The basic pathomechanism in both disorders is related to a conformational change of normally expressed proteins: amyloid-b Ab in AD and the prion protein PrP in Keywords Alzheimer’s dise
Trang 1Therapeutic approaches for prion and Alzheimer’s diseases Thomas Wisniewski1,2,3and Einar M Sigurdsson2,3
1 Department of Neurology, New York University School of Medicine, NY, USA
2 Department of Pathology, New York University School of Medicine, NY, USA
3 Department of Psychiatry, New York University School of Medicine, NY, USA
Introduction
Alzheimer’s disease (AD) and prion disease belong to
a category of conformational disorders showing
sub-stantial overlap in pathologic mechanism [1–3] The basic pathomechanism in both disorders is related to a conformational change of normally expressed proteins: amyloid-b (Ab) in AD and the prion protein (PrP) in
Keywords
Alzheimer’s disease; metals; mucosal
vaccination; prion; vaccine
Correspondence
T Wisniewski, New York University School
of Medicine, Departments of Neurology,
Psychiatry and Pathology, Millhauser
Laboratories, Room HN419, 560 First
Avenue, New York, NY 10016, USA
Fax: +1 212 263 7528
Tel: +1 212 263 7993
E-mail: thomas.wisniewski@med.nyu.edu
(Received 9 March 2007, revised 3 May
2007, accepted 4 May 2007)
doi:10.1111/j.1742-4658.2007.05919.x
Alzheimer’s and prion diseases belong to a category of conformational neu-rodegenerative disorders [Prusiner SB (2001) N Eng J Med 344, 1516–1526; Sadowski M & Wisniewski T (2007) Curr Pharm Des 13, 1943–1954; Beekes M (2007) FEBS J 274, 575] Treatments capable of arresting or at least effectively modifying the course of disease do not yet exist for either one of these diseases Alzheimer’s disease is the major cause of dementia in the elderly and has become an ever greater problem with the aging of Western societies Unlike Alzheimer’s disease, prion diseases are relatively rare Each year only approximately 300 people in the USA and approxi-mately 100 people in the UK succumb to various forms of prion diseases [Beekes M (2007) FEBS J 274, 575; Sigurdsson EM & Wisniewski T (2005) Exp Rev Vaccines 4, 607–610] Nevertheless, these disorders have received great scientific and public interest due to the fact that they can be transmis-sible among humans and in certain conditions from animals to humans The emergence of variant Creutzfeld–Jakob disease demonstrated the trans-missibility of the bovine spongiform encephalopathy to humans [Beekes M (2007) FEBS J 274, 575] Therefore, the spread of bovine spongiform encephalopathy across Europe and the recently identified cases in North America have put a large human population at risk of prion infection It is estimated that at least several thousand Britons are asymptomatic carriers
of prion infections and may develop variant Creutzfeld–Jakob disease in the future [Hilton DA (2006) J Pathol 208, 134–141] This delayed emer-gence of human cases following the near elimination of bovine spongiform encephalopathy in the UK may occur because prion disease have a very prolonged incubation period, ranging from months to decades, which depends on the amount of inoculum, the route of infection and the genetic predisposition of the infected subject [Hilton DA (2006) J Pathol 208, 134– 141] Therefore, there is a great need for effective therapies for both Alzhei-mer’s disease and prion diseases
Abbreviations
ACT, a1-antichymotrypsin; AD, Alzheimer’s disease; Ab, amyloid-b; apoE, apolipoprotein E; BBB, blood–brain barrier; BSE, bovine
spongiform encephalopathy; CAA, congophilic amyloid angiopathy; CNS, central nervous system; CWD, chronic wasting disease;
DC, dendritic cell; GSSS, Gerstmann–Stra¨usler–Scheinker syndrome; PrP, prion protein; sAb, soluble Ab; sCJD, sporadic CJD;
Tg, transgenic; vCJD, variant Creutzfeld–Jakob disease.
Trang 2prion disease (Fig 1) [4,5] This occurs without an
alteration in the amino-acid sequence of the proteins
Ab is a 40–43 amino acid peptide, which, in AD,
self-assembles into toxic oligomers and fibrils that
accumu-late in the brain, forming plaques and deposits in the
walls of meningocephalic vessels [6,7] The same
pep-tide can be detected in most physiological fluids, such
as serum or cerebrospinal fluid, where it is called
sol-uble Ab (sAb) [7] PrPC (C-cellular) is a 209 amino
acid, cell membrane anchored protein expressed at
highest levels by neurons and follicular dendritic cells
of the immune system In the setting of prion disease,
this protein undergoes a transformation to toxic PrPSc
(Sc-scrapie) [8–10] Fibrillar Ab and PrPSchave a high
b-sheet content which renders them insoluble, resistant
to proteolytic degradation and toxic to neurons
Neu-rological symptoms in AD and prion disease are
directly related to loss of neurons and synaptic
connec-tions Oligomeric and fibrillar Ab can be directly
neurotoxic and⁄ or can promote formation of
neuro-fibrillary tangles [7] Both fibrillar Ab and PrPSc are
capable of forming amyloid deposits The presence of
amyloid deposits is necessary for making the diagnosis
of AD [11,12] Abundant amyloid deposits composed
of PrPSc (full length or fragments) are a neuropatho-logical hallmark of variant Creutzfeld–Jakob disease (vCJD), Gerstmann–Stra¨usler–Scheinker syndrome (GSS), and kuru [13] They are also present in 10% of sporadic CJD (sCJD) cases [9]
A number of proteins may actively promote the con-formational transformation of these disease specific pro-teins and stabilize their abnormal structure Examples
of such proteins in AD include apolipoprotein E (apoE), especially its E4 isoform [13,14], a1-antichymotrypsin (ACT) [15] or C1q complement factor [16,17] (Fig 1)
In their presence, the formation of Ab fibrils in a solution of sAb is much more efficient [13,15] These
‘pathological chaperone’ proteins have been found histologically and biochemically in association with fibrillar Ab deposits [18] but not in preamyloid aggre-gates, which are not associated with neuronal loss [19] Similarly, in prion disease, extensive data points toward the existence of an unidentified protein X actively involved in the conversion of PrPCinto PrPSc[20]
AD and prion diseases exist as sporadic and inher-ited illnesses In addition, prion disease can be trans-mitted from one subject to another In experimental model settings, some evidence also exists for the infec-tivity of AD [21,22] An important event in the patho-mechanism of AD is thought to be reaching a critical concentration of sAb and⁄ or chaperone proteins in the brain, at which point the conformational change occurs [23] This leads to the formation of Ab aggre-gates, initiating a neurodegenerative cascade In sporadic AD, this occurs due to an age-associated overproduction of Ab, impaired clearance from the brain, and⁄ or influx into the central nervous system (CNS) of sAb circulating in the serum [24] Inherited forms of AD are associated with various genetic defects, resulting in overproduction of total sAb, or more fibrillogenic Ab 1–42 species [25]
Sporadic prionoses like sCJD are thought to result from the spontaneous conversion of PrPC into PrPSc [26] The mechanisms that stabilize PrPCstructure are largely unknown but, once PrPSc assumes its patholo-gical conformation, it can bind to PrPC and induce a conformation change This starts a self-perpetuating vicious cycle allowing PrPScto replicate without DNA, using the host cell’s PrPC as a template [9,26] Most inherited prionoses such as GSS or inherited forms of CJD are the result of a point mutation in PrPC that increases the propensity for it to assume an abnormal conformation Virtually all genetic defects implicated
in familial forms of AD and prionoses are inherited in
an autosomal dominant fashion Unlike AD, prionoses can be easily transmitted between subjects of the same
Protofibrils Fibrils Increased
Aggregated
Toxic
CONFORMATIONAL DISORDERS
Mainly
Random Coil
Monomers
Non-Toxic
Alzheimer’s Disease
A β Plaque Neurofibrillary
Tangle
Prionoses
Neuronal loss Spongiform changes
Pathological Chaperones
Metals
Fig 1 Conversion of sAb peptide or PrP C to their pathological
b-sheet conformers is a key step in the pathogenesis of AD and
pri-onoses, respectively In AD, these b-sheet rich structures consist
of oligomers, protofibrils and fibrils that form plaques within the
brain parenchyma or deposit in the cerebrovasculature A
compar-able entity in prion diseases consists of the proteinase K resistant
scrapie form of the prion protein (PrP Sc ) that, in certain prion
dis-eases, fibrillizes and deposits as plaques within the brain This
pro-cess is facilitated by various pathological chaperones as well as
several metals The aim of most therapeutic interventions for these
conformational disorders is to reduce the amount of the substrate
(sAb, PrP C ) and ⁄ or its availability for this structural alteration;
interfere with the conversion either directly or indirectly (via the
pathological chaperones or metals); and promote removal of the
disease-associated conformers.
Trang 3species Transmissibility of prionoses between different
species is generally ineffective due to differences in the
PrP sequence The phenomenon protecting one species
from acquiring a prion disease from another is called
‘the species barrier’ Therefore, scrapie (a prionosis
affecting sheep) is not transmissible to humans The
species barrier does not provide absolute protection;
therefore, transmission of scrapie to cattle and
trans-mission of bovine spongiform encephalopathy (BSE)
from cattle to humans results in the emergence of
vCJD In transmissible prionoses, exogenous PrPSc
present in the inoculum is responsible for the
conform-ational transformation of host PrPC Upon entering an
organism, PrPSc initially replicates within the
lym-phoreticular organs, including the spleen, lymph nodes
and tonsils, for months to years prior to neuroinvasion
and the onset of neurological symptoms Therefore,
infected but asymptomatic individuals are a reservoir
of infectious material This occurs because PrPC is
expressed by follicular dendritic cells and other
lym-phoid cells [27] Accumulation of PrPScin the
lympha-tic organs of presymptomalympha-tic humans infected with
BSE has been demonstrated by immunohistochemistry
[28] PrPSc replication is possible because it does not
elicit an immune response [29] This is related to the
inability of the immune system to distinguish between
PrPCand PrPSc
Vaccination approaches for AD
Vaccination was the first treatment approach
demon-strated to have genuine impact on disease process, at
least in animal models of AD Vaccination of AD
transgenic (Tg) mice with Ab1–42 or Ab homologous
peptides coinjected with Freund’s adjuvant prevented
the formation of Ab deposition and, as a consequence,
eliminated the behavioral impairments that are related
to Ab deposition [30–35] Similar effects on Ab load
and behavior have been demonstrated in AD Tg mice
by peripheral injections of anti-Ab monoclonal serum
indicating that the therapeutic effect of the vaccine
is based primarily on eliciting a humoral response
[36,37] The striking biological effect of the vaccine in
preclinical testing and the apparent lack of side-effects
in AD Tg mice encouraged Elan⁄ Wyeth to launch
clin-ical trials with a vaccine designated as AN1792 which
contained preaggregated Ab1–42 and QS21 as an
adju-vant This type of vaccine design was aimed to induce
a strong cell-mediated immune response because QS21
is known to be a strong inducer of Th-1 lymphocytes
[38] The initial safety testing of AN1792 in phase I of
the trial did not demonstrate any adverse effects The
phase II of the trial was prematurely terminated when
6% of vaccinated patients manifested symptoms of acute meningoencephalitis [38,39] An autopsy per-formed on one of the affected patients revealed an extensive cytotoxic T-cell reaction surrounding some cerebral vessels; however, analysis of the Ab load in the brain cortex suggested that Ab clearance had occurred [40] It appeared that the immune reaction triggered by AN1792 was a double-edge sword, where the benefits of a humoral response against Ab were overshadowed in some individuals by uncontrolled cytotoxicity [41] Not all patients who received AN1792 responded with antibody production The majority mounted a humoral response and showed a modest but statistically significant cognitive benefit demonstrated as an improvement on some cognitive testing scales compared to baseline and a slowed rate
of disease progression compared to patients who did not form antibodies [42] The follow-up data from the
‘Zurich’s cohort’, who are a subset of the Elan⁄ Wyeth trial followed by Dr Nitsch’s group [42,43], indicated that the vaccination approach may be beneficial for human AD patients but that the concept of the vaccine has to be redesigned
It appears that a humoral response elicited by the vaccine has at least two mechanisms of action and both
of these are thought to be involved in amyloid clearance [44,45] Conformational selective anti-Ab serum may target Ab deposits in the brain [43] leading to their disassembly [46,47] and elicit Fc mediated phagocytosis
by microglia cells The second mechanism by which anti-Ab serum likely prevents Ab deposition is the cre-ation of a ‘peripheral sink’ effect, where the removal of excess sAb circulating in the blood stream leads to sAb being drawn out from the brain [31,34, 47,48] This per-ipheral sink mechanism is likely to be the dominant means of reducing Ab peptides in the brain
The cause(s) for the toxicity in 6% of the Elan trial patients are not entirely known; however, from the available clinical and limited autopsy data, it is thought that an excessive Th-1 cell-mediated response within the brain was to blame [49] The concept of a redesigned AD vaccine puts emphasis on avoiding this cell-mediated response in the following ways: (a) avoiding stimulation of Th-1 lymphocytes so the vaccine could potentially elicit a purely humoral res-ponse; (b) using nontoxic and nonfibrillogenic Ab homologous peptides, so that the immunogen can not produce any direct toxicity; and (c) enhancing the peripheral sink effect rather than central action Passive transfer of exogenous anti-Ab monoclonal serum appears to be the easiest way to fulfill the goal of providing anti-Ab serum without risk of uncontrolled Th-1 mediated autoimmunity AD Tg model mice
Trang 4treated this way had a significantly reduced Ab level and
demonstrated cognitive benefit [36,37] The major
draw-backs of this approach are the high cost, limited half-life
of monoclonal antibodies (2–21 days depending on class
and isoform) and the potential for inducing serum
sick-ness with resultant complications such as renal failure
or lymphomas Nevertheless, clinical trials for passive
immunization trials are underway Alternative
approa-ches for passive immunization which are less likely to be
associated with toxicity, are use of Fv fragments or
mimetics of the active antibody binding site
Another potential source of toxicity in association
with passive immunization is cerebral hemorrhage The
mechanism of this hemorrhage is thought to be
inflam-mation in association with cerebral amyloid deposits
(congophilic amyloid angiopathy; CAA) that weakens
the blood vessel wall Several reports have shown an
increase in microhemorrhages in different AD mouse
models following passive intraperitoneal immunization
with different monoclonal antibodies with high affinity
for Ab plaques and CAA [50–52] The risk of
micro-hemorrhage following active immunization in animal
models has not been fully assessed It has not been
a problem in our own active immunization studies
[34,35], but has been reported in one study [53]
Furthermore, the clinical trial data from the limited
number of autopsied cases suggests that vascular
amyloid was not being cleared and that hemorrhage
may have been increased [54–56] In one of these
autopsies, numerous cortical bleeds, which are
typically rare in AD patients, were evident [55] In
addition, the association of T lymphocytosis and
cuffing with the cerebral vessel Ab in these autopsies
suggests a potential role of CAA and an excessive
Th-1 response in the genesis of the inflammatory
side-effects [57] This is an important issue because CAA is
present in virtually all AD cases, with approximately
20% of AD patients having ‘severe’ CAA [58]
Furthermore CAA is present in approximately 33% of
cognitively normal elderly, control populations [59–61]
Understanding the antigenic profile of Ab peptide,
allows engineering of modifications that favor a
humoral response and reduce the potential for a Th-1
mediated response This approach has been termed
altered peptide ligands Computer models have
predic-ted that Ab1–42 has one major antibody binding site
located on its N-terminus and two major T-cell epitopes
located at the central and C-terminal hydrophobic
regions encompassing residues 17–21 and 29–42,
respectively [62–64] Therefore, their elimination or
modification provides a double gain by eliminating
tox-icity, as well as the potential for T-cell stimulation
Sigurdsson et al [34] immunized AD Tg mice with
K6Ab1–30[E18E19], a nontoxic Ab-homologous peptide, where the first above mentioned T-cell epitope was modified and the second removed Polyamino acid chains coupled to its N-terminus aimed to increase the immunogenicity and solubility of the peptide AD Tg mice vaccinated with this peptide produced mainly an IgM class antibodies and low or absent IgG titer These animals showed behavioral improvement and a partial reduction of Ab deposits [34,35] One of the advantages
of this design is that IgM, with a molecular mass of
900 kDa, does not penetrate the blood–brain barrier (BBB) and therefore is unlikely to be associated with any immune reaction in the brain Like passive immun-ization, this type of vaccine focuses its mechanism of action on the peripheral sink Furthermore, the IgM response is reversible because it is T-cell independent; hence memory T-cells that could maintain the immune response are not generated Therefore, this vaccine method may potentially be safer than typical active immunization
Mucosal vaccination can be an alternative way to achieve a primarily humoral response This mechanism
is based on the presence of lymphocytes in the mucosa
of the nasal cavity and of the gastrointestinal tract This type of response produces primarily S-IgA antibodies but, when the antigen is coadministrated with adjuvants such as cholera toxin subunit B or heat-labile Escheri-chia coli enterotoxin, significant IgG titer in the serum may be achieved [65,66] A marked reduction of Ab bur-den in AD Tg mice immunized this way using Ab as an antigen has been already demonstrated [66,67] Interest-ingly, this type of mucosal immunization has recently been shown to be highly effective for prion infection [68,69,70] This promising approach requires further exploration, especially using nonfibrillar and nontoxic
Ab homologous peptides as an antigen Mucosal immunization offers a great potential advantage in that
a more limited humoral immune response can be obtained, with little or no cell-mediated immunity
Inhibition of Ab fibrillization Formation of Ab fibrils and deposition of Ab in the brain parenchyma or in the brain’s vessels occurs in the setting of increased local Ab peptide concentra-tions [71] Initially, condiconcentra-tions do not favor aggrega-tion of fibrils; however, once a critical nucleus has been formed, aggregation with fast kinetics is favored Any available monomer can then become entrapped in
an aggregate or fibril Several compounds, such as Congo red [71], anthracycline [73], rifampicin [74], anionic sulphonates [75], or melatonin [76], can inter-act with Ab and prevent its aggregation into fibrils
Trang 5in vitro, thereby reducing toxicity It has been further
identified that certain nonfibrillogenic, Ab homologous
peptides can bind to Ab and break the formation of
b-sheet structure [77–80] Therefore, these peptides
were termed b-sheet breakers Several modifications
were used to extend serum half-life and increase BBB
permeability of these peptides Permanne et al [81],
using a BBB permeable five amino-acid long peptide
(iAb5), were able to demonstrate a reduction of Ab
load in AD Tg mice that received this peptide compared
with age-matched control group which received placebo
Of interest, a similar concept of b-sheet breakers has
been shown to be applicable to prion disease [82]
Extensive evidence suggests that the most toxic forms
of Ab are oligomeric aggregates [83] There is also
evi-dence implicating oligomeric aggregates in the
medi-ation of PrPSctoxicity and infectivity [84,85] Recently,
compounds and antibodies have been developed that
specifically target Ab oligomers [86–88] Similar
approa-ches are being developed for prion oligomers
Ab homologous peptides can aggregate and form
fibrils spontaneously in vitro; however, in vivo this
pro-cess appears more dependant on the presence of Ab
pathological chaperones This group of proteins
promotes conformational transformation at certain
concentrations by increasing the b-sheet content of
these disease specific proteins and stabilizes their
abnormal structure [89,90] Examples of such proteins
in AD include apoE, especially its E4 isoform [18,91],
ACT [20] or C1q complement factor [21,22] In
their presence, the formation of Ab fibrils in a solution
of sAb monomers becomes much more efficient
[18,20] These ‘pathological chaperone’ proteins have
been found histologically and biochemically in
associ-ation with fibrillar Ab deposits [23,89,92,93] but not in
preamyloid aggregates that are not associated with
neuronal toxicity [24,94] Inheritance of the apoE4
isoform has been identified as the major identified
genetic risk factor for sporadic, late-onset AD [95] and
correlates with an earlier age of onset and greater Ab
deposition, in an allele-dose-dependent manner
[19,95,96] In vitro, all apoE isoforms can propagate
the b-sheet content of Ab peptides promoting fibril
formation [92], with apoE4 being the most efficient
[18] The critical dependence of Ab deposition in
plaques on the presence of apoE has also been
confirmed in AD Tg APPV717F⁄ apoE– ⁄ –
mice which have a delayed onset of Ab deposition, a reduced Ab
load, and no fibrillar Ab deposits Compared to
APPV717F⁄ apoE+⁄ +Tg mice, APPV717F⁄ apoE+⁄ – mice
demonstrate an intermediate level of pathology
[97–100] Neutralization of the chaperoning effect of
apoE would therefore potentially have a mitigating
effect on Ab accumulation ApoE hydrophobically binds to the 12–28 amino acid sequence of Ab, form-ing SDS insoluble complexes [101–103] Ma et al [104] have demonstrated that a synthetic peptide homolog-ous to 12–28 amino-acid sequence of Ab can be used
as a competitive inhibitor of the binding of full length
Ab to apoE, resulting in reduced fibril formation
in vitro and increased survival of cultured neurons The introduction of several modifications to Ab12–28
by replacing a valine for proline in position 18, making this peptide nontoxic and nonfibrillogenic, as well as end-protection by amidation and and acetylation of the C- and N-termini, respectively, to increase serum half-life, have allowed us to use this peptide therapeu-tically in the APPK670N⁄ M671L⁄ PS1M146L double Tg mice model Tg mice treated with Ab12–28P for
1 month demonstrated a 63.3% reduction in Ab load
in the cortex (P¼ 0.0043) and a 59.5% (P ¼ 0.0087) reduction in the hippocampus comparing to age-matched control Tg mice that received placebo [105,106] The treated Tg mice also had a cognitive benefit [105,106] No antibodies against Ab were detec-ted in sera of treadetec-ted mice; therefore, the observed therapeutic effect of Ab12–28P cannot be attributed to
an antibody clearance response This experiment demonstrates that compounds blocking the interaction between Ab and its pathological chaperones may be beneficial for treatment of Ab accumulation in AD [14,105,106] Whether similar approaches can be used for prion disease remains to be determined
Prion disease Interest in prion disease has greatly increased subse-quent to the emergence of BSE in England and the resulting appearance of vCJD in human populations BSE arose from the feeding of cattle with prion con-taminated meat and bone meal products, whereas vCJD developed following entry of BSE into the human food chain [107,108] Since the original report
in 1995, a total of 201 probable or confirmed cases of vCJD have been diagnosed, 165 in Great Britain, 21 in France, four in Ireland, three in the USA, two in the Netherlands and one each in Italy, Canada, Japan, Saudi Arabia, Portugal and Spain Most of the patients from these countries resided in the UK during
a key exposure period of the UK population to the BSE agent It has proven difficult to predict the expec-ted future numbers of vCJD Mathematical analysis has given a range from 1000 to approximately 136 000 individuals who will eventually develop the disease This broad range reflects a lack of knowledge regard-ing the time of incubation and the number of patients
Trang 6who could be infected from a given dosage of BSE
agent Because the vCJD agent is present at high levels
in the lymphatic tissue, screening for PrPSc was
per-formed on sections from lymph nodes, tonsils, and
appendices archives in the UK Three out of 12 674
randomly selected cases showed evidence of subclinical
infection, leading to a prediction that approximately
4000 vCJD further cases may occur in the UK [109]
However, there is much uncertainty about such a
pre-diction because it is not known whether all subclinical
infections will progress and also whether such
screen-ing of lymphoid tissue would capture all subclinical
cases The initially predicted epidemic of vCJD does
not seem to be materializing because the number of
cases in the UK has declined from a peak of 28 in
2000 to five cases in 2006 [107] A complicating factor
for estimating future numbers of vCJD is the
docu-mentation of several transfusion associated cases
These occurred after incubation periods of 6–8 years
One of these disease associated donations was made
more than 3 years before the donor became
sympto-matic, suggesting that vCJD can be transmitted from
silently infected individuals [110] The estimated risk
for new cases of vCJD in other European countries
looks more optimistic In the UK, 200 000 cases of
BSE were reported (it is estimated that four times this
number entered the food chain), compared to
approxi-mately 5600 BSE cases in other European countries
(with the highest numbers being 1590, 1030 and 986 in
Ireland, Portugal and Frances, respectively) This
sug-gests a significantly lower exposure of these
popula-tions to BSE prions A few cases of BSE have also
been reported in other parts of the world, such as
Japan, the USA and Canada
Of greater concern in North America is chronic
wast-ing disease (CWD) This disease is now endemic in
Colorado, Wyoming and Nebraska and continues to
spread to other parts in the USA, initially in the
Mid-west but now detected as far East as New York State
[111,112] Most vulnerable to CWD infection are white
tailed deer and the disease is now found in areas with a
large population of these animals, which indicates that
its prevalence can be expected to increase substantially
in the future The occurrence of CJD among three
young deer hunters from this same region raised the
spe-culation of transmission of the CWD to humans [113]
However, autopsy of these three subjects did not reveal
the extensive amyloidosis characteristic of vCJD and
CWD [114] However like BSE, CWD is transmissible
to nonhuman primates and transgenic mice expressing
human PrPC[115,116] Therefore, the possibility of such
transmission needs to be closely monitored CWD is
similar to BSE in that the peripheral titers of the prion
agent are high PrPSchas been detected in both muscle and saliva of CWD infected deer [117,118]
Vaccination as a therapeutic approach for prionoses
The prion protein is a self-antigen; hence, prion infec-tion is not known to elicit a classical immune response
In fact, the immune system is involved in the peri-pheral replication of the prion agent and its ultimate access to the CNS [29,68] This involvement is further supported by the observation that immune suppression with, for example, splenectomy or immunosuppressive drugs, increases the incubation period This interval, during which time the prion agent replicates peripher-ally, without producing any symptoms, is quite long, lasting many months in experimental animals and up
to 56 years in documented human cases associated with cannibalistic exposure to the prion agent [119] Lymphatic organs such as the spleen, tonsils, lymph nodes or gut associated lymphoid tissue contain high concentrations of PrPSc long before PrPSc replication starts in the brain [27,120,121] Cells found to be par-ticularly important for peripheral PrPScreplication are the follicular dendritic cells (DC) and the migratory bone-marrow derived DC [121,122] DC from infected animals are capable of spreading the disease [122] An emerging therapeutic approach for prion infection is immunomodulation [68,70,123]
Currently, there is no treatment that would arrest and⁄ or reverse progression of prion disease in non-experimental settings, although many approaches have been tried [124] Partly due to the success in AD models discussed above, similar experiments with anti-PrP serum were initiated in prion infectivity cul-ture models as well as active and passive immuniza-tion studies in rodent models Earlier in vivo studies showed that infection with a slow strain of PrPSc blocked expression of a more virulent fast strain of PrP, mimicking vaccination with a live attenuated organism [125] In tissue culture studies, anti-PrP serum and antigen binding fragments directed against PrP were shown to inhibit prion replication [126–128] Although we first demonstrated that active immunization with recombinant PrP delayed the onset
of prion disease in wild-type mice, the therapeutic effect was relatively modest and, eventually, all the mice succumbed to the disease [129] This limited therapeutic effect may be explained by the observa-tion that antibodies generated against prokaryotic PrP often do not have a high affinity towards PrPC [130], although, in our studies, the increase in the incubation period correlated well with the antibody
Trang 7titers against PrPC Our follow-up passive anti-PrP
immunization study confirmed the importance of the
humoral response, showing that anti-PrP serum is
able to prolong the incubation period [131]
Subse-quently, other investigators, using a much higher
anti-body dosage, were able to completely prevent disease
onset in mice exposed to PrPSc provided that passive
immunization was initiated within 1 month of
expo-sure [132] This type of approach could be used
immediately following accidental exposure in humans
to prevent future infection However, passive
immun-ization has not been found to be effective closer to
the clinically symptomatic stages of prion infection
Also, passive immunization would be an approach
that is too costly for animal prion diseases
In the development of immunotherapeutic
approa-ches targeting a self-antigen, designing a vaccine
avoid-ing auto-immune related toxicity is a major concern
The emerging data from AD targeting immunization is
that toxicity is due to excessive cell-mediated immunity
within the CNS, whereas the therapeutic response is
linked to humoral immunity In addition, toxicity
could be partially related to the immunogen and⁄ or to
the adjuvant used; in the human AD vaccination trial,
fibrillar Ab1–42 was used as an immunogen This
pep-tide is well characterized to be toxic Hence, we have
been promoting the use of nonamyloidogenic
deriva-tives as immunogens for protein conformational
disor-ders, including AD and prion disease [31,34,38] How
significant an issue direct toxicity of the immunogen
may be for prion vaccination remains unclear Unlike
the Ab peptide used for vaccination in AD models,
direct application of recombinant PrP has not been
shown to be toxic However, this issue has not been
investigated as thoroughly as in the Alzheimer’s field
and remains controversial Several lines of evidence
suggest that intracellular accumulations of PrPSc
pro-mote neurodegeneration [133]
A potential ideal means of using immunomodulation
to prevent prion infection is by mucosal immunization
One important reason for this is that the gut is the
major route of entry for many prion diseases such as
CWD, BSE and vCJD Furthermore, mucosal
immun-ization can be designed to induce primarily a humoral
immune response, avoiding the cell-mediated toxicity
that was seen in the human AD vaccine trial In
addi-tion, mucosal vaccination has the advantage that it is
unlikely to induce significant immune response within
the brain Although it has been shown that reduced
levels or absence of CNS PrPCby, for example,
condi-tional ablation by genetic manipulation of neuronal
PrPC [134] can prevent clinical prion infection, it is
likely that the immunological targeting of neuronal
PrP would be associated with inflammatory toxicity Recently, we have been developing prion vaccines that target gut associated tissue, the main site of entry of the prion agent One of our approaches is to express PrP in attenuated Salmonella strains as a live vector for oral vaccination, which has resulted in prevention
or significant delay of prion disease in mice [69] Live attenuated strains of Salmonella enterica have been used for many years as vaccines against salmonellosis and as a delivery system for the construction of multi-valent vaccines with a broad application in human and veterinary medicine [135] A main advantage for this system is that the safety of human administration of live attenuated Salmonella has been extensively con-firmed in humans and animals [136,137] Ruminants and other veterinary species can be effectively immun-ized by the oral route using attenuated Salmonella, to induce humoral mucosal responses [138,139] We are currently exploring ways to increase the efficacy even further In these studies, the mucosal IgA anti-PrP titer correlates well with the delay or prevention of prion infection, further supporting the importance of the humoral response for the therapeutic effect Salmonella target M-cells, antigen sampling cells in the intestines, which may also be important for uptake of PrPSc [27,68,121] Hence, this approach is more targeted than prior vaccination studies, likely explaining the improved efficacy By exploring other strains of attenu-ated Salmonella, using different bacteria or oral adjuvants, and⁄ or by altering the expression levels or sequence of the PrP antigen, it is likely that the percentage of uninfected animals can be improved Our recent work utilizing this approach indicates that complete protection to clinical prion infection via an oral route is possible Overall, this approach holds great promise as an inexpensive prophylactic immuno-therapy to prevent the spread of prion disease, partic-ularly in animals at risk and perhaps eventually in certain high risk human populations
Metal chelation for prion and AD Metal chelation is emerging as an important therapeu-tic approach for AD, which is currently in clinical trial [140,141] This approach for AD is reviewed elsewhere
in this minireview series Importantly, modulation of metal levels, in particular copper, has been shown to
be important for the conversion of PrPC to PrPSc, highlighting another similarity between AD and prion diseases [10] Copper binding is thought to be part of the normal function of PrPC[142–144] The binding of copper to PrPC gives the complex antioxidant activity [145,146]; hence, it has been suggested that the reduced
Trang 8copper binding of PrPSc with a consequent reduction
of antioxidant activity is part of the pathogenesis of
prion disease [147] This hypothesis has been supported
by the finding that copper is reduced up to 50% in the
brains of sporadic CJD patients [148] How copper
binding influences the PrPC to PrPSc conversion is
complex [10,149] We were the first to show that,
sim-ilar to studies in AD Tg models, metal chelation can
be used therapeutically [150] in prion infection Our
studies indicated that penicillamine, a copper chelator,
prolongs the incubation period of scrapie in mice
[150] Consistent with this observation, the presence of
copper has also been shown to stabilize the PrPSc
con-formation using preformed fibrils [151–158], as well as
to induce aggregation of the prion peptide 106–126
[159] Some tissue culture studies of prion infection
have also suggested that copper chelators are suitable
candidates for antiprion drugs [160] However, there
are conflicting reports indicating that the interaction
between copper and PrP is likely to be quite complex
For example, copper has been shown to inhibit the
in vitro conversion of recombinant PrP into amyloid
fibrils but, also in contrast, to enhance the
protein-ase K resistance of preformed fibrils [157] These
findings indicate that copper may have a dual and
opposite effect on prion propagation It may both
inhibit prion replication and prevent clearance of
potentially infectious forms of the prion protein
Furthermore, copper treatment has also been shown to
inhibit PrPSc amplification in reactions where brain
derived PrPCwas used as a seed [161], as well as
delay-ing the onset of clinical disease in scrapie infected
hamsters [162] In addition, it has been shown that
physiological levels of copper promote internalization
of PrPC[163] The interaction between PrPCand
cop-per was found to be the overriding factor in
stimula-ting the internalization response with other metals
showing no effect The decrease in detectable levels of
PrPC at the cell surface following copper treatment
was found to be the result of internalization rather
than loss into the surrounding environment [163]
Such internalization would limit the exposure of
PrPC to conversion from exogenous PrPSc; however,
because cytoplasmic forms of PrP have been linked to
neurodegeneration [133], increased internalization
could also be deleterious in some settings Copper has
also been shown to have immunomodulatory effects
[164] and, as discussed earlier, the immune system can
have profound effects on prion infection Hence, it
appears that the deleterious or beneficial role of copper
in prion infection might vary depending on which
function predominates under the distinct experimental
conditions being used Nevertheless, it is clear that a
greater understanding of the role of metal binding in prion infection presents a therapeutic opportunity
Conclusions Immunization appears to be an effective therapeutic method for prevention of Ab deposition and cognitive decline in AD, provided that cell-mediated auto-immune toxicity can be avoided The second genera-tion AD vaccines, which are under development, are based on nontoxic and nonfibrillar Ab homologous peptides that are modified to eliminate the potential for inducing cellular immunity, and elicit primarily a humoral response Other related approaches include direct administration of antibodies that target Ab These interventions would likely favor a peripheral sink effect, clearing soluble Ab from the blood stream and inducing efflux of Ab from the brain Additional potentially synergistic therapeutic approaches for AD would include blocking the interaction of Ab with its
‘pathological chaperones’ such as apoE, as well as use
of b-sheet breaker compounds Immunization approa-ches could be used for sporadic AD, familial AD, and
AD associated with Down’s syndrome The effective-ness of treatment would depend on its initiation early
in the disease course Therefore, such a treatment needs to coincide with the development of a procedure for the detection and monitoring of Ab deposits Both active and passive immunization appear to be effective in prevention of prion infections in animal models Further studies are needed to develop specific protocols applicable for human use Active immuniza-tion, using especially mucosal immunization could be used to prevent spread of BSE through the oral route, whereas passive immunization protocols would
be more appropriate for subjects accidentally infected with prion contaminated material (e.g blood transfu-sion or organ transplant) Effective immunization for prion infections works through prevention of entry of PrPSc via the gut and⁄ or neutralization of PrPSc replicating in the peripheral lymphoreticular system Metal chelation is another promising therapeutic approach for AD, which is currently undergoing clinical trials Similar approaches are just emerging for prion diseases However, a greater understanding
of the role of copper and other metals in the PrPCto PrPSc conversion is needed before this therapeutic strategy can be effectively harnessed for prion infection
Acknowledgements This manuscript is supported by NIH grants: AG15408, AG20245, AG20197 and the Alzheimer’s Association
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