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From: Terry S. Singeltary Sr. (216-119-144-43.ipset24.wt.net)
Subject: PRION DISEASES — CLOSE TO EFFECTIVE THERAPY?
Date: October 7, 2004 at 9:58 am PST

Nature Reviews Drug Discovery 3, 874-884 (2004); doi:10.1038/nrd1525

printable pdf

[384K]


PRION DISEASES — CLOSE TO EFFECTIVE THERAPY?


Neil R. Cashman1 & Byron Caughey2 about the authors popupWindow('/nrd/journal/v3/n10/biog/nrd1525_biog.html','abouttheauthors2','400','300')>


1 Centre for Research in Neurodegenerative Diseases, University of
Toronto, 6 Queen's Park Crescent West, Toronto, Ontario M553H2, Canada.
neil.cashman@utoronto.ca
2 Rocky Mountain Laboratories, National Institutes of Health, 903 South
Fourth Street, Hamilton, Montana 57840, USA.
bcaughey@niaid.nih.gov

The transmissible spongiform encephalopathies could represent a new mode
of transmission for infectious diseases — a process more akin to
crystallization than to microbial replication. The prion hypothesis
proposes that the normal isoform of the prion protein is converted to a
disease-specific species by template-directed misfolding. Therapeutic
and prophylactic strategies to combat these diseases have emerged from
immunological and chemotherapeutic approaches. The lessons learned in
treating prion disease will almost certainly have an impact on other
diseases that are characterized by the pathological accumulation of
misfolded proteins.

TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF1','glossary','200','300')>
(TSEs) are rapidly progressive, fatal and untreatable neurodegenerative
syndromes that are neuropathologically characterized by SPONGIFORM
CHANGE popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF2','glossary','200','300')>
(microcavitation of the brain), neuronal loss, glial activation and
accumulation of an abnormal amyloidogenic protein. Human TSEs include
classical CREUTZFELDT–JAKOB DISEASE popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF3','glossary','200','300')>
(CJD), which has sporadic, iatrogenic and familial forms. Since 1996
(Ref. 1
),
a new VARIANT CJD popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF4','glossary','200','300')>
(vCJD) has been identified in the United Kingdom, France, the Republic
of Ireland, Hong Kong, Italy, the United States and Canada that is
characterized by young age of onset, a stereotypical pattern of illness
progression and distinctive neuropathological features2
.
This disease, which probably derives from the consumption of cattle
neural tissues contaminated with the BOVINE SPONGIFORM ENCEPHALOPATHY
popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF5','glossary','200','300')>
(BSE) agent, has afflicted 150 individuals to date. Although some
studies suggest that the 'primary' vCJD epidemic is waning3
,
the report of two probable cases of blood-borne transmission4

raises concerns about a secondary vCJD epidemic resulting from
iatrogenic transmission through donation of blood, tissues or organs,
and contaminated surgical instruments5
.
Notably, in contrast to classical forms of CJD, vCJD infectivity is more
likely to accumulate in peripheral tissues and organs to levels that
could represent a substantial transmission hazard6
.

The timing of the vCJD epidemic, similarities in transmission
characteristics in experimental animals (mice and primates)7
,
and similar biochemical features indicate that vCJD almost certainly
represents interspecies transmission of the agent responsible for BSE8
.
In the United Kingdom alone between December 1986 and 31 March 2003, BSE
was confirmed in 179,973 cattle9
,
with up to 3 million infected cattle entering the human food supply
undetected10
.
The UK BSE epidemic, initially amplified by the now-proscribed
supplementation of cattle feed with meat-and-bone meal, is clearly in
decline. The possibility that BSE has entered the UK sheep population
cannot be ruled out at present11-13
.
In 1993, Canadian authorities reported North America's first case of
BSE, in a steer imported from the United Kingdom. The discovery of a
case of BSE in an Alberta cow fed locally rendered feed in May 2003, and
a second case in Washington State in December 2003, has drastically
altered awareness of this disease in North America. Yet another animal
TSE concern in North America is CHRONIC WASTING DISEASE popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF6','glossary','200','300')>
(CWD) of captive and wild cervids (deer and elk), which has been
insidiously emerging since initial reports in Colorado in the 1960s14
.
CWD, arguably the most contagious of TSEs, has now been reported in 12
US states as far east as Illinois, and in the Canadian provinces of
Saskatchewan and Alberta. Unlike sheep SCRAPIE popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF7','glossary','200','300')>,
with which humans have coexisted for at least 300 years15
,
CWD represents an uncertain threat whose impact on human health is as
yet unknown16
.

Novel form of infectivity

Clearly, there is an urgent need for effective and efficient animal
prophylactic therapies for prion diseases, to prevent the spread of BSE,
CWD and sheep scrapie throughout the world. Moreover, successful
therapies for human TSEs need development, particularly in view of the
uncertainty surrounding the extent of primary and iatrogenic vCJD in the
United Kingdom and other countries. However, the development of
prophylactic and therapeutic agents will not be a trivial challenge
because of the unusual biology of prions. The agents that transmit TSEs
differ from viruses and viroids in that no evidence for an
agent-specific nucleic-acid component has been reproducibly detected in
infectious materials17
.
According to the 'protein only' or PRION HYPOTHESES popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF8','glossary','200','300')>18-21
,
infectivity resides in an abnormal isoform of the host-encoded cellular
prion protein (PrPC popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF9','glossary','200','300')>
or PrP-SEN popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF10','glossary','200','300')>).
The abnormal isoform (PrPSC popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF11','glossary','200','300')>
or PrP-RES popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF12','glossary','200','300')>)
is beta -sheet rich22-24
,
insoluble and partially protease resistant, whereas PrPC is alpha
-helix-rich24
,
25
,
soluble in mild detergents and protease sensitive26
,
27
.
PrPSc is the most prominent macromolecule in preparations of prion
infectivity and a reliable marker of most TSE infections. PrPC is a
widely distributed glycosylphosphatidylinositol (GPI)-linked
cell-surface protein with a molecular mass of 33–35 kDa28
,
29
,
and is non-infectious.

Currently favoured versions of the prion hypothesis posit that PrPSc
propagates itself as an infectious agent by causing PrPC to convert into
PrPSc in a template-directed process catalysed by physical contact with
PrPSc18
,
21
,
30
,
31

(Fig. 1 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F1.html','fig_hires','630','600')>).
It has been shown that PrPC can be converted to a protease-resistant
form by contact with PrPSc in vitro in highly species- and
strain-dependent reactions32-34
.
This conversion can be made more continuous in crude brain homogenates
by protein misfolding cyclic amplification (PMCA), a process analogous
to the polymerase chain reaction for nucleic-acid amplification35
.
Although the conversion mechanism is not fully understood, most
available evidence is consistent with the idea that ordered PrPSc
aggregates serve as templates or catalysts for the conformational change
and ordered aggregation of PrPC. However, the formation of
protease-resistant PrP species in vitro has not yet been associated with
the generation of increased TSE infectivity36
.
Although PrPSc in most infected tissues and cells is partially
protease-resistant and insoluble, these properties can vary with
experimental handling, TSE strain and host species and, therefore,
should not be considered absolute prerequisites for infectivity even if
they are likely to help stabilize it37-39
.
Highly protease-sensitive molecules (sPrPSc) co-purify with
protease-resistant PrPSc (rPrPSc) and infectivity40-42
,
which raises questions as to whether sPrPSc has a role in infectivity or
neuropathogenesis. Attempts to separate sPrPSc and rPrPSc have shown
that infectivity fractionates with the latter43
.
Other studies indicate that accumulation of weakly protease-resistant
and PrPSc-like PrP can cause neurodegenerative disease without
infectivity44
.
Moreover, high levels of infectivity and neurological disease have been
reported in the absence of measurable PrPSc39
,
162
.
Collectively, the available evidence indicates that several
disease-associated forms of PrP exist, and that neurotoxic forms are not
necessarily infectious. Conversely, the most infectious forms need not
be the most neurotoxic.


popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F1.html','fig_hires','630','600')>
Figure 1 | PrPSc formation in scrapie-infected cells.

Cellular prion protein (PrPC; blue dots) follows the secretory pathway
through the endoplasmic reticulum (ER) and Golgi apparatus to the plasma
membrane, where it is anchored by a glycophosphatidylinositol anchor.
According to the prion hypothesis, the abnormal prion isoform (PrPSc;
shown as ordered red oligomeric clusters) binds to PrPC and causes it to
undergo a conformational change while being incorporated into the PrPSc
oligomer18
,
20
,
as has been suggested experimentally32
,
46
.
This occurs primarily on the cell surface or in endocytic vesicles. The
conversion is affected by association with raft membrane microdomains86

and cofactors such as sulphated glycosaminoglycans (GAGs; maroon beads)
or proteoglycans120-123

(rods with attached maroon beads). PrPSc can then accumulate in
lysosomes, in the plasma membrane or in extracellular deposits such as
amyloid fibrils and plaques. It is notable that the sites of PrPSc
formation are accessible to potential inhibitors in the extracellular
medium without having to cross cellular membranes.

Should the protein-only hypothesis of TSE infectivity be considered
'proven'? The most direct proof — that is, de novo conversion of PrPC
alone into a high-titred, serially transmissible infectious agent — has
been extremely difficult to achieve. However, synthetic fibrils of
mutant PrP peptides were recently reported to cause serially
transmissible diseases when inoculated into transgenic mice
overexpressing related mutant PrPC molecules45
,
46
.
These results provide tantalizing support for the protein-only prion
model, but with certain caveats. One is that the apparently synthetic
prions seem to be many orders of magnitude lower in infectivity per unit
PrP than is genuine TSE infectivity, raising the question of what is
necessary to produce reasonably potent prions. Second, additional
controls are needed to rule out the possibility of spontaneous prion
formation and neuropathology in the transgenic mice without the
inoculation of synthetic fibrils. Nonetheless, considerable, but less
direct, experimental data seem to support the prion model. Perhaps most
tellingly, PRNP popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF13','glossary','200','300')>-null
mice do not support the replication of TSE infectivity47
.
Moreover, antibodies directed against PrPC (see below) and chemical
inhibitors of PrP conversion48

can block propagation of TSE infectivity in vitro and in vivo. Whether
or not various aberrant forms of PrP are solely responsible for TSE
infectivity and/or neuropathology, their central role in the TSE
pathogenesis provides a cogent framework to approach drug discovery in TSEs.

Immunopathogenesis

In addition to approaching TSEs as a protein chemical problem of
conformational conversion, the role of the immune system in prion
infection provides other avenues for therapy (Fig. 2 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F2.html','fig_hires','630','600')>).
Prion infection typically occurs by the oral route in sheep scrapie, BSE
of cattle and human vCJD, and is necessarily followed by replication in
a peripheral compartment prior to brain invasion. Non-human primates can
also be experimentally infected orally49
.
CWD is probably also orally contracted50
.
In orally transmitted TSEs, prion infectivity and/or protease-resistant
PrP can be identified in gut Peyer's patches51
,
52
.
Prion propagation to splanchnic lymphoid tissue and spleen has been also
demonstrated for natural and experimental scrapie infection52
,
53
,
and humans with vCJD display high levels of infectivity in
gut-associated lymphoid tissue (GALT; including tonsil) and spleen54
.
From GALT and other lymphoid tissue, prions are transported by
splanchnic innervation to the brainstem and spinal cord52
,
53
.
This means that in early infection, replication occurs in compartments
accessible to immunotherapy and antibody neutralization.


popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F2.html','fig_hires','630','600')>
Figure 2 | Possible spread of scrapie infectivity from the gut lumen to
the nervous system following oral infection (route indicated by dotted
line).

Soon after ingestion, the abnormal prion isoform (PrPSc) is detected
readily within Peyer's patches on follicular dendritic cells (FDCs),
within macrophages, within cells with morphology consistent with that of
M cells and within ganglia of the enteric nervous system (ENS). These
observations indicate that, following uptake of scrapie infectivity from
the gut lumen, infectivity accumulates on FDCs in Peyer's patches and
subsequently spreads via the ENS to the central nervous system. FAE,
follicle-associated epithelium. Adapted, with permission, from Ref. 55

© Elsevier Ltd (2000).

A cell of particular importance in the peripheral propagation of prions
is the follicular dendritic cell (FDC), which resides in immune
follicles in the gut, lymph nodes and spleen. These cells acquire a
large burden of PrPSc in lymph nodes and spleens of scrapie-infected
mice55
.
Moreover, FDCs seem to be crucial for the propagation of prion
infectivity in these tissues56
.
Mutant or knockout mice lacking functional B cells, tumour-necrosis
factor-alpha (TNF-alpha
),
TNF receptor-1, lymphotoxin (LT) alpha +beta and LT beta -receptor,
which are all deficient in maturation and activation of FDCs, have also
been shown to be poorly permissive for peripheral inoculation of scrapie
prions for many experimental scrapie strains57-60
.
PrPC expression in FDCs is required for efficient infection of spleen55
,
59
,
whereas PrPC expression restricted to non-FDC lymphoid cells does not
permit scrapie replication in this organ. The mutation or depletion of
the complement component C3 also blocks peripheral prion propagation, a
result that supports a proposed role for FDC complement receptors in
prion infection of these cells61
.
PrPC expression, which is proven to be crucial for prion replication in
knockout mice47
,
is readily detected even in resting FDCs not infected with prions62
.
FDCs can therefore efficiently support prion replication because they
are long-lived cells that express high levels of PrPC (similar to
neurons), and are specialized to trap, retain and present unprocessed
antigens52
.
Other cells in the gut and follicle have also been implicated in prion
propagation in orally transmitted disease, including M cells63
.
Accessory-cell-dependent replication of prions in the brain is less
understood than that in the lymphoid follicle. However, brain microglial
cells express many FDC and myeloid markers, including receptors for TNF,
immunoglobulin G and C3 (Ref. 45
),
and have been implicated in neurotoxicity of prions64
.

Immune-active therapies for TSEs

It therefore seems possible that immune manipulation might affect
lymphoid prion replication, to block or slow neuro-invasion, as has been
shown with experimental depletion of TNF-alpha and complement56
,
58
,
61
,
65
.
Moreover, prions that replicate in peripheral compartments might be
vulnerable to circulating anti-prion protein antibodies; unfortunately,
however, prions do not naturally elicit protective immune responses
(reviewed in Ref. 66
).
Several strategies have emerged to test the possibility that immune
recognition of prion protein isoforms could prove to be of therapeutic
importance in treating prion infection. Several recent publications have
indicated that antibodies predominantly directed against PrPC can clear
scrapie-infected cells of PrPSc in vitro, and presumably scrapie
infectivity as well67-69
.
In addition, Aguzzi and colleagues have found that the transgenic
expression of the antibody 6H4, which is non-selective for prion protein
isoforms can block experimental scrapie in mice70
,
and Hawke and colleagues have shown that anti-PrP antibody infusion can
generate a similar effect with peripherally inoculated prions71
.
These data indicate that interference with the intermolecular
interactions of PrPC, or changes in compartmental cycling of this
protein, disrupt the conversion of PrPC to PrPSc.

However, antibodies directed against PrPC, a normal cell-surface
protein, could have adverse consequences if used as immunotherapies in
vivo. PrPC is ubiquitously expressed28
,
29
,
and therefore circulating antibodies against PrPC could trigger
widespread complement-dependent lysis of many cells. Moreover, it is
possible that anti-PrPC antibodies would cause a breakdown of
immunological tolerance of this molecule, with the consequent induction
of autoimmune disease. Furthermore, antibodies directed against PrPC
might impair its normal function, thereby triggering apoptosis in the
brain72

and causing inappropriate activation of signalling cascades73
.
Antibody-mediated cell-surface ligation of PrPC can also suppress T-cell
activation of human lymphocytes74
.
PrPSc-specific immune recognition would circumvent problems of
autoimmune recognition and impaired function of PrPC. A PrPSc-specific
immune response would be expected to opsonize infectious prions for
degradation in the reticulo-endothelial system, and could block the
production of PrPSc by impairing PrPC–PrPSc interactions that are
considered a prerequisite for the recruitment process75
.
As the conversion of prion isoforms occurs at the cell surface, or a
compartment close to cell surface76-79
,
PrPSc-specific antibodies are likely to interfere with the infectious
process, as does 6H4 (Ref. 67
)
and recombinant PrPC-specific antibodies and fragments68
.
It is also possible that anti-PrPSc antibodies might participate in the
immune recognition and destruction of prion-infected cells, possibly by
targeting them for antibody-dependent cellular cytotoxicity.

A recent report has shown that the prion protein repeat motif
Tyr-Tyr-Arg is accessible to antibody binding in the misfolded PrPSc
isoform, but not on the molecular surface of native PrPC80

(Fig. 3 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F3.html','fig_hires','630','600')>).
The incubation of scrapie-infected ScN2a neuroblastoma cells with
Tyr-Tyr-Arg monoclonal antibodies has also been shown to reduce the cell
content of PrPSc in a concentration- and time-dependent manner81
,
similarly to PrPC-directed antibodies62-66
.
PrPSc-specific antibodies have been generated that do not have toxic
effects through Tyr-Tyr-Arg peptide immunization using conventional
adjuvants in animals expressing endogenous PrPC80
.
Moreover, PrPSc-specific monoclonal and polyclonal antibodies do not
recognize antigens at the cell surface of normal dissociated splenocytes
and brain cells, despite the presence of Tyr-Tyr-Arg motifs in PrPC and
in other non-prion proteins. Tyr-Tyr-Arg motifs in the prion protein,
and in non-prion proteins, are therefore sequestered from antibody
recognition on normal cells that have not been infected by prions or
exposed to denaturing agents. The lack of immunological recognition of
PrPC or other native-structured cell-surface proteins by PrPSc-specific
Tyr-Tyr-Arg antibodies indicates that specific prion immunoprophylaxis
and/or immunotherapy could ultimately prove possible in animals and humans.


popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F3.html','fig_hires','630','600')>
Figure 3 | Tyr-Tyr-Arg antibodies selectively recognize PrPSc.

a | Monoclonal antibodies 1A12 and 17D10 selectively immunoprecipitate
the abnormal prion isoform (PrPSc) from experimentally and naturally
infected prion disease brain, but not normal prion (PrPC) from
uninfected brain. b | Efficiency comparison of proteinase K resistance
and Tyr-Tyr-Arg immunoprecipitation (monoclonal antibody 16A18) from
equivalent samples of frontal (1–4, 7, 8) and cerebellar (5, 6, 9, 10)
regions of a Creutzfeldt–Jakob disease (CJD) and a GERSTMANN–STRAUSSLER
SYNDROME popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF14','glossary','200','300')>
(GSS) brain. c | Tyr-Tyr-Arg antibodies recognize low concentrations of
PrPSc in ME7-infected mouse spleen. All panels: 6H4 (a and c) or 3F4 (b)
immunoblot detection of immunoprecipitated PrP. d | Tyr-Tyr-Arg
monoclonal antibody 9A4 recognizes a population of dendritic cells from
scrapie-infected sheep lymph nodes. CD58+ + CD45RO- retropharyngeal
lymph node cells from scrapie-infected and normal sheep stained with
9A4, or monoclonal antibody control 4E4. Adapted, with permission, from
Ref. 80

© Macmillan Magazines Ltd.

Notably, vaccine therapies are also now being organized with another
neurotoxic amyloid peptide, the Abeta fragment of amyloid precursor
protein

of Alzheimer's disease
. Preclinical
and clinical data seem to demonstrate that immune recognition of Abeta
could provide an effective therapy for Alzheimer's disease82
.
Human Abeta immunization trials have been halted because of the
development of acute encephalopathy in some patients83
,
which presumably results from T-cell immune recognition of brain plaque
Abeta , but further human strategies are being explored at present. It
is likely that antibodies directed against PrPSc in pre-symptomatic
cattle and humans would prevent neuro-invasion without causing
encephalopathy, as the cognate antigen is not yet present in the brain.

Chemotherapeutic targets

Depending on the type of TSE and the circumstances, a number of
different modes of intervention are possible: decontamination of sources
of infection; prophylaxis against initial infections; inhibition of
agent propagation in the periphery; blockade of invasion of central
nervous system (CNS) from the periphery; inhibition of pathogenic PrP
accumulation; destabilization of pathogenic PrP; blockade of direct or
indirect neurotoxic effects of pathogenic PrP; and compensation for
damage to brain cells. The first four of these modes will probably only
apply to TSEs that result from infections by peripheral routes. For
these situations, it would be worthwhile to identify compounds that are
protective against at least low-level sources of infections. Ideally,
they would be safe enough to be used prophylactically in foodstuffs or
other potentially contaminated materials that are consumed or inoculated
peripherally into humans or animals. Such compounds need not cross the
blood–brain barrier. However, once TSE infections have penetrated the
CNS, it will probably be necessary to target drugs directly to the CNS
(that is, the last four modes of intervention). This will be true in the
clinical and late preclinical phases of any TSE, with iatrogenic
transmissions into the CNS, and with sporadic CJD and familial TSEs in
which PrPSc formation might occur spontaneously in the CNS. These
scenarios will usually require drugs that can cross the blood–brain
barrier; however, it might also be possible — although cumbersome,
expensive and potentially risky — to inject or infuse drugs directly
into the brain.

At the molecular level, one primary chemotherapeutic target is the PrP
conversion reaction, and this has been the focus of the majority of TSE
drug discovery efforts to date. PrP conversion inhibitors can act
directly by binding to PrPC or PrPSc, and by affecting their
interactions with themselves or other influential ligands. Indirect
PrPSc inhibition mechanisms are also possible, such as those that affect
PrP expression84
,
turnover, trafficking85
,
membrane associations86

or ligand binding. One might also block initial infections or the spread
of infection by blocking interactions between PrPSc and
as-yet-unidentified receptor(s) on various cell types that might carry
the infection to the CNS.

Screens for potential anti-TSE drugs

In vivo testing of potential anti-TSE compounds tends to be very slow
and expensive. Most of the initial screening for potential anti-TSE
chemotherapies has therefore been done with surrogate in vitro tests.
The most common approach has been to test for inhibition of PrPSc
accumulation in scrapie-infected tissue culture cells. Several
chronically infected cell lines have been developed, including murine
neural (N2a87
,
88
,
SMB89
,
GT190
)
and fibroblast91

cell lines, and sheep scrapie-infected rabbit Rov epithelial cells
expressing sheep PrPC92
.
Unfortunately, little progress has been reported in developing human,
bovine or cervid cell lines infected with CJD, BSE or CWD, respectively.
Such cell lines would probably be helpful, because striking TSE strain-
and species-dependence has been observed with a few of the known
antiscrapie compounds and it cannot always be assumed that what works
against one TSE strain will be effective against another. Nonetheless,
the identification of antiscrapie therapeutics should provide at least
proof of principle that compounds of a given class can be beneficial
against TSE diseases. Of course, as is true with drug discovery in
general, not all compounds with in vitro activity are effective in vivo.
However, the fact that numerous classes of PrPSc inhibitors in
scrapie-infected cell cultures have also shown some antiscrapie activity
in animals (Table 1
)
indicates the utility of these cultures as initial high-throughput
screening tools. Recent progress in adapting mouse scrapie-infected N2a
cells93
,
94

(Fig. 4 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F4.html','fig_hires','630','600')>)
and sheep scrapie-infected Rov cells (D. Kocisko, A. Engel, K. Harbuck,
D. Villette and B. C., unpublished data) to higher-throughput formats
has allowed the screening of hundreds of compounds per week by a single
person. In the few examples that have been tested, long-term inhibition
of PrPSc accumulation in cultured cells has resulted in elimination of
scrapie infectivity as assayed by injecting cell lysates into animals48
,
95
,
96
.

popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_T1.html','tab_hires','630','600')>


Table 1 | Anti-transmissible spongiform encephalopathy compounds


popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F4.html','fig_hires','630','600')>
Figure 4 | High-throughput cell-based screen for inhibitors of PrP-res
formation.

Scrapie-infected murine N2a neuroblastoma cells are grown in 96-well
plates for several days in the presence or absence of test compounds.
The cultures are lysed, treated with proteinase K (PK) to eliminate the
normal prion protein isoform (PrPC), and assayed for accumulated PrP-res
content by a dot blot immunoassay. a | Dot blot of PrP content with and
without PK treatment of lysates of scrapie-infected or uninfected N2a
cells. b | Representative dot blot screen of multiple (unidentified)
test compounds for inhibition of PrP-res accumulation. The turmeric
component curcumin (10 mu M), a known inhibitor134
,
was included as a positive control. Adapted, with permission, from Ref.
94

© American Society for Microbiology (2003).

Other types of in vitro tests for potential anti-TSE compounds have been
devised as well. Cell-free PrP binding97-99
,
conversion94
,
97
,
100

and polymerization assays comprising purified PrP molecules, or
fragments of such molecules (Fig. 5 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F5.html','fig_hires','630','600')>)
(reviewed in Ref. 101
),
can provide evidence of whether or not PrPSc inhibitors act directly on
PrP molecules. PrPSc amplification reactions in crude brain or cellular
extracts35
,
102
,
103

provide more continuous, but less defined, cell-free assays that might
be adapted to high-throughput formats. PrPSc destabilization assays can
identify compounds that disinfect potential sources of infection or aid
infected hosts in reducing their burden of PrPSc104-107
.
Cell-culture assays of cytotoxicity induced by PrPSc or peptide
fragments of them can be used to screen for compounds that might protect
against the neuropathological consequences of TSE infections101
.
Some caution should be used in interpreting such assays, as it is not
yet clear which abnormal form or forms of PrP are the primary cytotoxic
species in TSE diseases.


popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F5.html','fig_hires','630','600')>
Figure 5 | Solid-phase cell-free PrP conversion assay for inhibition of
PrP-res formation.

a | Outline of assay as described previously94
,
97
.
b | Inhibition curves of three PrP-res inhibitors. Adapted, with
permission, from Ref. 94

© American Society for Microbiology (2003). BSA, bovine serum albumin;
PrP-res, protease-resistant prion protein; PrP-sen, protease-sensitive
prion protein.

Rodent models for testing anti-TSE drugs

For practical reasons, most in vivo testing has been done in mice and
hamsters inoculated with rodent-adapted TSE strains5
,
108-113
.
The rodent models allow for much faster and less expensive screening
than is possible in the natural, large-animal host species. Whereas
incubation periods tend to be years in sheep, cattle, deer and elk,
rodents can become ill within as little as 40-45 days after
intracerebral inoculation with high doses of an appropriate scrapie
strain114
,
115
.
The latter scenario is presumably suitable for testing potential
therapeutic activities in hosts with established CNS infections.
However, it is often also desirable to test for pre- or post-exposure
prophylactic effects against lower-dose TSE infections by peripheral
routes, such as oral exposure. However, under the latter circumstances,
rodent incubation periods can be much longer, and, in extreme cases,
extend nearly to the lifespan of the animal. Researchers are therefore
often torn between the conflicting goals of minimizing the duration of
experiments and reducing the infectious challenge to increase the
likelihood of detecting drug efficacy. To abbreviate the testing of
compounds against peripheral scrapie inoculations, some investigators
have opted to assay the accumulation of PrPSc in the spleen part way
through the incubation period, rather than waiting for the appearance of
clinical illness116
.

Pre- and post-exposure chemoprophylaxis

A growing list of compounds can prolong the lives of scrapie-infected
rodents, provided that drug treatment is initiated prior to or near the
time of infection (Table 1
). The
prophylactic agents fall into several different chemical classes and are
usually among the most potent inhibitors of PrPSc accumulation in
scrapie-infected cell cultures. Hundreds of other PrPSc inhibitors have
been identified in in vitro tests that await further testing in vivo,
including branched polyamines95
,
polyphenols, antipsychotics, antidepressants, analgesics and statins93
,
94
,
117
,
118
.

Sulphated glycans and other polyanions. In the 1980s, various compounds
with antiviral and/or immunological effects were tested against scrapie
in animals. Surprisingly, given that no virus or conventional immune
response has been associated with TSE infections, a number of large
polyanions, such as dextran sulphate, pentosan polysulphate and
heteropolyanion 23, were effective in protecting rodents against scrapie
infections108
,
109
.
In some cases, protection was observed by short treatments months before
peripheral scrapie infection108
,
119
.
This seemed to be related to sequestration of the polyanion in cells of
the lymphoreticular system. More recent studies showed that these
polyanionic compounds were potent inhibitors of PrPSc formation, which
provides a likely explanation for their prophylactic efficacy120
,
121
.
A number of lines of evidence indicate that sulphated glycans and other
polyanionic drugs act by affecting interactions between PrP molecules
and endogenous sulphated glycosaminoglycans that seem to be important in
PrPC trafficking and PrPSc formation85
,
120-123

(Fig. 1 popupWindow('/nrd/journal/v3/n10/fig_tab/nrd1525_F1.html','fig_hires','630','600')>).
Alternatively, polyanionic drugs might also interfere with PrP
interactions with RNA molecules (also large polyanions) that were
observed to support PrP conversion in brain homogenates and proposed to
be physiological cofactors124
,
125
.
Limitations of these large polyanionic drugs include potential
anticoagulant activity and poor bioavailability to the CNS (see below).

Sulphonated dyes. The first identified inhibitor of PrPSc accumulation,
Congo red126
,
is a sulphonated amyloid stain that has modest prophylactic activity
against scrapie in rodents127
.
In vitro studies have shown that Congo red can compete with sulphated
glycans for binding to PrPC, which is consistent with an inhibitory
mechanism that is similar to that of the large polyanions128
.
At high concentrations relative to those required to inhibit PrPSc
formation, Congo red can also overstabilize PrPSc, and this effect has
also been suggested as an aspect of its inhibitory mechanism129
.
Interest in Congo red as a potential drug was diminished by fears of
teratogenic and/or carcinogenic activity related to its benzidine
moiety. Since then, numerous analogues of Congo red and related
sulphonated dyes have also proven to be potent PrPSc inhibitors in
vitro93
,
130-132

and, in the case of suramin133
,
to have modest prophylactic activity as well. Structure–activity
analyses of Congo red analogues indicate that the central benzidine
moiety can be altered without neutralizing its inhibitory activity93
,
130
,
132
.
These observations raise hopes that safer and more effective analogues
of these types of inhibitors can be discovered. Curcumin — the major
yellow pigment in the spice turmeric, which has a structure that lacks
sulphonates and a benzidine moiety but is otherwise reminiscent of Congo
red — was recently shown to also be a very potent, yet non-toxic
(edible), inhibitor of PrPSc formation134
.
Unfortunately, efforts to show in vivo efficacy of curcumin have so far
failed.

Cyclic tetrapyrroles. Porphyrins and phthalocyanines are another diverse
group of PrPSc inhibitors100

that can delay the onset of disease in mice inoculated intraperitoneally
with scrapie, but only if treatment is initiated within several weeks of
infection111
,
135
.
Cyclic tetrapyrroles tend to have highly conjugated planar aromatic ring
systems that bind transition metal ions and can be circumscribed by
anionic, cationic or uncharged peripheral constituent groups. Several
cyclic tetrapyrroles have been shown to directly block cell-free PrP
conversion reactions, so it is assumed that their inhibitory mechanism
involves direct interaction with PrP molecules. None of the antiscrapie
tetrapyrroles that have been tested in vivo are likely to cross the
blood–brain barrier, and this presumably limits the therapeutic window
of opportunity. However, other tetrapyrrole inhibitors (in vitro) are
thought to penetrate the blood–brain barrier and could prove to be
efficacious later in the TSE incubation period.

Polyene antibiotics. Another promising group of compounds that inhibit
PrPSc formation and delay the onset of experimental scrapie in rodents
is the antifungal drug amphotericin B and its analogues136
,
137
.
The antiscrapie mechanism of action of the polyene antibiotics could
result from a perturbation of the raft membrane domains with which
PrP-sen is associated138
,
139
.
Unfortunately, although treatment can be initiated well after the point
of infection, it must begin before the onset of clinical disease. Other
drawbacks of the prototypic amphotericin B are its toxicity and scrapie
strain-specificity. However, less toxic and more broadly active
analogues have now been identified, strengthening hopes that more
effective therapies based on this type of compound might be possible140
.

Quinacrine, quinoline, acridines, phenathiazines and related molecules.
Quinacrine, chlorpromazine, quinine and related molecules have been
shown to be PrPSc inhibitors in vitro112
,
117
,
141
.
Although quinacrine has not delayed the onset of disease in rodents
infected intracerebrally5
,
113
,
quinine and biquinoline have shown some efficacy when administered
intraventricularly through osmotic pumps112
.
Quinacrine, the antimalarial drug, has nevertheless been tried
extensively with little success in human CJD patients (see below).

Dimethysulphoxide. The organic solvent dimethysulphoxide (DMSO) inhibits
the aggregation of PrPSc, reduces PrPSc accumulation, promotes PrPSc
excretion in the urine and modestly prolongs the lives of
scrapie-infected hamsters107
,
111
.
Accordingly, it has been suggested that DMSO might be useful
therapeutically, especially in combination with other potential drugs.

Copper chelators. Early treatment of scrapie-infected mice with the
copper chelator D-(-)-penicillamine delays the onset of clinical
disease142
.
As the proteinase K resistance of PrPSc was enhanced in vitro by
increasing copper in a dose-dependent manner, it is possible that the in
vivo effect of penicillamine relates to a decrease in the amount of
copper available to bind to PrPSc.

Incompatible PrPC molecules and PrP peptides. One clearly demonstrated
strategy for reducing susceptibility to TSE diseases is to express PrPC
molecules that are incompatible with conversion driven by particular TSE
strains143-145
.
This effect has been demonstrated by the natural resistance of certain
host species (for example, dogs), or PrP genotypes of host species (for
example, ARR sheep, or humans heterozygous at PrP codon 129), to
specific TSE diseases146
,
147
,
and by manipulation of the susceptibility of mice by transgenic
expression of various PrP genes148
.
In vitro experiments indicate that incompatible PrPC molecules both
resist conversion themselves and block conversion of compatible PrPC
molecules that might also be present144
,
145
.
Collectively, these data indicate that the introduction of incompatible
PrPC molecules or fragments of them149
,
150

— either directly, or indirectly via gene therapy methods — would be a
useful approach for prophylaxis against TSE diseases. In any case, the
breeding of scrapie-resistant genotypes into sheep flocks is moving
ahead quickly.

Therapeutic agents

As noted above, no chemotherapeutic treatments are known to be effective
against TSEs once the clinical symptoms have developed. A number of
compounds have been tested, with little success in clinically ill
patients137
,
151
.
Recently, following its discovery as a PrPSc inhibitor117
,
141
,
the antimalarial drug quinacrine was administered to rodents5
,
112
,
113
,
152

and human CJD patients153
,
154
,
but there is little evidence of anything more than transient benefit.
Flupirtine treatments of human CJD patients reduced their deterioration
in dementia tests155
.
Intracerebral treatments of at least one CJD patient with pentosan
polysulphate have been reported by the media, but it is unknown whether
this constitutes a safe and effective therapy. Similar pentosan
polysulphate intracerebral treatments of scrapie-infected rodents showed
promising effects152
.

Non-immune neutralization of TSE infectivity

Extensive studies of harsh methods for decontaminating TSE infectivity
have been reviewed elsewhere156
.
Here we will restrict our discussion to compounds that can be mixed with
infectious inocula just before inoculation to neutralize or destabilize
infectivity. Such compounds might be useful as additives to edible or
injectable substances at risk of being contaminated with TSE
infectivity. One type of compound that fits that description are the
'beta -sheet breaker' peptides which Soto and colleagues have shown can
destabilize PrPSc and reduce infectivity titres in brain homogenates104
.
Effective infectivity titres have also been reduced by mixing scrapie
inocula with 4'-iodo-4'-deoxy-doxorubicin105
,
tetracycline106

and phthalocyanine tetrasulphonate135
.

Future prospects for chemotherapeutics

In the absence of effective TSE therapeutics, it is pertinent to ask why
the numerous compounds that have prophylactic activity tend to have so
little effect after the infection has spread within the CNS. For many
compounds, such as the large polyanions and highly charged members of
the cyclic tetrapyrrole class, it is obvious that the drugs have little
if any ability to cross the blood–brain barrier when administered
peripherally. It might therefore be helpful to find ways to improve the
delivery of such compounds to the brain either by formulating them with
carriers that improve bioavailability to the brain or by pumping them
into the brain as Doh-Ura and colleagues have done with osmotic pumps112
,
152
.

At the same time, high-throughput screens have accelerated the
identification of new PrPSc inhibitors that can cross the blood–brain
barrier94
,
117
,
134
,
137
,
141
.
However, some apparently brain-permeable PrPSc inhibitors, such as the
polyene antibiotics137
,
curcumin134
,
quinacrine and quinoline5
,
112
,
113
,
152

and others157
,
have already been tested in vivo and are unable to halt or substantially
modify pathogenesis late in the course of disease. Although the
inhibition of new PrPSc formation is likely to be a major goal in TSE
therapeutics, this might not be sufficient in the clinical phase, once
significant PrPSc has accumulated and neuropathology has occurred. At
that stage, it might be important to also destabilize PrPSc and block or
reverse the neuropathological effects of the infection using other
drugs. For instance, compounds that reduce oxidative stress, apoptosis,
aberrant signal transduction or other pathological responses of neurons
and support cells might be helpful.

As the understanding of TSE pathogenesis and the functions of normal and
abnormal PrP isoforms improves, new therapeutic targets should be
revealed. The recent report that ablating PrPC expression in adult
scrapie-infected mice prolongs their lives and even reverses pathology84

indicates that it might be rewarding to search for compounds that can
downregulate PrPC expression. Such compounds might include small
interfering RNAs158

or antisense RNAs. Furthermore, advances in basic brain biology, neural
stem-cell biology and neural differentiation might ultimately suggest
treatments — for example, with stem cells and/or neurotrophic and
differentiation factors — that could aid the recovery of lost brain
functions or prevent further neurological decline in the clinical phase
of disease. In the meantime, it will remain important to develop
practical preclinical diagnostic tests so that potential therapeutic
treatments can begin as early as possible in the pathogenic process (Box
1
).

Conclusion

The prion diseases could provide a prototype for other disorders of
protein misfolding, including Alzheimer's disease, amyotrophic lateral
sclerosis
and Parkinson's disease
159
,
160
.
It is likely that a fuller understanding of the pathogenesis and
treatment of prion diseases will provide novel diagnostic and
therapeutic approaches to other diseases accompanied by neural
accumulation of misfolded proteins, and perhaps additional currently
unrecognized post-translational disorders of the proteome.


Box


Box 1 | Testing for prion infection


The treatment of human prion diseases, and the prevention of prion
contamination of the food supply, will be crucially dependent on the
sensitive and specific detection of prion infectivity. Unfortunately,
there is, to date, no universally accepted test for the ante-mortem
detection of prion infection, despite the availability of numerous
methods to detect prion infection in brain samples. The 'gold standard'
for the definitive diagnosis of prion disease in humans and animals
depends on the detection of histological features of prion infection in
affected brains, such as degeneration of specific populations of
neurons, regional spongiform change, GLIOSIS popupWindow('/nrd/journal/v3/n10/glossary/nrd1525_glossary.html#DF15','glossary','200','300')>
and abnormal deposits of prion protein. Biochemical tests for
protease-resistant prion protein can be conducted by enzyme-linked
immunosorbent assay (such as that marketed by BioRad) or immunoblotting
(as commercially pioneered by Prionics). The conformation-dependent
immunoassay (CDI; InPro) can sensitively distinguish between misfolded
and cellular isoforms of the prion protein. Affinity reagents for the
abnormal prion protein isoform (PrPSc) (for example, from Idexx
Laboratories) are also making the transition from the lab to the field.
However, the definite diagnosis of human prion disease is contingent on
post-mortem (or biopsy) analysis of brain, despite a number of clinical
and paraclinical laboratory features that can establish a 'probable'
diagnosis during life. Similarly, diagnosis of bovine spongiform
encephalopathy, scrapie and chronic wasting disease is dependent on
access to brain samples, although research for a non-invasive,
high-throughput, inexpensive test (for blood or other accessible
biofluids) is a goal sought by at least 100 academic and commercial
laboratories.


Links

DATABASES
Entrez Gene:
Amyloid precursor protein

| TNF-alpha

OMIM:
Amyotrophic lateral sclerosis
|
Alzheimer's disease
|
Parkinson's disease

FURTHER INFORMATION
Encyclopedia of Life Sciences: Prions

| prion diseases


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Acknowledgements

N.R.C.'s work is supported by Canadian Institutes of Health Research
(Institute of Infection and Immunity), Caprion Pharmaceuticals and
McDonald's Corp.

Competing interests statement. The authors declare competing financial
interests
.

http://www.nature.com/cgi-taf/DynaPage.taf?file=/nrd/journal/v3/n10/full/nrd1525_fs.html

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