1, C3, EIF2AK2, HLA-DRB1, HLA-DRB3, HLA-DRB4, IFNGR1,  IgG , IL6, MIF, PRKRA, PPIA, PSMA2, XRCC6	28% (10/36)	BECN1, CD59, EIF2AK2 EIF2AK3, HLA-DRB1, HLA-DRB3, HLA-DRB4, IFNGR1, IL6, PPIA	44%
Apoptosis	50% (6/12)	BAX, CYTC1, CASP3, CST3, SMAD3 TGFB1,	33% (4/12)	BAX, CAPS3, CST3,  SMAD3	40%
Translation	18% (2/11)	EEF1A1 EIF4A2	27% (3/11)	EEF1A1 EIF2S1, EIF4E	30%
Ubiquitin/Proteasome	17% (2/12)	PSMA2, UBC	8% (1/12)	UBC	17%
Nuclear	14% (12/86)	APEX1 Histone H1 Histone H2A Histone H2B Histone H4 HMGA1 HMGB1 HNRNPK JUND LMNA NFKB1 TP53	17% (15/86)	BRCA1 Histone H1 Histone H2A Histone H2B Histone H4 HNRNPK JUND KPNA2 NCL NUP358 PCNA SET SP1 SP3 TP53	26% (/86)
Replication repair	16% (3/19)	APEX1, PARP1, XRCC6	11% (2/19)	BRCA1 MCM2	26% (5/19)
Cell cycle	13% (1/8)	CDC42	13 % (1/8)	CDC2	25% (2/8)
Signalling	33% (3/9)	JNK, P38, PRKACA	63% (6/9)	CSNK2B, GRB2 SET, JNK, P38 PRKACA
Chromatin remodelling	0 (0/19)	-	5% (1/19)	CHMP2B	5%
RNA splicing	0 (0/5)	-	0 (0/5)		0%
Classes of viral proteins
Surface proteins	79% (26/33)	All Carriers and receptors (see above)	48% (16/33)	Carriers and receptors (see above)	82% (27/33)
Virion components	63% (32/51)	ACTB, ACTG1, APP, ARF1 ,ARF3,ARF4, ARF5 ,CFL1, CSRP1, HSPA1L,  PFN1, RAB2A, RAB2B, RAB4B, RAB5A, RAB6A, RAB7A, RAB10, RAB33B, RAB35,ANXA1,ANXA2, ANXA5, GAPDH, NME1,TPI1, PRDX1, UBC, PPIA, YWHAE, YWHAG, YWHAZ	25% (13/51)	ACTB, ACTG1, ANXA5, CD59, CFL1, CSNK2B GAPDH, GRB2, PPIA, PRDX1, PRDX2,  UBC, YWHAZ	71% 36/51
Capsid binding proteins	26% (5/19)	ANK2 DYNLL1,EEF1A1,  RAB3A, SNAP25	11% (2/19)	EEF1A1 NCL	25% (3/19)
Tegument binding proteins	25% (9/35)	BAD, CASP3, DYNLL1, GAP43, IFNGR1, PRKCA, RAB3A SLC25A5, SNAP25	21% (8/35)	BAD CASP3 CDK1 IFNGR1 NUP358, PRKCA SET SLC25A5	33% (12/35)
Transcription factors and genome binding	23% (10/43)	Histone H1 Histone H2A Histone H2B Histone H4 HMGA1, HSPA1A, IL6, JUND, NFKB1 XRCC6	16% (7/43)	Histone H1 Histone H2A Histone H2B Histone H4 JUND SP1 SP3	14%
Proteins binding to ICP8 (single stranded DNA binding protein)	3% (1/31)	PARP1	6% (2/31)	BRCA1 MCM2
Alzheimer’s disease susceptibility genes directly related to the viral life cycle.
Major genes: APOE, CR1, CLU, PICALM Minor genes: A2M, ACAN, APOA1, APP, BIN1, CST3, DNM2, EIF2AK2, gamma secretase (APH1B, NCSTN, PSEN1, PSEN2, PSENEN), F13A1, GAPDH, GSK3B, HLA-DRB1, HSPG2, IDE, LCK, LMNA, MAPT, MIF, NCL, PARP1, POU2F1, PVRL2, TAP2, TFCP2, TGFB1, TP53, XRCC1,  YWHAZ
Immune related susceptibility genes: RAGE receptor AGER; Complement components C4A, C4B, CR1; Chemokines CCL2, CCL3, CCR2; CD molecules CD14, CD33, CD36, CD86; C-reactive protein  CRP; Defensin DEFB122;  Immunoglobulin receptor FCER1G; HLA-antigens HLA-A2, HLA-DRB1, MICA; Interleukins  IL10, IL18, IL1A, IL1B, IL1RN, IL33, IL6, IL8; Lymphocyte tyrosine kinase LCK Cyclo-oxygenase PTGS2, Cytokines CSF1, TGFB1, TNF Toll receptor TLR4
Cholesterol and lipoprotein related susceptibility genes
Cholesterol: Transporters ABCA1, ABCA2, ABCC2, ABCG1, CETP, Cholesterol metabolism CH25H, CYP46A1, DHCR24, HMGCR, HMGCS2, SOAT1, Transcription factor SREBF1 Lipoprotein: A2M, APOA1, APOA4, APOA5, APOC1, APOC2, APOC3, APOC4, APOD, APOE, LPA Lipoprotein receptors LDLR, LRP1, LRP2, LRP6, LRP8, LRPAP1, OLR1, VLDLR; Lipases LIPA, LIPC,  LPL Sortilin SORL1
Functional classification of the amyloid plaque proteome according to Liao et al, VBP’s have been added. VBP+mitochondria (113); Metabolism (72); VBPs (64); Cytoskeleton (51); membrane trafficking (50); mitochondria (49); others (45) G-protein pathways (36) kinases/phosphatases and regulators (33); proteolysis (32); Cell adhesion (26); inflammation (24); nuclear activities (22); chaperones (16); channels/receptors (16) oxidative stress (10); Cell death (7)

 

 

 

Table 3

 

Primary viral receptors and carriers found in amyloid plaques or NFT’s. *HSV-1 binds to chondroitin sulphate or heparan sulphate proteoglycans, which are present in amyloid plaques, but which, apart from SDC1 and SDC2, have not been individually tested for HSV-1 binding. Viral associated proteins in this and all other tables are referenced in the online database at http://www.polygenicpathways.co.uk/herpeshost.html

 

 

Chemical receptors	Localisation in plaques and/or tangles
Heparan sulphate	Associated with amyloid plaques (Bruinsma, te et al. 2010)and NFT’s (Perry, Siedlak et al. 1991)
Chondroitin 4,6 sulphate	Antibodies to Chondroitin -4 or -6 sulphate label both plaques and NFT’s in AD (DeWitt, Silver et al. 1993)
Heparan sulphate proteoglycans
Syndecans SDC1, SDC2	SDC1,2,3 and 4 are found in amyloid plaques  and NFT’s(Verbeek, Otte-Holler et al. 1999)
Other receptors
ANK2 Ankyrin 2	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
F3 coagulation factor III (thromboplastin, tissue factor)	Immunoreactivity in plaques (McComb, Miller et al. 1991)
F8 Coagulation factor VIII	Occasionally observed in neutrophils surrounding vascular and brain amyloid plaques (Savage, Iqbal et al. 1994)
F12 coagulation factor XII (Hageman factor)	Immunoreactivity observed in senile plaques (Yasuhara, Walker et al. 1994)
FGFR1 fibroblast growth factor receptor 1	Increased immunoreactivity in reactive astrocytes surrounding senile plaques (Takami, Matsuo et al. 1998) and in tangles (Ferrer and Marti ,1998)
IGF2R insulin-like growth factor 2 receptor	Localised to Abeta-containing neuritic plaques and in NFT’s (Kar, Poirier et al. 2006)
M6PR mannose-6-phosphate receptor (cation dependent)	Increased expression in pyramidal neurones (Cataldo, Barnett et al. 1997)
IDE Insulin degrading enzyme	Present in amyloid plaques  (Bernstein, Ansorge et al. 1999)
MAG Myelin-associated glycoprotein (Suenaga, Satoh et al. 2010)	Found in amyloid plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
MAC-1 ITGB2 Integrin beta 2 ITGAM	Strongly expressed in microglia in amyloid plaques (ITGB2) (Eikelenboom, Zhan et al. 1994)
Vitronectin receptor integrin alpha 5./beta 3 (ITGA5*/ITGB3*)	Present in the microglial cores of amyloid plaques (Akiyama, Kawamata et al. 1991)
Viral transporters (ie soluble ligands that bind to receptors
A2M Alpha-2-macroglobulin	Associated with amyloid plaques  (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
APOA1 Apolipoprotein A1	Found in occasional senile plaques (Harr, Uint et al. 1996)
APOE Apolipoprotein E	Found in plaques, tangles and blood vessels (Namba, Tomonaga et al. 1991)
FGF2 fibroblast growth factor 2 (basic)	Present in senile  plaques and tangles (Schindowski, Belarbi et al. 2008)
LTF Lactotransferrin	Senile plaques and tangles (Leveugle, Spik et al. 1994)
Other Heparan sulphate proteoglycans*
Aggrecan ACAN	Present in plaques (Bignami, LeBlanc et al. 1994)
Agrin AGN	Plaques, tangles and blood vessels (Verbeek, Otte-Holler, van den, van den Heuvel, David, Wesseling, and de Waal ,1999))
Decorin DCN	Localised at the edges of amyloid plaques and in NFT’s (Snow, Mar et al. 1992)
Glypican 1 GPC1	Plaques and tangles(Verbeek, Otte-Holler, van den, van den Heuvel, David, Wesseling, and de Waal ,1999),
Perlecan HSPG2	Plaques tangles and blood vessels (Verbeek, Otte-Holler, van den, van den Heuvel, David, Wesseling, and de Waal ,1999)
SDC3	SDC1,2,3 and 4 are found in amyloid plaques  and NFT’s(Verbeek, Otte-Holler, van den, van den Heuvel, David, Wesseling, and de Waal ,1999)
SDC4
Other Chondroitin sulphate proteoglycans*
APP Appican	Beta-amyloid can be generated from appican (Shioi, Pangalos et al. 1996)
VCAN Versican	Found in amyloid plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
NCAN Neurocan	Found in amyloid plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Not found or not reported: CD209, PILRA, PVRL1, PVRL2, TNFRSF14

 

 

Table 4 HSV-1 binding proteins involved in viral transport and their presence in amyloid plaques or tangles.

 

 

Viral binding protein	Localisation in plaques and/or tangles
Actins ACTB1, ACTG1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
ADP-ribosylation factors ARF1 ARF3 ARF4 ARF5 ARF6	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Annexins: ANXA1 ANXA2 ANXA3
ANXA5	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004). Tangle component isolated by laser dissection(Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
ALIX =PDCD6IP : programmed cell death 6 interacting protein	A component of amyloid plaques (Rajendran, Honsho et al. 2006)
APP Amyloid precursor protein	Beta-amyloid  is the major plaque component(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
CANX Calnexin	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
CDC42 cell division cycle 42 (GTP binding protein, 25kDa)	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
CFL1 Cofilin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and tangles(Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
CHMP2B chromatin modifying protein 2B	Present in granulovacuolar degeneration (Pretangles) (Yamazaki, Takahashi et al. 2010)
CSRP1 cysteine and glycine-rich protein 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
DCTN1 Dynactin 1 (p150glued)	Observed in areas of granulovacuolar degeneration (Ateh, Hussain et al. 2008). These may be a form of autophagosome and contain phosphorylated tau suggesting they may precede tangle formation (Okamoto, Hirai et al. 1991)
DYNC1I1  dynein, cytoplasmic 1, intermediate chain 1	Component of amyloid plaques isolated by laser dissection: Plaques also contain DYNC1H1, DYNLL2  (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
DYNLL1	Component of amyloid plaques isolated by laser dissection: Plaques also contain DYNC1H1, DYNLL2  (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
GAP43 growth associated protein 43	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
PFN1 Profilin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
RAB proteins members of RAS oncogene family RAB/ RAB2A / RAB2B / RAB3A/ RAB5A /RAB6A / RAB7A / RAB10 /  RAB33B / RAB35 /	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
RAB4B	Increased RAB4 expression in vesicles of pyramidal neurones, pre-plaque (Cataldo, Peterhoff et al. 2000)
MAPT Microtubule associated protein tau	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) Major component of tangles.
Tubulins TUBA4A, TUBB2A, TUBB3, TUBB4 and TUBB4Q	Components of laser-dissected tangles(Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
SNAP25 synaptosomal-associated protein, 25kDa	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Nuclear transport
LMNA Lamin A	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
NCL Nucleolin	A CDC2-phosphorylated form is found in NFT’s (Dranovsky, Vincent et al. 2001)
Dynamins, dynactins, dyneins and kinesins: Do not specifically bind to the virus but are involved in its transport along microtubules (Lyman and Enquist ,2009)	Dynamin 1 (plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and tangles (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005) )DNM1L plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004).Dynamitin (DCTN2) plaques (Ateh, Hussain, Mustafa, Price, Gulati, Nickols, Bird, Greensmith, Hafezparast, Fisher, Baker, and Martin ,2008) Dynactin DCTN4, Tangles (Ateh, Hussain, Mustafa, Price, Gulati, Nickols, Bird, Greensmith, Hafezparast, Fisher, Baker, and Martin ,2008)Other Dyneins DYNC1H1, DYNC1I1, DYNLL2, DYNLL1 Kinesin : KIF5B (Plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)  )

 

 

 

Table 5 Host proteins associated with the HSV-1 virion and their localisation in amyloid plaques or neurofibrillary tangles.

 

Viral associated proteins	Localisation in plaques and/or tangles
Transport related
Actins ACTB, ACTG1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
Annexins ANXA1 ANXA2	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
ANXA5	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
ADP Ribosylation factors ARF1 ARF3 ARF4 ARF5	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
CFL1 Cofilin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
CSRP1 cysteine and glycine-rich protein 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
PFN1 Profilin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
RAB Proteins RAB2A RAB2B RAB5A RAB6A RAB7A RAB10 RAB33B RAB35	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Heat shock
HSPA1L	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Metabolic
GAPDH Glyceraldehyde 3-phosphate dehydrogenase NME1 non-metastatic cells 1, protein (NM23A) TPI1 Triosephosphate isomerase 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Immune related
CD59 CD59 antigen	Present in tangled neurones and dystrophic neurites (McGeer, Walker, Akiyama, Kawamata, Guan, Parker, Okada, and McGeer ,1991)
MIF Macrophage inhibitory factor	A component of  amyloid plaques (Bacher, Deuster et al. 2010)
PPIA Cyclophilin A	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
14-3-3 proteins
YWHAE 14-3-3 epsilon YWHAG 14-3-3 gamma	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
YWHAZ 14-3-3 zeta	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) amd of NFT’s (Umahara, Uchihara et al. 2004)
Free radical
PRDX1 Peroxiredoxin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Ubiquitin
UBC Ubiquitin C	Ubiquitin is observed in both plaques and tangles although specific isoforms were not examined (He, Delaere et al. 1993)UBC is found in neurofibrillary tangles (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)

 

Table 6

HSV-1 binding Histones and Heat shock proteins and their presence in amyloid plaques or neurofibrillary tangles

 

Viral binding Protein	Localisation in plaques and/or tangles
Histone H1	H1,  H2 H3  histones (HIST1H1E, HIST2H2AB, HIST3H2BB) are  Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004). H1 and H2 histones (HIST1H2AA , HIST1H2AB, HIST1H2AC, HIST1H2AD HIST1H2AI, HIST1H2AJ, H2AFJ, H2AFX, HIST2H2AA4, HIST2H2AC, HIST3H2A)are associated with NFT’s isolated by laser dissection (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
Histone H2A
Histone H2B
Histone H4	H4 histone HIST4H4 is a Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
HSP40	High expression in homogenates correlates with fibrillar tau levels (Sahara, Maeda et al. 2007)
HSP90AA1 heat shock protein 90kDa alpha (cytosolic), class A member 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
HSPA1A heat shock 70kDa protein 1A (HSP72)	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Hamos, Oblas et al. 1991)
HSPA8 heat shock 70kDa protein 8	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)

 

 

 

Table 7 Viral binding components related to the complement and immune systems and their presence in amyloid plaques or neurofibrillary tangles.

 

 

Viral binding protein	Localisation in plaques and/or tangles
C3 Complement C3 component	Complements C1q, C3b, C3c, C3d and C4 are components of amyloid plaques in Alzheimer’s disease (Eikelenboom and Stam ,1982) and C3 is observed in some tangles (McGeer, Akiyama, Itagaki, and McGeer ,1989)
BECN1 Beclin 1	Found in endothelial cells in the vicinity of amyloid plaques and in dystrophic neurites, correlating with phosphorylated tau (Ma, Huang et al. 2010).
HSV-1 glycoprotein E is an Fc receptor mimic that binds to the Fc domain of Immunoglobulin G (IgG) preventing its binding to complement component C1 (Friedman ,2003)	Immunoglobulin G is located in  the amyloid plaque corona (Eikelenboom and Stam ,1982).
CD59 CD59 molecule	Present in tangled neurones and dystrophic neurites (McGeer, Walker, Akiyama, Kawamata, Guan, Parker, Okada, and McGeer ,1991)
EIF2AK2 eukaryotic translation initiation factor 2-alpha kinase 2 (PKR: double stranded RNA activated protein kinase)	PKR immunoreactivity is associated with neuritic plaques and pyramidal neurons  Its distribution correlates with that of  phosphorylated tau (Peel and Bredesen ,2003)
PRKRA protein kinase, interferon-inducible double stranded RNA dependent activator (activates EIF2AK2)	Present in amyloid plaques (Page, Rioux et al. 2006)
HLADRB1 major histocompatibility complex, class II, DR beta 1 DRB3 DR beta3 DRB4 beta 4	HLA-DR molecules are found in activated microglia around plaques and tangles (Perlmutter, Scott et al. 1992)
IFNGR1 interferon gamma receptor 1	Increased expression in astrocytes in affected areas (Hashioka, Klegeris et al. 2009)
Kallikrein KLK1	Immunoreactivity associated with neuritic plaques (Bernstein ,1997)
MIF Macrophage inhibitory factor	A component of  amyloid plaques (Bacher, Deuster, Aljabari, Egensperger, Neff, Jessen, Popp, Noelker, Reese, Al Abed, and Dodel ,2010)
PPIA cyclophilinA	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
PSMA2  proteasome (prosome, macropain) subunit, alpha type, 2
XRCC6 X-ray repair complementing defective repair 6 (Ku antigen)

 

Table 8 Diverse HSV-1 binding proteins and their presence in amyloid plaques or neurofibrillary tangles.

 

Viral associated proteins	Localisation in plaques and/or tangles
Apoptosis related
BAD BCL2-associated agonist of cell death	Increased expression in brain homogenates (Kitamura, Shimohama et al. 1998)
BAX BCL2-associated X protein	Strongly expressed in plaques and tangles (MacGibbon, Lawlor et al. 1997)
CASP3 Caspase 3	Increased expression in plaques and tangles (Su, Zhao et al. 2001)
CST3 Cystatin C	Localises with beta-amyloid and occasionally in  tangles (Levy, Sastre et al. 2001)
CYTC1 Cytochrome C	CYS (cytochrome c, somatic) and CYC1 cytochrome c-1 present in amyloid plaques  (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
TGFB1 transforming growth factor, beta 1	Present in amyloid plaques (van der Wal, Gomez-Pinilla et al. 1993)
SMAD3 SMAD family member 3	Present in amyloid plaques  and NFT’s (Ueberham, Ueberham et al. 2006)
Metabolic
ATP5J ATP synthase, H+ transporting, mitochondrial F0 complex, subunit F6	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
GAPDH Glyceraldehyde 3-phosphate dehydrogenase.	A component of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005) and of amyloid plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
NME1 NME2 Nucleoside diphosphate kinase A/B	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
SLC25A5 solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 5	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004). Component of  Neurofibrillary tangles isolated by laser dissection (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
TPI Triose phosphate isomerase	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Free radical related
PRDX1 Peroxiredoxin 1	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and tangles (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
PRDX2 Peroxiredoxin 2	Component of Neurofibrillary tangles isolated by laser dissection (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005)
TXN Thioredoxin	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Haemoglobins
HBA1 Haemoglobin alpha HBB Haeomoglobin beta HBE1 Haemoglobin epsilon	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
Translation initiation
EEF1A1 eukaryotic translation elongation factor 1 alpha 1	A component of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005) and of amyloid plaques (Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
EIF4A2 eukaryotic translation initiation factor 4A2	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
EIF4E eukaryotic translation initiation factor 4E	High levels of phosphorylated EIF4E correlate with hyperphosphorylated tau (Li, An et al. 2004).
EIF2S1 eukaryotic translation initiation factor 2, subunit 1 alpha, 35kDa	Over-expressed in cells containing NFT’s (Ferrer ,2002)
Transcription factors and others binding to viral genome
IL6 Interleukin 6	Expressed in early stage amyloid plaques and associated with tangles (Thal, Schober et al. 1997)
JUND jun D proto-oncogene (AP-1)	Overexpressed and activated in brain homogenates (Vukic, Callaghan et al. 2009)
NFKB1 nuclear factor of kappa light polypeptide gene enhancer in B-cells 1	Activated in cells surrounding amyloid plaques (Ueberham, Ueberham, Gruschka, and Arendt ,2006)
SP1 Sp1 transcription factor	Expressed in NFT’s, and dystrophic neurites of senile plaques  (Santpere, Nieto et al. 2006)
SP3 Sp3 transcription factor	Highly expressed in association with NFT’s (Boutillier, Lannes et al. 2007)
STAT1 signal transducer and activator of transcription 1	Increased levels in brain homogenates  (Kitamura, Shimohama et al. 1997)
Nuclear proteins
APEX1 APEX nuclease (multifunctional DNA repair enzyme) 1	A component of senile plaques (Tan, Sun et al. 1998)
BRCA1 breast cancer 1, early onset	Intensely expressed in NFT’s (Evans, Raina et al. 2007)
HMGA1 high mobility group AT-hook 1	Highly expressed in hippocampal pyramidal neurones in damaged areas (Manabe, Katayama et al. 2003)
HMGB1 high-mobility group box 1	Highly expressed in amyloid plaques (Takata, Kitamura et al. 2003)
Heterogeneous ribonucloprotein K HNRNPK	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
LMNA Lamin A
KPNA2 karyopherin alpha 2 (RAG cohort 1, importin alpha 1)	Increased expression in Hirano bodies (contain actins and related cytoskeletal elements) (Lee, Ueda et al. 2006)
MCM2 minichromosome maintenance complex component 2	A phosphorylated form is associated with neurofibrillary tangles and dystrophic neurites (Bonda, Evans et al. 2009)
NCL Nucleolin	A CDC2-phosphorylated form is found in NFT’s (Dranovsky, Vincent, Gregori, Schwarzman, Colflesh, Enghild, Strittmatter, Davies, and Goldgaber ,2001)
NUP358 Nucleoporin 358	Present in tangle bearing neurones along with karyopherin beta 2 (Sheffield and Mirra ,2008)
PARP1 poly (ADP-ribose) polymerase 1	Overexpressed in healthy pyramidal neurones and also in some amyloid plaques, but not in NFT’s (Love, Barber et al. 1999)
PCNA Proliferating cell nuclear antigen	Highly expressed in tangle bearing neurones (Busser, Geldmacher et al. 1998)
SET SET nuclear oncogene: phosphatase 2A inhibitor I2PP2A	Increased hippocampal expression related to NFT’s and tau phosphorylation (Tanimukai, Grundke-Iqbal et al. 2005)  which is regulated by SET (Chohan, Khatoon, Iqbal, and Iqbal ,2006)
TP53 Tumor suppressor p53	Found in tau positive neurites and in dystrophic processes around amyloid plaques(de la Monte, Sohn et al. 1997)
14-3-3 proteins
YWHAE tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
YWHAG  tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, gamma polypeptide	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
YWHAZ tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and of NFT’s (Umahara, Uchihara, Tsuchiya, Nakamura, Iwamoto, Ikeda, and Takasaki ,2004)
Diverse
EIF2AK3 eukaryotic translation initiation factor 2-alpha kinase 3 (PERK)	Not seen in plaques or tangle but increased expression in pretangle neurones with high levels of  phosphorylated tau  (Hoozemans, van Haastert et al. 2009)
CDC2 (=CDK1) cyclin-dependent kinase 1	Phosphorylates the microtubule protein tau and is expressed in neurones with tangle-like inclusions (Pei, Braak et al. 2002)
CSNK2B Casein kinase 2 beta	Phosphorylates tau (Greenwood, Scott et al. 1994)and is present in NFT’s (Baum, Masliah et al. 1992)
CST3 Cystatin 3	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004)
GRB2 growth factor receptor-bound protein 2	Expression correlates with cytoskeletal abnormalities (tangles) (McShea, Zelasko et al. 1999)Binds to Dynamin (Vidal, Goudreau et al. 1999).
JNK kinase	Activated in granular bodies , tangles and occasional plaques (Lagalwar, Guillozet-Bongaarts et al. 2006)
P38 MAP Kinase	Activated in neuritic plaques and tangles (Hensley, Floyd et al. 1999)
PRKACA protein kinase, cAMP-dependent, catalytic, alpha	Component of laser-dissected plaques(Liao, Cheng, Wang, Duong, Losik, Gearing, Rees, Lah, Levey, and Peng ,2004) and associated with tangles : Phosphorylates tau (Jicha, Weaver et al. 1999)
UBC Ubiquitin C	Ubiquitin is observed in both plaques and tangles although specific isoforms were not examined (Perry, Friedman et al. 1987)UBC was identified in a proteomics study of NFT’s (Wang, Woltjer, Cimino, Pan, Montine, Zhang, and Montine ,2005;He, Delaere, Duyckaerts, Wasowicz, Piette, and Hauw ,1993)

 

 

Figure legends

 

Figure 1

 

Herpes simplex binding proteins found in amyloid plaques in Alzheimer’s disease. Proteins are represented by HUGO approved gene symbols and corresponding definitions can be found in Table 3-8. All proteins in this figure bind to HSV-1 except for those marked with* (clusterin, dynamin DNM1,dynactin DCTN2, Dyneins DYNC1H1,  DYNLL2,  Kinesin KLC3, and PICALM) although these are likely to be, or are, implicated in the viral life cycle. All proteins except for those marked by # are plaque components (PICALM). Increased BAD immunoreactivity was observed in brain homogenates. 49 mitochondrial proteins and 18 glycolytic enzymes may reflect mitochondrial clustering around the virus and increased glucose consumption in infected areas (see text). Proteins are organised with respect to their cellular functions. ER = endoplasmic reticulum. βAmy = beta-amyloid. Linked diamonds (CLU/M6PR) represent binding between components. Genes in bold have been implicated as risk factors in Alzheimer’s disease and those underlined (APOE, CLU, PICALM) as major genetic risk factors (see text for details).

 

Figure 2

 

Herpes simplex binding proteins found in neurofibrillary tangles in Alzheimer’s disease. Proteins are represented by HUGO approved gene symbols and corresponding definitions can be found in Table 3-8. All proteins in this figure bind to HSV-1 except for those marked with* (DNM1, DCTN4,  MAPT (=tau) although these are implicated in the viral life cycle. All proteins except for those marked by # are recorded tangle components (PRKCA). Increased BAD immunoreactivity was observed in brain homogenates Proteins are organised with respect to their cellular functions. Genes in bold have been implicated as risk factors in Alzheimer’s disease and those underlined (APOE, CLU) as major genetic risk factors.(see text for details).

 

Figure 3

 

The distribution of viral associated proteins found in amyloid plaques or neurofibrillary tangles, grouped in relation to function. The classes are ranked in relation to plaque containing proteins and the number of proteins in each class is indicated on the X-axis. The types of viral component (glycoprotein, virion, capsid, tegument, Latency transcript (LAT), viral genome or ICP8) interacting with host proteins are also shown. The classification is based on Table 2, Figs 1, 2 and 5 and is detailed in the text and at to  http://www.polygenicpathways.co.uk/herpeshost.html

 

 .

Figure 4

 

The distribution of viral associated proteins found in amyloid plaques or neurofibrillary tangles, grouped in relation to function. The classes are ranked in relation to tangle containing proteins and the number of proteins in each class is indicated on the X-axis. The types of viral component (glycoprotein, virion, capsid, tegument, Latency transcript (LAT), viral genome or ICP8) interacting with host proteins are also shown. The classification is based on Table 2, Figs 1, 2 and 5 and is detailed in the text and at to  http://www.polygenicpathways.co.uk/herpeshost.html

 

 

Figure 5

 

The relationships between herpes simplex and various aspects of APP physiology.

 

HSV-1 entry is modified by beta-amyloid (βAmy) and the APP intracellular protease complex, gamma-secretase, cleaves the viral entry receptors syndecans SDC1 and 2 (top). HSPG and CSPG (heparan and chondroitin sulphate proteoglycans, the latter including appican, an isoform of APP, are putative viral receptors. Thrombin, the coagulation factor activated protease cleaves APP.

Intracellular APP processing (de novo synthesis and transport to the plasma membrane) employs the same pathways as those used by herpes simplex and is dependent upon the viral associated proteins, SNAP25 and RAB3A. APP endocytosis is mediated via dynamin and RAB5A (top left).

Beta-amyloid (βAmy) degradation and clearance (right) is mediated by insulin degrading enzyme (IDE) and by APOE, APOA1, clusterin, and complement C3 and their respective receptors/transporters, as illustrated. Clusterin delivery via its receptor, LRP2, influences its utilisation as a complement membrane attack complex (MAC) inhibitor (bottom right).

APP intracellular signalling (bottom) is mediated by a gamma-secretase cleaved intracellular domain of APP (AICD) that complexes with tip60 and APBB1 or TFCP2 and APBB1, which control the expression of APP, BACE1 and GSK3B, leading to tau phosphorylation (tau-P) as shown. AICD is cleaved by caspase 3. HSV-1 nuclear egress is via crm1 (XPO1) dependent pathways and PICALM binds to crm1. HSV-1 infection degrades PML3 which binds to tip60, which is also connected to the viral binding protein lamin A (bottom left). Linked diamonds represent binding between components and components of the HSV-1 virus that bind to the various proteins are represented by. Genes implicated as Alzheimer’s disease risk factors are in black boxes with white lettering and major genes (APOE, CLU, CR1 and PICALM) are underlined.

 

 

 

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