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PRIONSPRIONS

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Definition

PRION: Infectious protein particles thought to be

responsible for a group of transmissible and/or inherited

neurodegenerative diseases.

"Prion" is short for "Proteinaceous Infectious particle"

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StructurePrions are infectious agents composed exclusively of a

single sialoglycoprotein called PrP 27-30.

They contain no nucleic acid.

PrP 27-30 has a mass of 27,000 - 30,000 daltons and is

composed of 145 amino acids with glycosylation at or near

amino acids 181 and 197.

This protein polymerizes into rods possessing the

ultrastructural and histochemical characteristics of amyloid.

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Humans diseases Evidence suggests that a prion is a modified form of a

normal cellular protein known as PrPc (for cellular), a

single copy gene.

This protein is found predominantly on the surface of

neurons attached by a glycoinositol phospholipid anchor,

and is protease sensitive. Thought to be involved in

synaptic function.

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Humans diseases The modified form of PrPc which may cause disease i.e.

the prion is known as PrPsc (for scrapie) which is

relatively resistant to proteases and accumulates incytoplasmic vesicles of diseased individuals.

It has been proposed that PrPsc when introduced into a

normal cell causes the conversion of PrPc into PrPsc.

Process is unknown but it could involve a chemical or

conformational modification.

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Humans diseases PrPc serves a function that is not apparent in a

laboratory setting

Current possibilities involve shuttling or sequestration

of synaptic Cu(II) via binding to N-terminal octapeptide

residues and/or signal transduction involving the fyn

kinase.

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ReplicationThe prion is a product of a human gene, termed the PrP

gene, found on chromosome 20.

This gene contains two exons separated by a single intron.

Exon I and Exon II are transcribed and the two RNAs ligated

into a single mRNA.

This mRNA contains an open reading frame (ORF) or protein

coding region which is translated into the PrP protein.

The PrP protein is a precursor of the prion protein. It is

termed PrP 33-35.

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ReplicationThe PrP 33-35 undergoes several post-translational events to

become the prion protein (PrP 27-30):

1. Glycosylation - at two sites.

2. Formation of a disulfide bond between two cysteine

residues.

3. Removal of the N-terminal signal peptide.

4. Removal of the C-terminal hydrophobic segment.

5. Addition of a phosphatidylinositol glycolipid at the C-terminal.

6. Removal of the N-terminal first 57 amino acids.

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Replication

In normal cells only the PrP 33-35 protein is synthesized.

It is found in the neural cell membrane where it's function is

to sequester Cu++ ions.

In abnormal ("infected") cells, the PrP 27-30 is produced from

the PrP 33-35 protein.

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ReplicationThe PrP 27-30 triggers a series of reactions that produce

more PrP 27-30 proteins, i.e., PrP 27-30 induces its own

synthesis.

In addition to the post translational modifications, the PrP 27-

30 protein differs from the PrP 33-35 protein in a single

amino acid residue.

Residue 178 in the PrP 27-30 contains an asparagine

residue whereas the PrP 33-35 protein has an aspartate

residue at this position.

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Replication

This causes a conformational change in the PrP 27-30

protein from an -helix to a -sheet. This conformational

change in the PrP 27-30 protein has three effects:

1. It imparts to the PrP 27-30 protein the ability to induce

the same -helix to -sheet conformation in the PrP 33-35

protein. This is a permanent conformational change. It thus

induces its own "replication."

2. The -sheet-forming peptides aggregate to form amyloid

fibrils.

3. The amyloid fibrils kill thalamus neurons through

apoptosis.

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Pathologies induced by prionsAll diseases known to be of prion etiology, in animals and

humans, are neurodegenerative diseases. In the human this

includes:

Creutzfeldt-Jakob disease (CJD)

Fatal Familial Insomnia

Gerstmann-Straussler syndromeKuru

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Pathologies induced by prions

The pathological and clinical signs of these diseases suggest

that they are closely related.

In fact they may be variants of the same disorder.

All pathological features are confined to the central nervous

system.

The prion protein accumulates selectively and abnormally in

CNS nerve cells during the course of the disease.

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Pathologies induced by prions

PrP 27-30 accumulates within the neuronal system where it

causes:

1. Astrocyte gliosis (an increase in the number of

astrocytes).

2. Depletion of dendritic spines in neurons.

3. Formation of numerous vacuoles in the cerebellar cortex

(spongiform encephalopathy).

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Pathologies induced by prions

4. Amyloidosis - deposition of amyloid in the cerebellar

cortex, thalamus, brain stem and in the lumen of bloodvessels within the brain.

These amyloid plaques consist of discrete eosinophilic

glassy-appearing masses, often having radiating amyloid

fibrils at their periphery.

The plaques are primarily subependymal, subpial and

perivascular.

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Pathologies induced by prionsNote that the pathology does NOT include any signs of

inflammation or fever.

This is evidence that the immune system does not respond to

the prion protein.

Since the prion protein is derived from self this is what youwould expect.

These pathologies give rise to the clinical symptomology

seen in these patients.

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Pathologies induced by prionsThese are:

1. A long incubation period (several years) which has given

rise to the term "slow infection."

2. Loss of muscle coordination which leads to a difficulty in

walking, indicating a functional disorder of the cerebellum.

3. Dementia characterized initially by loss of memory,

diminished intellect and poor judgment.

4. Progressive insomnia characterized by a marked

reduction or loss of the slow-wave and rapid-eye-movement

phases.

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TransmissionSpread of the disease is via horizontal transmission, i.e.,

transmission from one person to another, either directly or

by ingestion of contaminated meat.

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DiagnosisIn the past, diagnosis of prion disease was made through

examination of brain biopsies taken from patients inadvanced stages of the disease or, more commonly, after

they had died.

In January of 1999 it was found that the prion protein

accumulated in the tonsils and could be detected by an

immunofluorescence test on tonsilar biopsies.

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DiagnosisA second test was simultaneously developed which was

based on a Western blot.

Later that year a third test was developed that had the high

sensitivity necessary to detect the prion protein in blood.

This test is based on capillary electrophoresis with laser-induced fluorescence. It detects as little as 10-18 mole.

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Prion DiseasesPrion diseases are often called spongiform encephalopathies

because of the post mortem appearance of the brain withlarge vacuoles in the cortex and cerebellum.

Probably most mammalian species develop these diseases.

Specific examples include:

Scrapie: sheep

TME (transmissible mink encephalopathy): mink

CWD (chronic wasting disease): muledeer, elk

BSE (bovine spongiform encephalopathy): cows

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Humans are also susceptibleto several prion diseases:

CJD: Creutzfeld-Jacob Disease

GSS: Gerstmann-Straussler-Scheinker syndrome

FFI: Fatal familial Insomnia

Kuru

Alpers Syndrome

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Humans are also susceptible

to several prion diseases:

The incidence of sporadic CJD is about 1 per million peryear.

GSS occurs at about 2% of the rate of CJD.

It is estimated that 1 in 10,000 people are infected with CJD

at the time of death.

These figures are likely to be underestimates since prion

diseases may be misdiagnosed as other neurological

disorders.

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Humans are also susceptibleto several prion diseases:

The diseases are characterized by loss of motor control,

dementia, paralysis wasting and eventually death,

typically following pneumonia.

Fatal Familial Insomnia presents with an untreatable

insomnia and dysautonomia. Details of pathogenesis arelargely unknown.

Visible end results at post-mortem are non-inflammatory

lesions, vacuoles, amyloid protein deposits and

astrogliosis.

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Humans are also susceptible

to several prion diseases:

GSS is distinct from CJD, it occurs typically in the 4th-5th

decade, characterized by cerebellar ataxia and concomitant

motor problems, and less common dementia.

(Originally thought to be familial, but now known to occur

sporadically as well).

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Humans are also susceptibleto several prion diseases:

CJD typically occurs a decade later has cerebral

involvement so dementia is more common and patient

seldom survives a year (originally thought to be sporadic, but

now known to be familial as well).

FFI pathology is characterized by severe selective atrophy ofthe thalamus.

Alpers syndrome is the name given to prion diseases in

infants.

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Humans diseases Kuru is the condition which first brought prion diseases to

prominence in the 1950s. Found in geographically isolated

tribes in the Fore highlands of New Guinea.

Established that ingesting brain tissue of dead relatives for

religious reasons was likely to be the route of transmission.

They ground up the brain into a pale grey soup, heated it and

ate it.

Clinically, the disease resembles CJD. Other tribes in the

vicinity with same religious habit did not develop the disease. It

is speculated that at some point in the past a tribe member

developed CJD, and as brain tissue is highly infectious thisallowed the disease to spread. Afflicted tribes were encouraged

not to ingest brain tissue and the incidence of disease rapidly

declined and is now almost unknown.

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Humans might be infected byprions in 2 ways:

1. Acquired infection (diet and following medical procedures

such as surgery, growth hormone injections, corneal

transplants) i.e. infectious agent implicated.

2. Apparent hereditary Mendelian transmission - where it is

an autosomal and dominant trait. This is not consistentwith an infectious agent.

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Humans might be infected by

prions in 2 ways:

This is one of the features that single out prion diseasesfor particular attention.

They are both infectious and hereditary diseases.

They are also sporadic, in the sense that there are also

cases in which there is no known risk factor although it

seems likely that infection was acquired in one of the two

ways listed above.

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Prion diseases:Scrapie was the first example of this type of disease to be

noticed and has been known about for many hundreds of

years.

There are two possible methods of transmission in sheep:

1. Infection of pasture with placental tissue carrying the

agent followed by ingestion,or direct sheep-lambtransmission i.e. an acquired infection.

2. Parryshowed considerable foresight by suggesting that

it is not normally an infectious disease at all but a genetic

disorder. He further suggested that selective breeding

would get rid of the disease.

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Several lines of evidence support protein only

model of infection:1. Nucleic acid is not necessary for infectivity:

failure to identify a scrapie specific nucleic acid either in prion

preparations or infected brains using a variety of sophisticatedtechniques.

unusually small target size for ultraviolet and ionizing radiation:

the low ratio of nucleic acid to infectious material.

resistance of infectivity to agents which modify or damage nucleic

acids but infectivity is susceptible to reagents which destroy proteins:

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Stabilities of the scrapie agent and viriods (PSTV):

Chemical Treatment: Concentration PSTV Scrapie

Et2PC 10-20mM (-) +

NH2OH 0.1-0.5mM + -

Psoralen 10-500g/ml + -

Phenol Saturated - +

SDS 1-10% - +

Zn2+ 2mM + -

Urea 3-8M - +Alkali pH 10 (-) +

KSCN 1M - +

Enzymatic Treatment: Concentration PSTV Scrapie

RNAse A 0.1-100g/ml + -

DNAse 100g/ml - -

Proteinase K 100g/ml - +

Trypsin 100g/ml - +

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2. PrPsc is associated with scrapie infectivity:

purification of scrapie infectivity results in preparationshighly enriched for PrPsc

purification of PrPsc results in enrichment of scrapie

activity

purification of PrPsc by SDS-PAGE also recovers

infectivity

PrPsc can be denatured and renatured without loss of

infectivity

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3. Susceptibility of a host to prion infection is co-determined by the prion inoculum and the PrP gene:

disease incubation time for a single prion isolate varies

between mouse strains,

this variation depends on the PrP gene,

suggesting some forms of PrPc may be more easily

converted to PrPsc than others.

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3. Susceptibility of a host to prion infection is co-

determined by the prion inoculum and the PrP gene:

when prions are transmitted from one species to anotherdisease develops only after a very long incubation period, if

at all, but on serial passage in the new species the incubation

time often decreases dramatically and then stabilizes.

This species barrier can be overcome by introducing a PrP

transgene from the prion donor i.e. hamster PrPc but not

murine PrPc is a suitable substrate for conversion to hamster

PrPsc by hamster prions and vice versa:

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is co-determined by the prion inoculumand the PrP gene:

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4. Mutated gene can cause susceptibilityto disease without apparent infection:

Homozygosity at the polymorphic amino acid position 129 of PrP

protein predisposes an individual to acquired and sporadic CJD

2 unrelated GSS families have the same double mutation i.e. 178

D-N; 200 E-K

disease tightly linked to a P-L mutation at 102 in some familial

GSS cases

100 controls did not have this mutation nor did another 15

sporadic victims of CJD

other familial cases have been shown to carry this or several

other mutations e.g. 117-A-V; 198 F-S; N178; V129 = CJD; N178

M129 = FFI

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4. Mutated gene can cause susceptibility todisease without apparent infection:

One possibility, the genetic explanation, was that these

mutations enhance the rate of spontaneous conversion of

PrPc to PrPsc which permits disease manifestation within the

lifetime of an individual.

Suggests that some sporadic incidents can be accounted for

by somatic mutation of the PrP gene.

An alternative explanation is that these mutations confer

susceptibility to infection

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5. Crucial experiment:

Mice carrying a murine transgene with the 102P-L GSS

mutation spontaneously develop a lethal scrapie like disease.

Brains also contain infectious prions because transmission to

recipient animals has been demonstrated.

Mice lacking the PrPc gene develop normally, no evident

physiological or behavioural problems.

Suggests that loss of PrPc function is unlikely to be the

cause of disease rather accumulation of PrPsc is

responsible.

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5. Crucial experiment:

When inoculated with prions they do not develop disease.

There are obvious implications here for the livestock and

pharmaceutical industries and treatment of familial cases.

Animals expressing reduced levels of PrPc are also resistant

to infection - perhaps more immediately relevant to human

disease.

Animals overexpressing PrPc are more likely to develop

prion diseases.

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Evidence against the prion model:The existence of many different strains of scrapie (at least

15: latency, lesion patterns differ) which can be propagated

in the same inbred mouse strain.

These can transmit serially without changes in properties in

the same mouse strain homozygous for a single PrP

genotype.

Cannot argue that there a distinct PrPc is converted to

distinct PrPsc.

Must mean that a common PrPc is corrupted in a different

way, seems improbable but there is now experimentalsupport for it.

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So how can a protein be infectious?A likely possibility is that the difference between PrPc and

PrPsc is conformational.

It is proposed therefore that this protein can adopt two quite

different stable conformations.

The safe PrPc form is normally adopted but rarely it can

switch to the PrPsc form.

Mutations favour this switch.

It is proposed that PrPsc is transdominant and converts

PrPc to PrPsc in an exponential fashion.

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The structure of part of the hamster and mouse

PrPc molecules superimposed

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The position of various mutations important forprion disease development in humans.

Many of these mutations are positioned such that they

could disrupt the secondary structure of the molecule.

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TreatmentPrPc overexpression facilitates the development of prion

diseases.

It may therefore follow that agents which reduce PrPcexpression will delay the onset of prion diseases.

One can speculate that chemicals which bind to and stabilize

the PrPc conformation may be beneficial.

Similarly agents destabilizing the PrPsc conformation may be

effective. In this regard several vaccines to Alzheimers

amyloid plaques are in clinical trials.

Agents which interfere with the putative PrPc-PrPsc

interaction might similarly be effective.

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Treatment

A number of reagents showing affinity for amyloid proteins

are known e.g congo red.

As our knowledge of the structure of PrPc increases, the

chances of rationally deducing effective therapeutics based

on these ideas increases.

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Treatment

Finally we have seen that PrPc expression is required for

pathology.

Chemicals affecting the endocytosis, exocytosis, intracellular

trafficking and degradation of proteins and in particular PrPc

may also be effective.

Amphotericin for instance is reported to delay prion disease

in hamsters (although it apparently has little effect in

humans).

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References:

Prions.http://www.cartage.org.lb/en/themes/

Sciences/LifeScience/GeneralBiology/Micr

obiology/PrionsViroids/Prions/Prions.htm