Toll-like Receptors (TLRs) and Prion Disease: Relevance to ...pathogenesis may largely rely on our increased understanding of both the nature of the exact infectious agent(s), and

Article ID: WMC002410 ISSN 2046-1690

Toll-like Receptors (TLRs) and Prion Disease:Relevance to Pathology and Novel TherapyCorresponding Author:Dr. Okom Ofodile,Research Scientist, Center for Cardiovascular Research (CCR ), Institute for Phamacology and Toxicology, AG :THEURING, , Hessische Strasse 3-4, 10115 Berlin, Germany, 10115 - Germany

Submitting Author:Dr. Okom Ofodile,Research Scientist, Center for Cardiovascular Research (CCR ), Institute for Phamacology and Toxicology, AG :THEURING, , Hessische Strasse 3-4, 10115 Berlin, Germany, 10115 - Germany

Article ID: WMC002410

Article Type: Review articles

Submitted on:03-Nov-2011, 05:48:43 PM GMT Published on: 04-Nov-2011, 07:17:25 AM GMT

Article URL: http://www.webmedcentral.com/article_view/2410

Subject Categories:IMMUNOLOGY

Keywords:Innate Immune system, Pathogenic Microorganisms, Inflammation, Immunotherapy, TNF1-TLRInhibitor

How to cite the article:Ofodile O . Toll-like Receptors (TLRs) and Prion Disease: Relevance to Pathology andNovel Therapy . WebmedCentral IMMUNOLOGY 2011;2(11):WMC002410

Copyright: This is an open-access article distributed under the terms of the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the originalauthor and source are credited.

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This work is supportedsolely by Okom Ofodile and Children

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Toll-like Receptors (TLRs) and Prion Disease:Relevance to Pathology and Novel TherapyAuthor(s): Ofodile O

Abstract

Transmissible spongiform encephalopathies (TSEs) orprion diseases are a group of chronic, fatalneurodegenerative disorders of humans and animals,which have the unique property of being infectious,sporadic or genetic in origin. Creutzfeldt-Jakobdisease (CJD) in humans, scrapie (Sc) in sheep andgoats, and bovine spongiform encephalopathy (BSE)in cattle are typical prion diseases. Classical CJD canbe considered as sporadic, infectious or familial,whereas the new variant of CJD (nvCJD) isconsidered a BSE derived human disease. The exactpathogenic mechanisms and the exact nature of theinfectious agent of this disorder remain uncertain,however, it is largely believed that an abnormal form (PrPSc) of a host cellular prion protein (PrPc) maycompose the substantial parts of the infectious agentand that various factors such as oxidative stress,inflammation, perturbation of glutamatergichomeostasis, over-reactivity of the localized innateimmune system of the brain, and microbial attack areimplicated in the pathogenesis of PrD. Until veryrecently the signal-transducing receptors that triggerthe acute inflammatory cascade have been elusive.However, this enigma has been recently elucidatedwith the discovery of a transmembrane receptorprotein family, designated, Toll-like receptors (TLRs).TLRs are a fami ly o f h igh ly conserved,germline-encoded transmembrane receptors thatrecognize conserved products of a variety ofpathogen-derived molecular patterns (PAMPs), suchas lipoteichoic acids(LTA), lipopolysaccharide (LPS),peptodoglycan (PGN), flagellin, unmethylated DNAwith CpG motifs, viral double- stranded (ds) RNA andother components of microbial cell walls. In the lastdecade, advances in understanding mammalian hostimmune responses to microbial invasion suggest thatthe first line of defense against microbes is therecognition of pathogen-associated molecular patterns(PAMPs) by the Toll-like receptors (TLRs). TLRsmediate the recognition of PAMPs and inflammatoryresponses to a wide range of microbial products andthey are crucial for effective host defense. Recentdiscoveries revealed that TLRs also have importantrole in recognizing and regulating responses to

endogenous stimuli, such as heat shock proteins,necrotic cells, extracellular matrix breakdown products,and small synthetic molecules. Importantly, rapidlyaccumulating data have implicated the TLRs in thedevelopment and resolution of pathology in a widerange of neurological conditions. With regard to theaforementioned observations, and coupled, with themargins of what we now understand about the biologyand activities of Toll-like receptors, it is conceivable tosuggest that these receptor proteins, the TLRs, mayplay important role to play in the pathology of priondisease. Hence, elucidation and understanding thecellular and molecular basis responsible for both thebiochemical and molecular alterations associated withthe interactions between the TLRs and pathogenicagent(s), and the key processes of the pathogenicpathways in TSE pathology, and interactions between,and the interdependence of, the innate and adaptiveimmune responses may open a new dimension tounderstanding prion diseases.

Introduction

Transmissible spongiform encephalopathies (TSEs) orprion diseases are fatal neurodegenerative diseases inmammalian species that are sporadic, but also havebeen traced to mutations and to infectioustransmission, including iatrogenic transfer. TSEsinclude kuru, Creutzfeldt-Jakob disease (CJD),Gerstemann-Sträussler syndrome (GSS), and fatalfamilial insomnia (FFI) in human beings, as well asscrapie in sheeps and goats, bovine spongiformencephalopathy (BSE) in cattle, and encephalopathiesin mink, cats, mule, deer, elk, and several exoticungulates [1,2]. Neuron loss, spongiform degenerationand glial proliferation are the main pathologicalconsequences of TSEs [3, 4, 5]. Amyloid plaques areabundant in Kuru, Gerstermann-Sträussler-Schenkerand certain forms of sporadic CJD. Fluoride plaques,particularly common in new-variant CJD (nvCJD) arecomposed of a central core of amyloid surrounded byprominent microvasculation [6]. Abnormal PrPScaccumulation occurs in the majority of, but not all,prion diseases [7, 8]. The pathogenic mechanismunderlying these pathological conditions is largelybelieved to be a conformational conversion of thecellular prion proteinPrPc into disease-specific,



beta-sheet-rich forms (PrPSc or Prp-res) that possessabnormal physiological properties such as detergentinsolubility and protease resistance, PrPSc or PrP-res[9].Evidence indicates that PrPSc is substantiallyinvolved in the pathogenesis of brain changes, and inpropagation of the transmissibility of the diseaseprocess, by converting PrPc into a likeness of itself [3,9, 10]. The “protein only” hypothesis, which was laterrefined into “prion hypothesis” holds that TSEs aredistinct from infectious diseases caused by bacteria,viruses, fungi , or viroids in that the origin of thedisease is related to conformational alterations of anubiquitous protein, the prion protein, PrP, and thatnucleic acids are not essential for the propagation ofthe infectious agent [9,11,12,]. Thus, according toprion hypothesis: 1) the normal cellular form ofPrP(PrPc) is transformed into disease-related, andpossibly in itself infectious, scrapie form, 2) thereforePrPSc is solely responsible for the infectivity andtransmissibility of prion diseases, for it it is the solecomponent of the infectious particle, which is termedprion, [12, 13] and the variations in the tertiarystructures of PrPSc would account for the existence ofprion strains with distinct biological properties [14].However, despite the above data, the” protein only”hypothesis and the associated protein-only modelcannot explain all the existing data. The prionhypothesis has not yet been experimentally proved [5,15]. There are, at least, four hypotheses regarding theidentity of the infectious agent in prion disease: virus[16], virino [17], Bacterium [18] and prion [19]. Indeed,it has been shown in vivo that PrPSc deposition inneuronal tissue not expressing PrPc has nopathological consequence [20]. In addition, in bothinfectious and genetic models of TSEs typicalsymptoms of disease outcome and neurodegenerationhave been detected in the absence of observablePrPSc [21, 22]. Additionally, in commensurate to this,indeed, in vitro conversion experiments of PrPc toPrPSc, in which protease-resistance was, achieved bya denaturation/renaturation procedure, resulting inprotease-resistant beta-sheet rich PrPSc but notinfectious PrPSc [23, 24]. Second, the “prionhypothesis cannot explain the presence of many prionstrains that retain and inherit unique incubation period[15, 25].The cellular prion protein, PrPc, is a normal cellularglycophosphatidyl inositol (GPI) -anchoredsialoglycoprotein encoded by the PRNP gene [26].PrPc is found predominantly in CNS, but a loweramount of it is also found in the other tissues [27, 28].Despite a wide range of postulations, the physiologicalfunctions of PrPc have not yet been fully understood.Nevertheless, strong compelling evidence exist

suggesting that PrPc is essential for the developmentof prion disease serving as a template forconformational change [29].This notion is evidencedby Bueler and associates [30]. These authorsdemonstrated that PrPc knockout (PRNP-/-) mice areresistant to scrapie infection. The finding that PrPc ismultiply glycosidated indicates that PrPc may belinked to signal tranduction pathway [31]. BecausePrPc has been found to be localized in the synapse,some investigators suggested that the absence ofPrPc may alter synapse formation suggesting aninvolvement of PrPc in neurotransmitter system ofCNS [32, 33]. There is also increasing evidence thatsuggests functional roles for PrPs in the coppermetabolism [34, 35, 36]. Recent evidence indicatesthat PrPc controls the survival of the challenged cellsby governing the induction of pro-and anti-apoptoticsignaling pathways [37].Together, the above data suggest that disturbingfundamental uncertainties remains in the pathology ofTSEs. For instance, microbial pathogen agent(s),whose exact nature have yet to be fully resolved,should be substantially involved in driving priondisease. Hence the etiology of prion disease isunknown. This presents a substantial obstacle to thedevelopment of effective diagnosis, therefore, to date;neither preventive strategies nor long-term effectivetreatment modalities are available for these diseases.Nevertheless, abundant evidence now exists thatneuroinflammation, and oxidative stress-damagecontributes to the pathogenesis of severalneurodegenerative disorders including Alzheimer’sdisease (AD) [ 38, 39] and prion diseases [ 40,41, 42,43, 44, 45], and all these processes have been shownto be engaged and mediated by Toll-like Receptorsignaling actions. Therefore, at the margins of what wenow understand, a major event in elucidating aplethora of fundamental uncertainties in TSEpathogenesis may largely rely on our increasedunderstanding of both the nature of the exactinfectious agent(s), and the nature of the receptorsintimately involved in mediating the inflammatoryactivities in the pathogenesis of prion diseases.Until very recently, the signal-transducing receptorsthat trigger the inflammatory cascade have remainedelusive. However, this uncertainty has been a fewyears ago elucidated with the discovery of atransmembrane receptor protein family, designated,toll-like receptors. These advances in understandingthe molecular basis for mammalian host immuneresponses to microbial invasion suggest that the firstline of defense against microbes is the recognition ofpathogen-associated molecular patterns (PAMPs) bythe TLRs (reviewed in [46, 47]. TLRs have been



identif ied as being part of a large family ofpathogen-recognition receptors that play crucial rolefor the induction of both innate and adaptive immunity.Most recent studies have implicated TLRs inrecognition of proteinaceous molecules such as heatshock proteins, and other endogenous ligandsincluding extracellular matrix breakdown products,chromatin-IgG, pulmonary surficants, necrotic cells [48,49,50] and endogenous mRNA [51]. Heat shockproteins have been implicated in a plethora of centralevents in prion disease pathogenesis [52, 53].Johannes van Noort and co-workers [54] have recentlydemonstrated the presence and activities of TLRs inthe central nervous system, thereby indicating theimplication of these receptors in executingphysiological and pathophysiological processes in theCNS. Furthermore, Schluesener and colleagues [55]have reported that that astrocytes and microglia areactivated upon intracranial injection of CpG ODN(synthetic oligodesoxynucleotides containing theunmethylated 5’-CpG-3’ dinucleotides motif). Inaddition to this, Hemmi and associates [56] disclosedearlier that Toll-like receptor 9 (TLR9) was responsiblefor the transduction of inflammatory intracellularsignals upon stimulation with bacterial DNA and CpGODN. Supports for the aforementioned notion havebeen evidenced by further studies [57, 59, 59].Together, these data indicate that TLR9 is expressedon astrocytes and microglia, and points to animportant role for TLR9-activated glial cells inCpGODN-induced neuropathological conditions. Inthis respect, I am tempted to suggest that TLRs mayhave an important role in the development andresolution of TSE pathology because of their criticalrole in initiation of inflammatory responses, andmediating a wide range of signal transductionpathways intimately associated in the developmentand resolution of pathology in a wide range ofneuropathological conditions. Here I summarize thecurrent state of knowledge on TLR-associatedsignaling, and interactions, alongside discuss thebroad implications of these interactions for thepathology of prion diseases, and concluding thatadvances in understanding of the biology and TLRpathways may allow us to tackle a wide range ofchallenges in immunology and medicine.

TOLL-LIKE RECEPTORS (TLRs)The finding that the CNS is not immunologicallyprivileged as was previously believed represents amajor breakthrough and a milestone in the history ofimmunological sciences [60]. This recent discovery ofthe immune response in the brain revives the idea thatimmunological challenges might well be etiologicalfactors in sporadic cases of neurodegeneration, and

also indicates that primary causes of suchdegeneration could originate outside the centralnervous system.Immunity to infectious agents is mediated by twogeneral systems, innate and acquired immunity(reviewed in ref: [61, 62]. In contrast to adaptiveimmunity , the innate immunity appears early duringevolution and has the ability to recognize pathogenicmicroorganisms by germ-like encoded receptors,equipped with defined specificities for highlyconserved structures present on most pathogenicorganisms [63]. The innate immunity, in contrary toadaptive immunity, is activated immediately afterinfection and rapidly controls microorganismreplication, until adaptive immunity takes over.The innate immune response is activated by a fewhighly conserved structures present in mostpathogenic microorganisms, instead of by recognitionby a wide range of various antigens.These structuresare defined as pathogen-associated molecularpatterns (PAMP) sensed by pattern-recognitionreceptors (PRP).Among the well-defined PAMPs arebacterial lipopolysaccharide (LPS), peptidoglycan,mannans, bacterial DNA and double-stranded RNA.PAMPs are believed to be produced almost solely bybacterial pathogens and are are essential for thesurvival or pathogenicity of microorganisms[64, 65,66]Patter-recognition receptors are mainly expressedon effector , such as dendrit ic cells (DCs),macrophages and B cells. All PRRs are displayed by agiven cell type and have identical specificities.When aPAMP is recognized, all cells are immediatelytriggered to perform their effector functions, therebyleading to a rapid innate immune response [67, 68].

Review

Toll-like ReceptorsNow, research over the last few years has greatlyadvanced our understanding of the mechanisms bywhich the immune system functions, and especially,the innate immune system [69, 70, 71]. In keeping withthis, recent advances in understanding the molecularbasis for mammalian host immune responses tomicrobial invasion suggest that the first line of defenseaga ins t m ic robes i s the recogn i t i on o fpathogen-associated molecular patterns (PAMPs) bythe Toll-like Receptors (TLRs). In addition, the findingsof Lemaitre B and colleagues [72] and Poltorak A andcolleagues [73] led to the realization that the proximalinnate innate immune sensing tool of insects andmammals are related by descent, and thus furtherindicates a key pivotal role for TLRs in the primary



recognition of infectious pathogens by mammals. Themammalian Toll-like receptors, major integralcomponent of the innate immune system, are a familyo f h igh ly conserved, germl ine-encodedtransmembrane receptors that are critically involved inmammalian host defense. It is now known that thereare 13 mammalian TLRs, which can sense molecularpatterns that are common constituents of a widevariety of pathogens but are rarely found in the host;10 in humans and 12 in mice [74,75]. Structurally,TLRs are characterized by the presence of aleucine-rich repeats domain in their extracellularregions and a Toll/IL-1R (TIR) domain in theintracellular regions. In respect of the amino acidsequence and genomic structure, TLRs can be dividedinto five subfamilies: TLR2, TLR3, TLR4, TLR5, andTLR9. The TLR2 subfamily is composed of TLR1,TLR2, TLR6, and TLR10, and TLR9 subfamily iscomposed of TLR7, TLR8, and TLR9. TLR1 and TLR6form heterodimers with TLR2 [67].TLRs serve to identify conserved products of microbialmetabolism (PAMPs), such as lipoteichoic acids(LTA), lipopolysaccharide (LPS), peptidoglycan (PGN),other components of microbial cell walls [76, 77, 67],which enables the innate immune system to recognizeinvading microorganisms and to induce a protectiveimmune response.Recent discoveries disclosed that TLRs do not onlymediate recognition and inflammatory responses to awide range of microbial products but also tonon-microbial endogenous proteinaceous molecules,heparan sulfate and RNA, DNA and small molecularsynthetic products [50, 78,79,80, 51]. The TLRs,hence, play a key central role in innate immunity byrecognizing conserved molecular patterns andgenerating signals leading to the initiation of anadaptive immune response, thereby serving as animportant link between the innate- and the adaptivearms of the immune system. Recent discoveriesdisclosed the identification of a human TLR11. Thiswas originally isolated from murine. Murine TLR 11appears to be closely related to TLR5 and it isexpressed abundantly in kidney and bladder.TLR11-deficient mice are reported to be highlysusceptible to infection of the kidney by uropathogenicbacteria [81], suggesting that TLR11 play importantrole in urinary tract infection. The function of thehuman TLR11 is not known because of the presenceof a stop codon in the gene [81]. Important updatessuggested that a microbial profilin-like moleculeisolated from the protozoan parasite Toxoplasmagondii (T. gondii) functions as a ligand for TLR11 [ 82].This profilin-like molecule (a protein) was shown totrigger IL-12 through TLR11.

TLR signaling pathwaysTLRs activate distinct signaling cascades via fourdifferent Toll/IL-1 receptor (TIR) domain-containingadapter proteins. The four adapter proteins are MyD88(myeloid differentiation factor 88), MAL/TIRAP(MyD88-adapter-like/TIR-associated protein), TRIF orTICAM-1/ Toll-receptor-associated activator ofinterferon) and TRAM (Toll-receptor-associatedmolecule). The aforementioned four adapter proteinstransduce signals from all of the TIR domains,activating protein kinases and then the transcriptionfactors that lead to inflammatory effects. Recentdiscoveries disclosed the identification of a possiblyfifth TIR adapter protein, designated, Sarm [83].However, the function of Sarm is presently completelyunknown.Despite divergent PAMP ligands, all TLRs with theexception of TLR3 activate MyD88-dependentpathways to induce a core of stereotyped responsessuch as inflammatory events. The pathways thattransduce TLR signals in mammals appear to haveboth similar and dissimilar characteristics from those inDrosophila [84]. In Drosophila the Toll-IMD-pathwaysare crucial for antifungal and anti-Gram negativebacterial responses, respectively. In mammals thehost defense against microorganisms mainly relies onpathways that originate from the common TIR domainof TLRs. The TLR family signaling pathway is highlyhomologous to that of IL-1R family. Both TLR andIL-1R interact with an adapter protein MyD88, whichhas a TIR domain in its C-terminal segment but adeath domain (DD) in its N-terminal segment insteadof the transmembrane domain found in TLRs. MyD88associates with both the TLRs and IL-1R viainteraction between the respective TIR domains. Uponstimulation by a ligand, MyD88 recruits a deathdomain-containing serine kinase, the IL-1R–associated kinase (IRAK (IRAK 1 and IRAK4). IRAKis activated by phosphorylation through its N-terminaldeath domain and then activates TNFR associatedfactor 6 (TRAF6) to stimulate IKappaB Kinase (IKK)complex and MAP kinase. Phosphorylation of IkappaBby IKK complex induces the degradation of Ikappa Bthrough the ubiquitin-proteasome pathway, andsubsequent nuclear translocation of liberatedNF-kappaB mediates transcription of pro-inflammatorycytokine gerne [85, 86].The TLR3 ligand, double-stranded RNA has beenreported to induce the activation of NF-kappaB inMyD88 knockout (KO) mice, thereby buttressing thenotion that TLR3 signaling is independent of MyD88[87] Furthermore, signaling can occur independently ofMyD88 for TLR4, which also activates NF-kappaBthrough the adapter protein TIRAP and for TLR3, as



indicated above, which induces an antiviral interferon(IFN)-? response through TICAM-1[67; 88]. Recently,a germline-induced mutation in TRIF led toidentification of another adapter molecule with a TIRdomain; TRIF-related adopter molecule (TRAM),shown to be required by TLR4, but not TLR3-mediatedIFN response. In Tram-deficient mice, LPS inducedpersistent NF-kappaB activation, whereas theexpression of IFN-inducible genes was defective,thereby strongly pointing to a crucially pivotal role forTRAM in the Toll- l ike receptor 4-mediatedMyD88-independent signaling pathway[ 89, 90] .Thesecompellingly indicate that studies designed todetermine the activities of TLRs in any specificpatholog ica l condi t ion, whereby s t r ic t lyMyD88-deficient model was tested (solely puttingMyD88-dependent pathway into consideration), couldalmost impossibly lead to findings, which could beenough to categorically rule out the involvement ofTLRs in the development and resolution of thepathology in the concerned model, strongly suggestingthat the earlier reports by Prinz et al [ 91 ] that Toll-likereceptors were not involved in the pathology of TSEdisease should not be considered as very accurate.Thus, individual TLR signaling pathways are divergent,although MyD88 is common to almost all TLRs.Nevertheless, it has become increasingly clear thatthere are MyD88-dependent and MyD88-independentpathways. Further support for this notion is evidencedby a huge body of important updates [92, 93, 94, 95,].Judging from the enormous interest in research onthese transmembrane receptor proteins, the TLRs, it isvery likely that more Toll-like receptor ligands andsignaling pathways will be identified in the future. It isalso of note that signaling pathways mediated byToll-like Receptors have also been revealed to be celltype-specific [96, 97].This indicate that , under certainconditions, some cells and tissues might be prone tofavor this pathway to the other. The molecularmechanisms underlying this have yet to be fullyresolved.

EVIDENCE ARGUING IN FAVOR OF MICROBIALINVOLVEMENT IN DRIVING PRION DISEASEThe realization that a ubiquitous protein called, prionprotein, plays an important role in the pathology oftransmissible encephalopathies is a milestone, andrepresents one of the most gratifying aspects ofresea rch ca r r i ed ou t on t ransmiss ib leencephalopathies during the last five decades. Thisdiscovery, concomitantly led to the birth of the “proteinonly” hypothesis [11], which was later refined intoprion hypothesis [12, 9]. Albeit, recent intensiveresearches and significant advances in the province ofprion diseases in the last ten years, fundamental

uncertainties remain; the true nature of the causativeagent (unconventional virus, virino, bacterium, fungusor “prion”?), mechanisms underlying the conversion ofPrPc to PrPSc, the preference of PrPSc accumulationin glia and neurons or both, accurate pathogenicmechanisms of neurodegeneration, precise modes ofinfection, transmission, definite physiological functionsof PrPc and exhaustive structural characterization ofPrPc and its pathogenic isoform, PrPSc [98, 99]Hence, a plethora of fundamental uncertainties stillremains in prion disease biology and pathology, asprevious described. Among all these, the mostpressing and conflicting question is that of the truenature of the causative agent in prion diseases.Support for this notion is evidenced by the followingdata:Earlier biophysical studies carried out by Eigen M.[100] conclude that the infectious unit in vivo does notconsiderably correspond with an in vitro form ofaggregated prion proteins, thus strongly indicative ofthe involvement of a non-prion protein identicalpathogenic agent in the orchestrated network ofevents intimately associated with the pathogenesis ofprion diseases

Bacterial InvolvementIn spirited and thorough studies, coupled withenormous patience and determination, Bastian F.O.and associates [101, 102, 103, 104, 18, 105] followedup progressively the involvement of a bacterium,named, Spiroplasma, which belongs to a large familyof mycobacterium, in the pathogenesis ofCreutzfeldt-Jakob disease. Support for this notion hasalso been evidenced by studies carried out by others[106, 107, 108]. Spiroplasmas are hel icalmycoplasmas that play a significant role in insects andplants diseases [109] and they are also found inarthropods that are likely to bit animals and humans,such as such as ticks and mosquitoes [110]. Thissubject (Spiroplasmas and their characteristics) hasbeen recently reviewed by Gasparich G. andassociates [111, 112] and comprehensive and detaileddisclosure of the morphological and the ultrastructuralaspects of spiroplasmas have been earl ierdemonstrated by Cole and associates (109). Theearlier report of Bastian FO [101] demonstrating theinclusion of Spiroplasma-like molecules in CJD, wasfollowed by a plethora of data from both Bastian andassociates,and other various laboratoriessubstantiating and strengthening the notion, thatSpiroplasma-associated inclusions have a role inTSEs pathogenesis [101, 102, 103, 18, 104, 105, 106,107, 108] The most recent work of Bastian andassociate on this subject, overwhelmingly and mostcompell ingly implicated Spiroplasma sp (a



Mycobacterium) in the pathology of TSEs [18].Sequencing of the amplified PCR products ofSpiroplasma 16S rDNA robustly confirmed thepresence of Spiroplasma –like DNA (Spiroplasma 16SrDNA) in all the tested (5 in number) of TSE brains.This result presents a very clear and reproducibleevidence for the involvement of a bacterium in TSEpathology. Consistent with this notion, Ebringer andco-workers earlier suggested the involvement ofanother bacterium, Acinrtobacter calcoaceiticus, in thebrain of a BSE-affacted animal ([113]. Additionally, incommensurate to this, several lines of data havedemonstrated the ability of various anti-bacterial drugs( antibiotics) to suppress the progression of TSEs andin some cases , also to significantly hinder theconversion of PrPc (PrPsen) into PrPSc (PrPres) [ 114,115]. Now, as we have been thought, the cell wall isresponsible for many of the characteristic properties ofbacteria (e.g. acid fastness, slow growth, resistance todetergents, resistance to common antibacterialantibiotics, antigenicity). In agreement with this, themycoplasma, a bacterial family to whom Spiroplasmasp belongs, do not have cell wall, and as a result, themycoplasma are resistance to penici l l ins,cephalosporins, vancomycin, and other antibiotics thatpredominantly interfere with the synthesis of the cellwall [116]. Theses findings are consistent with earlierreport showing that spiroplasmas were highly sensitiveto antibiotics such as tetracycline, and other antibiotics[117]. Furthermore, neither penicillins, nor very closelyrelated antibacterial antibiotics, as previous described,have been reported to confer proctection to TSE[116,117]. Hence, essentially, the above data stronglysuggest involvement of microbial pathogenic agent(s)in the pathology of TSEs [101, 102, 103, 18, 104, 105,106, 107, and 108]. In addition to this, Seya andMatsumoto [118] have recently disclosed that thefunctions of three forms of a mycoplasmal i p o p e d t i d e / p r o t e i n , d e s i g n a t e d ,macrophage-activating lipopeptide 2(MALP-2), P48,and M161Ag, which were isolated from a mycoplasma,M fermentans ( which thus belongs to the same familyas its close relative, Spiroplasma sp), were largelymediated by Toll-like receptor 2. These proteinmolecules exert similar immunomodulatory effects onmacrophages and dentritic cells. Immunomodulatoryeffects such as cytokine induction, NO production andmaturation of antigen-presenting cells. M161Ag hasalso been associated with the capability to induce invitro apoptotic cell death and to induce complementactivation by binding macrophages via complementelements C3b/C3bi and their receptors [119]. Together,essentially, the above experimental evidence stronglysuggests that; 1) microbial pathogen agent(s) should

be implicated in driving prion disease, 2) that the TLRsmay have critically important role in mediating thepathology of prion disease, and finally 3) the TLRs,indeed, represent attractive target for pharmacologicalinterventions in the development of novel approachesfor the management of TSE diseases and a variety ofother pathological conditions.

C-reactive ProteinC-reactive protein (CRP) is a serum protein that ismassively induced as a part of innate immuneresponse to infection or tissue injury. The ability ofCRP to recognize pathogens and to mediate theirelimination by recruiting the complement system andphogocytic cells makes CRP and importantcomponent of the innate arm of the immune system,whict is solely responsible for the first line of hostdefense. According to the prion hypothesis, the mainproteinaceous component in TSEs is the prion protein,PrPc, whose misfolded isoform, PrPSc, is suggestedto be solely responsible for the infectivity andtransmissibility of TSE diseases. Aside from prionprotein, however, a large number of other proteinshave been localized to the TSE-affected brain. Theseinclude, for instance, serum amyloid component (SAP)in mice Coe J. and associates [120] and C-reactiveprotein (CRP) and IL-6 in CJD (121). Equally, in otherrelated neurodegenerative disease such as AD, acutephase reactants including CRP have been located inthe diseased brain tissues [122, 123, 124, 125, 126].Mouse serum amyloid component (SAP), a majoracute phase protein, is a physiologic and functionalcounterpart of C-reactive protein (CRP) in humans.These proteins belong to the member of pentraxinfamily of proteins and they are potent acute phasereactants, characterized by by the cyclic pentamericstructure; these proteins interact with their ligands in acalcium-dependent manner [127, 128, 129]. They aremainly serum constituents and are not formed in thebrain under normal circumstances. On the other hand,under pathological conditions, associated withmicrobial attack, the concentrations of C-reactiveprotein, for instance, rises dramatically in host defenseaga ins t the m ic rob ia l a t tack . CRP hasCa2+-dependent b ind ing spec i f i c i ty fo rphosphocholine (PCh), a constiruent of many bacterialand fungal polysaccharides and most biological cellmembranes [130, 131]. Consistent with the primaryfunction of CRP, CRP was discovered and namedbecause of its reactivity with the PCh residues ofC-polysaccharide (PNC) , the teichoic acid ofStreptococcus pneumonia [132], pointing to animportant role of TLR in mediating the actions of CRP.This notion is recently evidenced by Greenhalgh andco-workers [133]. These investigators provided



evidence indicating the two components of the LPSreceptor complex, CD14 and TLR4 should participatein the cellular and molecular events controlling theinduction of acute phase proteins, SAA and SAP, inthe lever after burn injury.. CRP is also bind otherconstituents, which do not contain phosphocholine(PCh), such as small ribonucleaoprotein particuiles[134]. Ligand-complexed CRP is recognized by C1qand efficiently activates the classical pathway ofhuman complement system [135, 136]. It has alsobeen proposed that CRP working in concert with thecomplement component plays a role in the clearanceof apoptotic and necrotic cells, thereby contributing torestoration of normal structure and function of injuredtissues [137]. However, as with most other elements ofimmunity, CRP has the lineage, the appropriatereceptors, and the capacity to participate in bothpotentially destructive inflammatory responses andpotentially protective responses. Thus recentdiscoveries have implicated C-reactive inatherogenesis [138, 139], mediation of tissue damagein acute myocardial infarction [140] and in the processof “autotoxicity” in neurodegenerative disorders [141,142]. Despite the above mentioned observations,however, the functions of CRP are yet fully to beelucidated: Nevertheless, it is now well establishedthat CRP, in host defense against pathogenic agents,exerts three major functions: activation thecomplement system; opsonization and the induction ofphagocytosis [143]. Therefore, a very importantfunction of CRP that could hardly be overlooked is itsability to offer protection against microbial agents. Thisis believed to be the primary function of CRP, whoseblood levels increases from almost zero to severalhundred micrograms per milliliter during inflammatoryevents in a variety of pathological conditions [144,145]. In addition to this, recently, Szalai AJ [143] lentfurther support to this notion and extended on it byreporting that the CRP primarily functions in hostdefense against microbial pathogen agents of largelybacterial, in the first instance, and fungal origins. Thisnotion was consistent with earlier studies carried outby Du Clos and associate [146, 147]. .Xia et al.reported the ability of CRP to offer protection againstheightened inflammatory response mediated by toxinssuch as lipopolysaccharide, which are released by theinfectious agents [147]. This finding may beconsidered consistent with the reports of Coe andcolleagues [120], Peyrin and colleagues [40] andSharief and associates [42]. These investigatorsdemonstrated, totally, independent from one anotherthat systemic inflammatory responses have pivotalrole in the pathophysiology/pathogenesis of TSEs.Conclusively, 1) CRP is usually over-expressed as a

result of microbial attack largely of bacterial and fungalorigins and this represents the fundamental function ofCRP, 2) the expression of pentraxin in TSE stronglyindicates the involvement of systematic inflammatoryevents in the pathophysiology and /or pathogenesis ofTSEs, 3) the generally believed “Marker” function ofCRP is , indeed, purely a secondary function, which isof mere diagnostic importance, 4) the reportedexpression- and up-regulation of CRP in TSEs [121],could , therefore, very conceivably, be indicative of thepresence of exogenous microbial agents, largely , ifnot, solely , of bacterial and fungal origins, consistentwith the reports previously described [143, 145, 147,146]. Finally, the upregulation of CRP in the context offulfilling host defense function againt pathogenicmicroorganisms has been reported to be alsoimplicated in Aspergillus fumigatus conidia-inducedattack [148] and in malaria-parasite-related attack , aswell [149]. The above observations, indeed, do notreconcile with the “prion hypothesis”. Taken togetherwith the evidence that Toll-like receptors mediate thebiological effects of CRP [133,150], coupled with themargins of our present understandings of the biologyand actions of TLRs, the above observations stronglystrengthens the notion that Toll-like receptors shouldhave important role in the pathphysiology andpathogenesis of prion disease,

TOLL-LIKE RECEPTORS SHOULD HAVE A ROLEIN THE DEVELOPMENT AND RESOLUTION OFPRION DISEASE PATHOLOGYCross-Talk with Bacterial CpG-DNA and TLR9 linksInnate and Adaptive ImmunityIt has been well established for decades that bacterialDNA is immunogenic in vertebrates. Introduction ofbacterial DNA in mice induces anti-DNA antibody andNK cell activation. . In 1995, Krieg et al. [151] using aB cel l prol i ferat ion essay discovered that5´-CpG-3´dinuclöeotides with selective flanking basesare important for the immunogenicity of bacterial DNA,thereby disclosing the sequence –specif icimmunogenic characteristics of bacterial DNA. Inaddirtion to this, in 1998, Jakob and associates [152]demonstrated that bacterial DNA induces strong Th1-like inflammatory responses .The usage of thissequence is suppressed in mammals and thateukaryotic CpG motifs are preferentially methylated,which then abolishes their immunogenic potential.Following the work of [153], in 2001, Hemmi et al. [56]disclosed that a member of the Toll-like receptor family,TLR9, is the true receptor for the CpG motif-containingoligodeoxynucleotide (CpG-ODN). Accordingly, theability of the TLR9 to satimulate the vertebrate innateimmune system , and subsequently that of theadaptive immune system, presents TLR9 agonists as



attractivetarget for the development of highly effectivevaccines for infectious diseases, and as well as astand-alone therapies or in combination with othertherapies in other diseases such as cancer. Advancesin this field of research have led to the discovery ofdifferent types of CpG -ODNs, and disclosed that thebiological activity of CpG - ODNs is not restricted tostimulatory cells of the primary. Recent advancesrevealed that CpG- ODNs also activate cells in thecental nervous system. For instance, particularlymicroglia and astrocytes present in the brain wereinduced to upregulate their expression of cytokine andchemokines following exposure to CpG -ODNs [57].These observations strongly suggest that CpG- ODNscan directly activate immunologically relevant cells inthe central nervous system. Now, in the relentlessefforts to eventually work out effective therapeuticapproaches for TSE disease management, HansKretzschmar and co-workers [154] recently revealedt h a t a p p r o p r i a t e e m p l o y m e n t o f C p Gdeoxyoligonucleotides may have a role in significantlydelaying the progression of TSE disease and may, atleast, be beneficial as a potent post-exposureprophylactic measure. Thus, essentially, the abovedata widely imply that pathogenic agent of bacterialorigin consistent with the compelling findings ofBastian et al [101, 102, 103, 18, 104, 105, 106, 107,108] may have a role in driving the prion disease, andthat endosomal –lysosomal compartmen, earlierdisclosed as the major location for PrPSc synthesis[155], may represent a potential compartmental targetfor TLR-associated therapeutic interventions.Furthermore, the above data are consistent with theconcept of protective role of innate immune system.Therfore, on the basis of the aforementionedobservations , it is tempting to suggest that innateimmune system is an important player in TSEpathology, and that TLRs may have important role indriving the pathology of TSE disease. Hence,increased understanding of the activities, and biologyof TLRs may bear relevance to pathology andmanagement of TSE diseases, and otherneurodegenerative diseases .

PrP106-126 (a peptide mimetic of PrPSc) canmature and activate Dendritic Cells (DCs)The activation of monocyte-derived cells is largelybelieved to play a pivotal role in the inflammatoryprocess leading to the pathogenesis of manyneurodegenerative diseases such as Alzheimer’sdisease and prion diseases [156, 157, 158, 159, 160].Denditic cells have been shown to mature andbecome highly activated in response to severaldifferent mediators, including inflammatory cytokines[161] oligo CpG motifs, LPS and other bacterial

products, and dsRNA, and also by endogenous mRNA,as previously described. Support for this notion isevidenced by recent studies carried out by Bacot andassociates [162], for instance. These investigatorsdemonstrated, in a set of series of thorough andelegant experiments, the activation of NF-?B signalingpathway in monocyte-derived DCs, which led to theproduction of inflammatory cytokines and furtherdifferentiation of DCs by PrP106-126. These findingindicates that PrP106-126 (a peptide mimetic of thepathogenic PrPSc) has the ability to mature andactivate DCs as determined by the increasedexpression of MHC classII (HLA-DR), costimulatorymolecules CD40 and CD80, and the maturationmarker, CD 83. This study also lends support to thenotion that a proteinaceous molecule, in this case,pathogenic prion protein, PrPSc, (its mimetic) is in afull position to mature and activate DCs. However,despite the elegancy and the convincing strength ofthe study, essential questions were left open. Theseinclude the following points: 1) did PrP106-126 binddirectly to the DCs or was the binding activity, as itshould be expected, mediated by appropriate cellsurface receptors(s). In this context, it is reasonable toassume that the activities of PrP106-126 could be bestcarried out with the help of appropriate cell surfacereceptors, which could then effectively mediate, andpossibly regulate the activities of the PrP106-126peptide fragments. In the face of this problem, andbased on our present understanding, it is reasonableto contemplate that coordinated association ofPrP106-126 with surface receptor proteins should berequired to enable a successful interaction betweenthe PrP106-120 and the DCs. Now, at the margins ofwhat we now understand about the biology and theactions of TLRs, albeit that, there are also otheravailable surface receptor proteins [48], it isreasonable to contemplate these family oftransmembrane receptors may participate directly orindirectly in this event. As earlier reported by Lee et al.[163, 164], a G protein-coupled receptor formylpeptide receptor-like 1 (FPRL1) has been suggestedto mediate the binding activities of PrP106-126, butwith low interacting affinity to PrP106-126. Thisstrongly indicates that other receptor proteins shouldbe required in the mediation of PrP106-126binding-activities, to enable the protein PrP106-126achieve its objective effectively. Furthermore, Lee et al[163, 164] also concluded that FPRL1 also mediatethe activities of amyloid beta peptide (Abeta 42) andthat of almost all amyloidogenic molecules. Now,George Perry and associates [165] recently disclosedt h a t T L R 4 m e d i a t e s t h e m i c r o g l i a lactivation-associated interaction between amyloid beta



peptide and HSP70, thereby , pointing that a memberof the Toll-like receptor family of proteins, could alsowork in concert with FPRL1 in mediating the biologicaleffects of an amyloidogenic molecule( peptides). Inthis respect, coupled with the margins of what wepresently now about the biology and activities of TLRs,and the findings concluding that neurodegenerativedisease such as AD share various pathogenic factorswith CJD [166, 167, 168, 169] , I am tempted tosuggest that TLRs should have a role in mediating thisprocess. The TLRs might be or might not be the cellsurface receptors primarily and/or largely responsiblefor the mediation of PrP106-126 peptide fragmentsactivities in the previously described process [162].However, at least, TLRs, working in concert, with otherreceptor proteins including FPRL1 should beimplicated, in one way or the other, in this event. Itmay also be noteworthy, that under certaincircumstances the TLR signaling might not be requiredfor particle binding or internalization. However, formediation of inflammatory and immune responsesTLR activation and signaling are indispensable [170]The TLRs should be directly or indirectly involved inthe complex cascade of events that finally terminate ininflammatory responses, such as production of TNF-?and activation of NF-?B [171, 172, 173]. Therefore, theabove observations lend further support to the notionthat the TLRs may have important role in thedevelopment and resolution of TSE pathology. Hence,therapeutic strategies designed to inhibit theappropriate TLRs, and FPRL1 receptors and theircorresponding intracellular signaling pathways mayhold great promise for the management of TSEdiseases and related cases. However, to this end,appropriate measures should be taken to see that thepositive biological effects of the concerned receptorproteins are not excessively compromised.

The Variability of TLR-signaling PathwaysIt is now generally accepted that all TLRs can utilizethe adapter protein MyD88 to propagate signals togene targets to and generate a relatively rapidprotective response, by activating NF kappaB orthrough other routes. However, at least two Toll-likereceptors (TLR3 and TLR4), as previously described,can use alternative adapters such as Toll/IL-1receptorresistance (TIR) domain –containing adapter inducingIFN-beta /TRIF) and TRIF-related adapter molecule(TRAM); used by TLR4 than can activate responsesdifferent from those elicited by MyD88. Therefore, they(TLR3 and TLR4) utilize the adapter proteinMyD88-independent pathway to propagate signals togene targets. Now, previous report that prionpathogenesis is unhampered in MyD88-/- mice [91]simply told us indirectly that the TLRs can utilize both

the MyD88 adapter protein dependent-pathway anda l s o ( u n d e r c e r t a i n c o n d i t i o n s ) t h eMyD88-independent pathway to propagate signals togene targets [174, 175 ]. Aside from previouslymentioned data, further support for this notion hasbeen evidenced by very recent discovery, whichrevealed that the TLR3 efficiently mediated the entryof a microbial pathogen agent from the peripheralblood system into the brain [176].TLR3, notably, doesnot utilize the MyD88 dependent pathway for itsactivities. In addition to this, at the margins of what wenow know about the biology and activities of TLRs inhealth and disease states, studies designed toinvestigate the involvement of Toll-like receptorsignaling in the pathogenesis of a particular or anyspecific pathological condition, using a model,whereby only MyD88-dependent pathway, for instance,is strictly put into consideration, could hardly lead toresults, which could generate enough data tocategorically rule out the involvement of TLR signalingin the development and resolution of the concernedpathologic condition. In the face of the aboveconsiderations, the previous report that Toll-likereceptors are not involved in prion diseasepathogenesis [Prinz et al., 2003] should not beconsidered as very accurate. It is now generallyrecognized, supported by a rich vein of data [85, 93,95, 174 ], that TLRs, working alone or in concert, caneffectively carry out their biological functions throughMyD88 –dependent, and/or MyD88-independentpathways, as well. Therefore, based on a plethora ofdata previously described, and, coupled with a hugebody of compelling data persuasively arguing in favorof the involvement of pathogenic microorganisms indriving prion diseases, [18, 101, 102, 104, 105, 106,107,108 113, 177, 178 ], and strong implication of theactivated microglia -and activated complement system(both represent integral components of the innateimmune system) in TSE pathogenesis [41, 179, 180,181, 182,183], I am led to strongly contend thatToll-like receptors should have important role in thedevelopment and resolution of TSE pathology.

MICROGLIA ACTIVATION and PRION DISEASEIt is now largely recognized that the accumulation ofreactive microglia in the degenerative areasrepresents the major cellular evidence pointingunequivocably to the presence of neuroinflammation inTSE disease and other neurodegenerative disorders,as well. Microglia are macrophage-like cells residentwithin the CNS, which can perform APC andproinflammatory effector functions following activation.These cells are derived from bone marrow stem cellsand populate the CNS early during development and



remain in the CNS as resident macrophage population.Microglial cells are quiescent in the CNS unable toperform effector and APC functions until activated byinjury or infection, and have been suggested torepresent the first line of defense for the CNS, whichnormally lacks professional APCs until they arerecruited to the CNS by inflammatory stimuli [184,185].The neuropathology of Alzheimer`s disease and priondisease has many common features and it has beensuggested that microgial cells play a causative role inthe pathogenic cascade of neuroidegeneration inAlzheimer`s disease and TSE disease [ 180, 186].Support for this notion has been evidenced byhistological studies demonstrating that microgial cellsare associated with the accumulation of abnormal ,disease-associated isoforms of prion protein, PrPSc,in in the central nervous system in prion disease [187].The findings that fibrillar forms of amyloid –beta , orPrP peptides , stimulate microgial cells in vitro [188,189] buttress the notion that the deposition ofamyloidogenic peptides induces microgialcell-mediated inflammatory responses that largelycontribute to neurodegeneration and the concomitantcognitive decline observed in TSE disease andAlzheimer’s disease.Furthermore, Microglial cells maybe activated to produce directly neurotoxic substancesand inflammatory mediators in experimental priondisease and are very likely involved in phagocytosingPrPSc and/ or apoptotic neurons [157, 179, 190, 191].The toxic effect of prion peptide PrP106-126 has beenreported to require the presence of microglia, whichare activated to release reactive oxygen species.Therefore, PrP106-126 reduces neuronal reistance tooxidative stress [179, 192]. The aforementionedobservations points to a critical role for activatedmicrog l ia as one o f the mechan isms o fneurodegeneration in prion disease. Support of thisnotion is evidenced by the pioneering studies ofMeyermann and associates [193].These authorsconcluded that the actions of activated microgliacontribute largely to the cascade of events that finallyculminate in neurodegeneration in CJD. Now, aspreviously described, a wealth of scientific evidencehas shown that TLRs engage and mediate both theactivation and actions of microglia in CNS [54, 55, 57].In addition to this, TAK 1 ( TGFbeta-activated kinase1), is a member of the MKKK family that is increasinglybeing accepted to play a pivotal role in TLR signaling [194]. TAK 1 ( TGFbeta-activated kinase 1) is acommon upstream kinase that mediates the signalt ransduct ion fo r in f lammatory ce l l s v iamitogen-activation protein kinase(MAPK) andNF-kappaB pathways , after being activated byinflammatory cytokines and engagement of TLRs by

bacterial and viral pathogens. Recent works thatLPS/IFN-gamma induction of gene expression utilizesTAK1 as the major signal molecule in glial cells [195]and Bhat and associates [196] have seriouslyimplicated TAK 1 in the induction of nitric oxidesynthase gene expression in glia cells. These findingsstrongly implicate Toll-like receptor protein familyactivities in modulating the activities of glial cells. Inline with this, the discovery that TLR 2 and TLR4signal through TAK1, and that these two Toll-likereceptors are expressed in astrocytes and microglia invivo and potentially very strongly participate inmediating neuroinflammatory responses to infectionand disease processes [ 54, 197], strongly reinforcesthe notion that TLR-signaling participates in mediatingthe activities of microglia in CNS ,and this suggeststhat TAK1 may represent a potent target fora n t i - i n f l a m m a t o r y s t r a t e g i e s a g a i n s tneuroinflammatory diseases including TSE disease.Futhermore, microglia, which express TLR9, releaseTNF and IL-12 when stimulated with non-methylatedCpG DNA [198]. Chronic gl ia l act ivat ion,neurodegeneration and IL-1beta induction in rat brainhas been reported after administration of dsRNA, theligand of TLR3 [199]. Now, in addition to this,interestingly, the important updates from Olson andMiller [200], most persuasively buttress this notion.These investigators, based on a set of elegant seriesof experiments, demonstrated a critical role formicroglia in the innate immune response to CNSpathogens, leading to the activation of adaptiveimmune functions, in particular, antigene presentingcapacity. The microglia hence represent importantcomponent of both the innate and adaptive immuneresponse. Recent work by Ebert et al. [201] buttressesthis notion. Together and coupled with the fact that theactivation of microglia plays a pivotal in thepathogenesis of TSE disease, I am led to propose thatToll-like receptors should play important role in thepathology of TSE disease.

Toll-like Receptors mediate and regulate OxidativeStress and Apoptotic CascadesA wealth of scientific data has implicated TLRs inmediating and regulating the pathogenic cascades ofoxidative stress damage [ 202, 203, 204, 205, 206,207] , and apoptotic cell death [95, 208, 209, 210, 211]. Apoptosis is an active type of cell death. It differsfrom necrosis in its programmed manner, complexregulatory mechanisms, distintive morphologicalchanges and lack of inflammation [212, 213].Oxidative stress represents the imbalance betweenbiochemical processes leading to production ofreactive oxygen species (ROS) and the cellular



antioxidant cascade. The consequence of thisimbalance causes molecular damage that can lead toa critical failure of biological functions and ultimatelycell death. Hence, in line with the aforementioned,oxidative stress, although largely a secondary event,emerges as an important driving force in thepathogenic cascade of events responsible forsustaining and exacerbation of the disease process ina plethora of chronic degenerative diseases. Thesefindings broadly implicate that toll-like receptoractivities-based therapeutic interventions directed atthe key processes of these two pathogenic factorsmay positively influence the clinical outcome in themanagement of a plethora of pathological condiutionsincluding TSE diseases. To this end, importantly, thereis now strong evidence that oxidative stress [180, 214,215] and apoptotic cascades [180, 216, 217, 218,219]are key pathogenic factors involved in thedevelopment and progressiong of TSE disease, andthat these features contributes strongly in facilitatingthe processes of neurodegeneration in prion diseasebrain. Furthermore, the aforementioned obcervations,indeed, do not seem to argue against the involvementof Toll-like receptors in the pathophysiology andpathogenesis of prion disease.

PrPc Conversion to PrPSc and Lipid RaftsA key feature of prion diseases is the conversion ofthe normal, cellular prion protein, PrPc, into thebeta-sheet –rich disease-associated isoforms ,PrPSc,and the deposition of PrPSc is thought to lead toneurodegeneration [ 9]. The molecular mechanismsunderlying the aforemention processes areincompletely understood. However, a convergence ofscientific evidence strongly suggest that lipid rafts areintimately involved in the process of PrPc conversionto PrPSc [220, 221]. In this light, an important findinghas reported that both PrPc and PrPSc have beenfound in detergent –resistant microdomains (DRMs),also named lipd rafts [222]. Both PrPc and PrPSc aret h u s a s s o c i a s t z e d w i t h D R M s i n acholesterol-dependent. Cholesterol depletion of cellsleads to decreased formation of PrPSc from PrPc. Asreported by Baron et al. [ 220] the conversion of PrPsto PrPSc occurs when microsomes containing arefused with DRMs containing PrP. Lipid rafts have beendefined as liquid-ordered cholesterol and spingolipidrich microdamains within the plasma membrane thatare thought to function as platforms for signaling,internalization, and intracellular trafficking [ 223, 224] .They have been described to play a role in multipleprototypical cascades, such as the lipopolysaccharidepathways , and to host multiple signal proteins,including kinases and low molecular weight G-proteins.Little data currently exist regarding the role of lipid

rafts in LPS involved-signaling within the macrophages.To this end , important updates by Olsson and Sundlerreveal that lipid rafts may play important role in TLRsignaling [225]. These authors investigated thesignificance of lipid rafts in LPS signaling inmachrophages. They found that CD14, and MAPkinases (ERK2 and p38) are involved in lipidrafts-associated LPS-mediated signaling inmacrophages and that they become translocated tolipid rafts after stimulation with LPS, thereby, pointingto an important role for lipid rafts in LPS-inducedTLR4-driven signaling. The findings by Olsson andSundler[225], buttress the earlier report that within theimmune system , lipid rafts play important roles incoordinating and mediating the signaling cascadesemanating from multichain receptor complexes suchas Fc? receptor, T cell and B cell receptors, andpattern recognition receptors such as the TLRs [226,227, 228]. Additionally, emerging evidence indicatesthat Toll-like receptor proteins may represent potentialfactor in regulating cholesterol metabolism [229]. Thissuggests a functional role for these transmembranereceptor proteins in influencing the pathogenesis ofprion disease, for cholesterol depletion has beendemonstrated to influence the conversion of PrPc intoP rPSc [ 230 ] . Fu r t he rmore , P rPc i s aglycosylphosphatidylinositol (GPI) –anchored proteinlocated in lipid rafts or DRMS [231, 232]. Like othermembers of this class, many engulfing receptorsincluding CD36 (a sensor of diacylglycerides:TLR-2ligands [233]),CD44, and CD14 (a prominentc o - r e c e p t o r w i t h T L R 4 , a n d aglycosylphosphatidylinositol (GPI) –anchored protein)are present in lipid rafts [234], and ( GPI)-anchor isnotably a prominent TLR ligand [ 235], coupled withthe fact that GPI-anchors of vasrious parasites havebeen reported to be associated with a role similar tothat of bacterial LPS in the innate immune response,and that despite some structural differences notedamong GPI-anchored proteins, all GPI anchors have acommon core structure [236], I am tempted to suggestthat Toll-like Receptors might have an important role inthe process of PrPc to PrPSc conversion in priondisease pathogenesis. Moreover, the ability ofcholesterol to mudulate the conversion of PrPc toPrPSc combined with ability of TLRs to regulatecholesterol metabolism suggest that Toll-like receptorprotein family may be a fertile avenue of research forthe management of TSE diseases. This hypothesisunderscores the importance and urgency toproactively address this subject..Collectively, the aforementioned considerations are,by all means, in agreement with the accepted value ofscientific evidence, which generally revolves around



the probability and chance. Nevertheless, additionally,in agreement with the elementary lessons in statistics,the probability or chance that all these findings are dueto an indirect pathological effects or due tocoincidental circumstances related to the conundrumsand/or controversies, which prevail in the pathology ofTSEs, seems extremely far-fetched. The sheermagnitude of the insidious nature of TSEs, coupledwith the fact that neither preventive measures norlong-term treatment modalities are available for themanagement of this lesion, underscore the urgentneed to proactively address the above-mentionednotions by researchers, clinical investigators, andindustry.

Conclusion(s)

The recent advances in our understanding of Toll-likereceptor functions in the innate immune responseshed new light on how immunoinflammatoryresponses are initiated and mediated within the centralnervous system.. It has been increasingly recognizedthat Toll-like receptors (TLRs) play a major role ininnate immunity to recognize specific molecularpatterns derived from pathogens including lipids,protein, DNA, RNA, and also recognize endogenousligands and small molecular synthetic compounds.These observations that TLRs are also able to senseendogenous ligands such as heat shock proteins [49],surfacted protein A [240] and hyaluronan [80]buttresses the “danger theory” of immune activation[241, 242], which holds that the induction of immunityrequires stimulation of cells by not only non-selfderived ligands but also the so-called “endogenousdanger signals” constituting factors released by deador dying cells as result of tissue damage, for instance.Now; quite irrespective of the evidence compellinglyimplicating pathogenic microorganisms in driving TSEdisease. For instance, microglial activation andinflammatory mediators are detected in plaques inTSE patients as previously described [179, 180, 182,183], and in Alzheimer’s disease patients [ 243], aswell, in CNS inflammation. It is thus conceivable that“endogenous danger signal” exposed in the damageincurred in the brain during the progression of thedisease, stimulates viable glia cells through TLRs,thereby initiating inflammatory responses.Theseevents could well be extrapolated to the orchestratednetwork of events intimately involved in thepathogenesis of TSE diseases. For instance, heparansulfate, a products of the degradation of heparansulfate proteoglycan, suggested by a plethora of datato be intimately implicated in TSE pathogenesis [ 244,

245,246], can induce the maturation of DCs throughthe TLR4, as well, [79, 80,]. The maturation of DCs isan immunologically important process by which thedendritic cells acquire an ability to present antigensand to induce cellular response efficiently.Furthermore, putting into consideration that DrosophilaToll was originally identified as essential molecule forembryonic patterning in Drosophila [247] subsequentlyshown to be of key importance in antifungal immunityby Lemaitre B. et al. [72], and the Toll-like receptorsare the mammalian homologue of the Drosophila Tollreceptor [ 248], it is conceivable that certain membersof the TLR family expressed on glia cells may beimpl icated in both the physio logical andpathophysiological functions in the brain. To this end,importantly, a huge body of scientific data hascompellingly prion protein in embyronic development [249; 250; 251]. Therefore, it seems very difficult, if notalmost impossible, to exclude the involvement of TLRreceptors in the development and resolution ofpathology in neuropathological conditions includingthat of TSE diseases.The ability of TLR-activated APC to activate CD4+ Tcells and shape a TH 1-associated immune responsehas been well described elsewhere [252; 253]. In thisrespect, as previously described, TLRs activatemultiple steps in the inflammatory reactions toeliminate the invading pathogens and subsequentlycoordinate the systemic defenses, which involves themodulation of multiple dendritic cell functions and theactivation of signals that are of critical importance ininitiation of adaptive immune responses, unequivocallypointing to a protective function for the innate immunesystem. On the other hands, when the innate immunesystem, or a component of the innate immune system,is inappropriately stimulated or activated, the immuneresponses driven by this inappropriate activation maylead to severe consequences. In this respect, forinstance, excessive TLR signaling caused by microbialinfection and/or other challenges can lead todetrimental inflammation, tissue /cell damage, andoccasionally to death. Now, CpG-containingoligodeoxynucleotides (CpG-ODN) are known asstrong stimulators of innate immunity by mimicking theeffects of bacterial DNA. Thus, CpG-ODNs, aspreviously described, are recognized by Toll-likereceptor 9 (TLR9). They can act as immune adjuvants,accelerating and boosting antigen-specific antibodyresponses by up to 500-fold. CpG motifs promote theproduction of T-helper 1 and pro-inflammatorycytokines and induce the maturation/activation ofprofessional antigen-presenting cells includingmacrophage and dendritic cells. The therapeuticimportance of these products are now being studied,



and tested in a wide range of pathological conditions[254; 255; 256; 257; 258] including prion disease.Importantly, employloyment of this agent in themanagement of TSE disease by Hans Kretzschmarand co-workers [154] led to a protective effect on miceinfected with scrapie agent, consistent with thewell-established ability of the innate immune system toconfer protection to cells. Interestingly, further studiescarried out to replicate the efficacy CpG-ODN on theprion disease [259] in a different laboratory byHeikenwalder and associates surprisingly yieldeddisastrous results. These observations [ 259], however,may not be easy to explain, for very few studies arepresently available on this subject with regard to priondiseases. Nevertheless, CpG-ODN employment aswas documented by Heikenwalder and associates[259] apparently led to negative clinical outcome,because, very probably, the immune system in thebrain of the concerned TSE-infected models musthave been already over- stimulated. Therefore,judging from the results obtained by concernedcolleagues [ 259], it might be reasonable to concludethat repeated application of the concerned agent to theinfected models, whereby the infectious dosis,apparently, must not have been necessarily lowenough, could almost impossibly lead to a positiveresult. Prion protein has now been realized to have theability to induce the production of antibodies [98, 113]and autoantibodies [260], and the presence of PrPSc(or PrPres), can lead to activation of the complementsystem as well. In this respect, PrPres can activate thecomplement system, leading to production ofmembrane attack complex (MAC) among othercomplement proteins [41]. Now, as it can be the caseunder pathological condition, the complement systemcould be inappropriately stimulated in the process ofprion disease progression, and over-stimulation of thecomplement leading to sustained inappropriateproduction of the membrane attack complex (MAC) inTSE pathogenesis. This, in turn, can initiate andsustain the process of autodestruction of neuronalcells and viable tissues, by host defense system, aspreviously described in the cases of Alzheimer`sdisease and Amyotrophic lateral sclerosis [141, 142],and, very recently, in the case of CJD by Budka andassociates [41]. Furthermore, the mere fact that thepresence of PrPres could result to production ofautoantibodies; that the structure of microglia (thepotent professional APCs in the CNS) could beimpaired in TSEs pathogenesis, leading to dysfunctionof APCs; and that the homeostasis of theglutamatergic system could be significantly perturbedin TSE pathogenesis [261]; all these events possessthe ability to elicit production of autoantibodies in the

brain, which could invariably lead to a situation, where“Horror autotoxicus” becomes commonplace. Thesenotions, therefore, strongly indicate that administrationof CpG-ODN, under certain conditions, could alsoresult or lead to premature hyperimmunity ofautoimmunity, Horror autotoxicus = autoimmunereaction.”Horror autotoxicus,” a Latin expression, is aterm coined by Paul Ehrlich at the turn of the lastcentury to describe autoimmunity to self, or the attackof “self” by immune system, which ultimately results toautoimmune condition. For instance, dysfunction ofglutamatergic system could lead to over-expression ofglutamate receptors, which can in turn result toproduction of physiologically active autoantibodies[262]. These autoantibodies could be directed towardsexcitatory ionotropic glutamate receptors in the brain.Hence, it is well-established that appropriateemployment of an agent (pharmaceutical agent), inmost cases, result to amelioration of the concerneddisease state or condition with minimal side-effect.The aforementioned consideration seems to beconsistent with appropriate employment of CpG-ODNagents. Support for this notion is evidenced by thework of Zimmermann et al. [263] and that of Schetterand associaste [198]. Additionally, and importantly,this notion has been very recently stengthened in thecase of prion disease by Kretzschmar and co-workers[154], who demonstrated positive clinical outcome as aresult of CpG-ODN agent employment. The aboveobservations further strengthen the concept that innateimmunity may be an important player in TSEpathology, and that TLRs are crucial receptors for theactivation of innate immune mechanisms, andtherefore; points to a critical role for TLRs in thepathology and therapy of a wide range of diseasesincluding prion diseases. Therefore, essentially theabove notions strongly argue that the previous report[259] might not necessarily represent a negation of thewell-established protective properties of the innateimmune system, nor the efficacy of CpG-ODN in themanagement of diseases including TSE diseases.Nevertheless, it is of importance to point out thatstandard conditions for effective administration ofCpG- ODN as therapeutic agent have yet to be fullydetermined. The aforementioned conflictingobservations underscore urgent need for increasedstudies in this direction. To this end as recentlysuggested in a closely -related case by McGeer andMcGeer [264], such studies could lead to less severeconsequences, if two major conditions are put intoconsideration,:1) if the problems of autoimmunereaction were substantially and generally put undercontrol, 2) if appropriate measures are taken tosubstantially or almost completely prevent specifically



the over-reactivity of the complement. Moreover,importantly, the recent discovery disclosing thatconcerted actions of tumor necrosis factor receptor 1(TNFR1)-and TLR are may imply that antagonists ofTNFR1-and TLR , as well as inhibitors of theirintracellular signaling pathways might be effectiveanti-neuroinvasion agents for the management of aplethora of neurological diseases, where the processof neuroinvasion plays a central role in the diseaseprocess. Whilst neuroinvasion is undoubtedly acomplex process that may include other mechanisms,these findings by Wang et al. [174] provide importantinsights into the signaling pathways and processesthat result in neuroinvasion. It should, however, not beruled out, that these observations [174] may bearenormous relevance to TSE pathology, and may holdgood promise for novel therapies for a plethora ofdevastating neurodegenerative diseases includingTSE diseases. It is reasonable to speculate that moreTLR proteins other than TLR3 may be involved or mayhave the capacity to mediate this particular process.Therefore, the mere fact that there are currently nopreventive measures nor long-term treatmentmodalities for TSEs, underscores the urgent need forfurther investigations into this particular issue.Human Toll-like receptors are transmembranesignal-transducing receptor proteins with anextracellular leucine-rich repeat domain andintracellular domain homologous to the IL-receptor.TLRs act as key receptors responsible not only for thedetection of a variety of micobial cell-wall componentsand bacterial DNA, ds-RNA, endogenous m-RNA,endogeneous proteinaceous molecules, extracellularmatrix breakdown particules, but also for the initiationand mediation of signal transduction events eventuallyleading to the production of various pro- inflammatorymediators. There is no longer any doubt that TLRs arecapable of sensing microbial organisms ranging fromprotozoa [235] to bacteria, fungus and viruses.Therefore; the above observations strongly strengthenthe notion that Toll-like receptors may play animportant role in the development and resolution ofTSE disease pathology. To our knowledge, this is thefirst work compellingly arguing in favor of theinvolvement of TLR activities in the development andresolution of TSE pathology. The message is simple;the innate immune system is a complex body. Whatstarted originally as the study of fruit flies andcaterpillars has become the basis of our hopes for newcures for diseases as lethal as cerebral malaria,sepsis, and systemic lupus erythematosus, andpossibly, Creutzfeldt-Jakob diseases, as well. Hence,enhanced understanding of the Toll-like receptorbiology , the nature of their various ligands, and the

molecular underpinnings responsible for theirbiochemical effects will allow a model to beconstructed in which the type of immune response toany type of infection associated with prion disease, forinstance, could be viewed as a function of manydifferent determinants, including the transmembraneprotein, TLRs, the form and the nature of the microbeand the cytokine microenvironments. Knowledgegained from these studies, could be employed intargeting appropriately the pathways associated withTLR signal-transducing activities for either inhibition oraugmentation, thereby opening novel avenues infuture approaches to TSE disease therapy and othervarious pathological conditions.

Acknowledgement(s)

The author; Dr. Ofodile, is exceedingly grateful toObiamaka, Ekene and Ifeanyi for their love, support,and understanding, and for the immense strength,which these brave and wonderful Girls gave to him(their Father) in the last nine years, without which itcould not have been possible for him to successfullycomplete his investigations and bring forward thismanuscript.The author, also, greatly acknowledged immensecollegial assistance given to him by Franz Theuring,PhD. during the preparation of this manuscript.

Authors Contribution(s)

OKOM NKILI-BALONWU F.C.OFODILE, PhD.CENTER FOR CARDIOVASCULAR RESEARCH(CCR), INSTITUTE OF PHARMACOLOGY andTOXICOLOGYAG: THEURING, CHARITE-UNIVERSITÄTSMEDIZINBERLINHESSISCHE STR. 3-4, 10115 BERLIN, GERMANY

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