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From: TSS ()
Subject: A field on fire The biochemistry of mad cows
Date: August 2, 2005 at 8:22 am PST

A field on fire

The biochemistry of mad cows

Prion diseases are neurodegenerative diseases1 that have been linked

together because they may potentially have the same cause.These

include the diseases scrapie of sheep and BSE (bovine spongiform

encephalopathy) of cattle, and also several human diseases that include

sporadic CJD (Creutzfeldt-Jakob) disease and a variety of inherited

forms.The inherited forms of prion diseases are linked to mutations

within the gene for the prion protein.Around 85% of all human cases of

prion disease are sporadic CJD, which is a disease affecting people of

around 60 years of age. The cause of this disease remains unknown.

Unfortunately, the name of this disease causes some confusion, as it is

similar to vCJD (variant CJD), a related disease of much younger people.

David R. Brown

(Bath, UK)

The Biochemist — August 2005. © 2005 Biochemical Society 6

vCJD first emerged in the mid-1990s

and has been linked to the BSE

epidemic that reached its peak a

few years earlier. It is now largely

accepted that BSE is the cause of

vCJD. The number of deaths from

vCJD stands at 150 and the number

of deaths in 2004 was the lowest since

the disease was first diagnosed 10

years ago. The BSE epidemic arose as

a result of the feeding of rendered

animal remains to dairy cattle.

As cattle were consuming BSEcontaminated

meat, the number of

cases escalated rapidly to reach a

maximum of around 32 000 in one

year. Despite the bans that came into

force to prevent the feeding of animal

remains to cattle, BSE persists,

although the numbers are much

lower. BSE occurs in many countries

outside of the UK, ranging across

Europe and including Japan and

North America. Currently there is no

clear explanation for the persistence

of BSE. Given the reduction of vCJD

cases, many researchers feel that this

disease is disappearing along with

BSE. Others researchers do not necessarily

agree. The link between BSE

and vCJD is not fully proven and

there is a view held that vCJD and

BSE are related diseases but not

causally linked. The most widely

accepted alternative hypothesis for

the cause of vCJD (and the low levels

of BSE seen in many countries) is the

effect of an environmental factor,

possibly altered trace-element

absorption. However, at present

this remains only a hypothesis.

Research into prion diseases has

always been controversial. Stanley

Prusiner did much to advance the

protein-only hypothesis, i.e that

the abnormal isoform of the PrP

(prion protein) can be transferred

between individuals and initiate

prion disease. Despite its initially

contentious nature, this theory has

largely been accepted, and Stanley

Prusiner won the Nobel Prize for

his work in 1997. One of the

strongest pieces of evidence was the

finding from Charles Weissmann’s

group that PrP-knockout mice

are resistant to mouse-passaged

scrapie2. Again, many researchers

still don’t accept that only abnormal

PrP is required for the prion disease.

Recently, Prusiner’s group published

a paper in Science in which

recombinant PrP in a fibrillar form

was introduced into transgenic mice

and caused a prion-like disease3.

This should have been the final

proof, but the argument rages on

because the mice were ‘transgenic’.

The most widely confirmed finding

regarding the biochemistry of

prion proteins in recent years is the

discovery that the PrP binds metals.

More than 50 research groups have

now confirmed that PrP is a copperbinding

protein4. Links between

TSEs (transmissible spongiform

encephalopathies) and copper date

back to the 1960s, but the first biochemical

evidence came from two

papers looking at copper binding to


peptides. My own work, published in

1997 in Nature, initiated the upsurge

in interest in this idea, as it showed

that Cu binding to PrP could have

effects in vivo5. Recent work on this

aspect is reviewed in the accompanying

article by John Viles. The metalbinding

capacity of PrP has recently

been used to extract large amounts

of PrP from the brains of cows. This

has allowed the first NMR structural

analysis of native PrP.

In contrast to the expanding role

of metals in prion research, other

directions in the field have often been

faddish, with new ideas being taken

up and hotly explored for a number

of years and then largely abandoned.

In 1999, a protein homologue of PrP

was discovered and termed doppel.

It was thought that this protein might

play a key role in disease progress,

but now this has largely been

dismissed. Similarly, transmembrane

forms of PrP were identified in in

vitro translation systems and it was

suggested these might occur in vivo.

Again, this lead has failed to provide

any real insight and the finding has

largely been dismissed.

Central to understanding how the

disease process works is determining

the mechanism of protein conversion

from the cellular form to the diseasespecific

form. It has been known for

some time that the disease-specific

form is highly resistant to proteases,

can polymerize to form either fibrils

or complex aggregates and is high in

B-sheet content. Much work has

focused on creating in vitro methods

for generating the abnormal form of

PrP, from either recombinant or

native sources6. Many assays that

involve the use of recombinant protein

also use high concentrations of

denaturants, such as 2 M guanidine,

and although producing elegant

models of fibril formation, they are

highly artificial and are unlikely to

reflect the normal mechanism of PrP

polymerization. Thus new methods

using native conditions have been

generated. In particular, refolding

PrP with Mn can generate PrP aggregates

able to form the nucleus of PrP

polymerization. Using native PrP,

Claudio Soto’s group have generated

a polymerization technique that

involves the addition of PrPSc

extracted from brain and sonication7.

This technique vastly amplifies the

amount of protease-resistant PrP

present in the samples. Promising

studies have suggested that this technique

could also amplify trace

amounts of PrPSc in blood.

Transgenic mice have been used

as a powerful tool in prion disease

research. In particular, mice expressing

either bovine or human PrP

genes without background mouse

PrP were used to verify the link

between BSE and vCJD. In addition,

PrP knockout mice have been used

to investigate the cellular relevance

of PrP expression. In more recent

years, transgenic approaches have

been used in an attempt to understand

the pathway of peripheral

infection in prion disease and, in

particular, the role of the immune

system8. Lymphoid tissues can also

accumulate infectious prions and, in

particular, follicular dendritic cells

of the spleen are crucial to the initial

amplification of prions when they

first enter the body. Following this

initial step, prions progress into the

central nervous system in mice via

the sympathetic innervation. Other

studies have suggested that complement

components in mice are

involved in the progression of prions

towards the brain through the

immune system. It has also recently

been shown that, in mice, chronic

inflammatory conditions in the

periphery alter the distribution of

prions in peripheral tissues, and

this is dependent on expression of

lymphotoxin-. There is, however, a

slight problem with such elaborate

schemes. Mice do not normally contract

a prion disease and the immune

systems of humans and mice may

have subtle differences that would

make the findings in mice irrelevant

to disease progression in humans.

In such a short article it is not

possible to summarize fully the

extraordinary extent to which PrP

and prion diseases are being studied

in terms of basic biochemistry and

cell biology. Important advances

have been made into the mechanisms

of cellular internalization of the protein,

factors that regulate its expression,

the structural content (and how

the structure changes with metal

binding), potential binding partners

for the protein, and the protein’s

function. The evidence that the

prion protein normally acts as an

antioxidant continues to mount, but

there are other theories that are also

important. In particular, PrP expression

modifies intracellular signaltransduction

pathways and cellular

adhesion. In the study of prion

diseases, a major concern for biochemists

is how the conversion to an

abnormal isoform, rich in B-sheets,

makes the protein more resistant to

processes that should degrade it.

The resistance of abnormal PrP to

degradation by heat or other harsh

conditions is controversial (biochemically)

because the data suggest

that the bonds in the protein cannot

be hydrolysed in the same ways as

the bonds in other proteins. As some

of the findings proposed are chemically

impossible, then the supposed

super-resistance of this protein to

extreme conditions needs to be reinvestigated.

However, interactions

with metal surfaces have been found

to increase the potential infectivity

of the protein, making ways to

detect the protein on metal surfaces

and to clean surgical instruments a

high priority.

A developing field of interest is

the possible link between the cause

of sporadic TSEs and potential risk

factors in the environment. This

has led to a major programme of

research, funded by the European

Commission under the title

‘FatePride’. The link between trace

elements in the environment and

scrapie is a central interest of this

project. This was initiated by a

finding that manganese substitution

in the prion protein metal-binding

domain can cause protein conversion

to an abnormal isoform. This project

requires the comparison of highly

accurate geochemical maps that

indicate the level of bioavailable metals

with regional levels of TSEs, such

as scrapie. Unfortunately, this project

has been greatly inhibited

by officials of DEFRA selectively

withholding data on the location of

scrapie-infected farms from the geochemists

involved in the project.

Investigation of the cell-death

mechanism of prion disease follows

a circular path. In 1994, it was first

shown with cell culture experiments

that expression of native PrP by cells

was necessary for the neurotoxicity

of the toxic form of PrP. In 1996,

studies with transplantation of

brain tissue into transgenic mice

showed that PrPSc generated in the

brain could not kill neurons lacking

cellular PrP. Recently, conditional

PrP-knockout mice were developed.

When expression of PrP was

switched off during the time-course

of the disease, production of abnormal

PrP halted, and the mice recovered,

showing no further signs of

neuronal cell death9.

It is now 10 years since the panic

created by the first cases of vCJD

were identified. Has work in this field

really advanced? The major advances

have been in terms of cell biology and

biochemistry, particularly related to

the normal activity of the PrP. There

has also been a great improvement in

the diagnostic tools used to verify

that a demented person has vCJD and

not some other neurodegenerative

disorder, but this diagnosis still only

occurs after the patient is beyond

help. It is perhaps fortunate that there

has been no massive epidemic of

vCJD, as the field has not advanced

quickly enough to deal with such an

epidemic if it emerged. The research

in this field is plagued, more than in

many other fields, by obsessive

rivalry, dirty politics, miscommunication,

the withholding of data, poor

collaboration and bullying of young

researchers. Perhaps much of this is

driven by the fear of research funding

disappearing faster than new cases of

vCJD appear, but it is not helped by

the current trend among the science

funders to divert funding away from

basic research and into diagnosis and

therapy. Although these tools will

ultimately be needed10, breaking the

back of successful basic research

on the prion protein will not assist

anyone. As far as we know, the true

vCJD epidemic could be seen in

people of the same age as most CJD

patients, placing the true epidemic

20 years in the future.

Figure 3. PrP is expressed on the surface of cells as

a glycosylphosphatidylinositol-anchored protein.

Confocal images of cells expressing green-fluorescentprotein-

labelled PrP are shown.

References

1. Brown, D.R. (2005) Neurodegeneration and

Prion Disease, Springer, New York

2. Weissmann, C. (2004) Nat. Rev. Microbiol. 2,

861–871

3. Legname, G., Baskakov, I.V., Nguyen et al.

(2004) Science 305, 673–676

4. Brown, D.R. (2002) Prion Disease and Copper

Metabolism, Horwood Publishing, Chichester

5. Brown, D.R., Kefeng, Q., Herms, J.W. et al.

Nature (London) 390, 684–687

6. May, B.C., Govaerts, C., Prusiner, S.B. and

Cohen, F.E. (2004) Trends Biochem. Sci. 29,

162–165

7. Soto, C., Saborio, G. P. and Anderes, L. (2002)

Trends Neurosci. 25, 390–394

8. Aguzzi, A. and Sigurdson, C.J. (2004) Nat. Rev.,

4, 725–735

9. Mallucci, G., Dickinson, A., Lineham, J. et al.

(2003) Science 302, 871–4

10. Mallucci, G. and Collinge J. (2005) Nat. Rev.

6, 23–34

The Biochemist — August 2005. © 2005 Biochemical Society 8

David Brown was born in

Australia, where he completed

his university

training at Sydney

University, graduating in

1990 with a Ph.D. in neuroscience.

In 1993 he left

Australia and subsequently

worked in the US,

Germany and the UK. He

has worked on prion diseases for the last 12 years.

In 2001 he established a laboratory at the

University of Bath, where he is currently Reader in

Biochemistry. David Brown is a member of the

Spongiform Encephalopathy Advisory Committee.

bssdrb@bath.ac.uk

FULL TEXT WITH GRAPHICAL CHARTS;

http://www.biochemist.org/bio/02704/0006/027040006.pdf

TSS




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