PRIONSSahil Kulkarni

Department of Zoonosis HAFFKINE INSTITUTE

PRIONSWhat is a PRION? A prion is an abnormally folded

protein that acts as an infectious particle

A prion arises from a normal protein in the body to become a deadly agent when it undergoes a shape change that prevents it from being broken down by normal degradative activities of the body

Background

• Prions are not viruses - They don’t contain genetic material .

• “Prion” - short for proteinaceous infectious particle (coined by Prusiner), made ONLY of protein. Therefore, they are resistant to nucleases .

• The specific protein that the prion was made of was named PrP, an abbreviation for "prion-related protein".

DISCOVERY

The radiation biologist Tikvah Alper and the mathematician John Stanley Griffith developed the hypothesis during the 1960s that sometransmissible spongiform encephalopathies are caused by an infectious agent consisting solely of proteins.

This theory was developed to explain the discovery that the mysterious infectious agent causing the diseases scrapie and Creutzfeldt–Jakob disease resisted ultraviolet radiation (UV radiation damages nucleic acids).

Stanley B. Prusiner of the University of California, San Francisco announced in 1982 that his team had purified the hypothetical infectious prion, and that the infectious agent consisted mainly of a specific protein – though they did not manage to isolate the protein until two years after Prusiner's announcement.

5

Stanley Prusiner , M.D. from UCSF

Nobel Prize in Medicine 1997 (1982-1997, 15 years for Nobel Prize Award)

Prusiner coined the word "prion" as a name for the infectious agent. While the infectious agent was named a prion, the specific protein that the prion was composed of is also known as the Prion Protein (PrP), though this protein may occur both in infectious and non-infectious forms. Prusiner was awarded the Nobel Prize in Physiology or Medicine in 1997 for his research into prions .

PRION termed by Prusiner “Proteinaceous Infectious Particles Suffix with - ON = elementary particle

History1738: First clinical manifestation of scrapie described .1955: Vincent Zigas begins clinical study of kuru in Papua New Guinea .1956: Dr. Carleton Gajdusek begins investigation of kuru : Noble Prize in 19761959: WJ Hallow observes the similarity between kuru and scrapie . 1962: H.B. Parry believed that scrapie can be eradicated by breeding methods . 1965: First chimpanzees injected with brain extracts of both kuru and CJD patients developed similar symptoms to the respective diseases . 1982: Stanley Prusiner et al: develop animal model for studying prion infectivity. 1986: BSE epidemic in England. Believed to arise from contaminated feed. 1988: N. Hunter observes fibrils in BSE infected cows that are similar to scrapie protein. 1990: J. Hope determines two alleles of protein gene linked to scrapie in sheep. 1991: Stanley Prusiner elucidates the molecular biology of prion proteins. 1993: T.G.F. Esmonde determines possible links to CJD caused from BSE.(vCJD) 1997: Stanley Prusiner : Nobel Prize for work in Prion concept 1999: Discovery of the PrP homolog (Moore et al.,1999)2001: Complement involved in prion pathogenesis ( Klein et al.,2001)2003: Transgenic expression of soluble PrP inhibits prion replication (Meier et al.,)

7

PrPC Structure

• Cellular prion protein 50% alpha-helix content 20% beta-sheet content Completely sensitive to

proteinase-K digestion “ PrP-sen ” GPI-linked surface

glycoprotein t 1/2 ~ 3-6 hr

Normal cellular protein (PrPc)• PrPc which is a normal cellular host

protein encoded by a single axon of a single copy gene.

• Found predominantly on the surface of neurons attached by a glycoinositol phospholipids anchor, and is protease sensitive.

• Involved in synaptic function.

9

PrPSC Structure• Scrapie prion protein

High in beta-sheet content (>40%)

Partial resistance to proteinase K digestion “ PrP-res ”

Can form aggregated fibrous or amyloid structure

Intracellular/cell surface /extracellular space

t 1/2 > 24 hr

Prion: a modified form a PrPc• Prion is known as PrPsc (for scrapie) which

is relatively resistant to proteases and accumulates in cytoplasmic vesicles of diseased individuals.

• Insertion of PrPsc into a normal cell causes the conversion of PrPc to PrPsc. The nature of process involves conformational modification.

• Then these mis-shapen or abnormal proteins collect up in nerve cells and eventually crowd out normal functions of individual brain cells.

• Apparently prions are able to recruit and convert normal proteins into pathogenic form.

One protein: Two structuresPrPc “NORMAL” conformation

PrPsc “BAD” conformation

PROPOSED MECHANISM OF PRION PROPAGATION

Prion video http://highered.mcgraw-hill.com/sites/dl/free/0072835125/126997/animation44.html

Transmissible Spongiform Encephalopathies

• Prion diseases are often called transmissible spongiform encephalopathies (TSE).

• Because of the post mortem appearance of the brain with large vacuoles in the cortex and cerebellum. Probably most mammalian species develop these diseases.

Prion diseases

HOW PRPSC ATTACK THE BRAIN?

• Brain consists of a mass of nerve tissue (Hundred billions of neuron) and neuroglia, supporting neural tissue .

• When enough PrPSc proteins have been made they form long filamentous aggregates that gradually damage neuronal tissue.

• When neuron in the brain are all dead, the appearance of the brain will become sponge-like appearance. And this eventually lead to death.

• The harmful PrPSc form is very resistant to high temperatures, UV-irradiation and strong degradative enzymes.

DIFFERENT PRIONS AFFECT DIFFERENT REGIONS OF THE BRAIN

• Cerebral cortex: the symptoms include loss of memory and mental acuity, also visual imparement (CJD).

• Thalamus: Fatal Familial Insomnia (FFI). • Cerebellum: lose the control of body movements and difficulties to walk (kuru, GSS). • Brain stem: In the mad cow disease (BSE), the brain stem is affected.

Prions are contracted in a few different ways:

• Eating tissue infected with PrP-res.

• An inherited mutation in the

gene that encodes for normal PrP.

• Spontaneous formation of PrP-

res (rare).

The following diseases are caused by prions :

In animals:

• Scrapie in sheep .• Bovine spongiform encephalopathy (BSE) in cattle (known

as mad cow disease) .• Transmissible mink encephalopathy (TME) in mink .• Chronic wasting disease (CWD) in elk and mule deer .• Feline spongiform encephalopathy in cats .• Exotic ungulate encephalopathy (EUE) in nyala, oryx and

greater kudu .

SCRAPIE:

• This disease is of sheep (and goats) .• The first TSE to be studied.• Known for several hundreds yrs . • Transmitted from animal to animal in

feed contaminated with nerve tissue. • Loss of motor control, paralysis

wasting and death.

20

Bovine Spongiform Encephalopathy (BSE)• Mad cow disease • Inc. period 2-8 y• Sensitive • Weight loss• Decrease in milk product• Died in 2 wk - 6 M (after symptom)

• Although no. of cattle with BSE (UK) till 2003 is >180,000, the total estimated number is excess of 2 million.

• Between 1980-996 ,about 750,000 BSE-infected cattle's have been slaughtered and consumed by millions of U.K. residents.

mad cow disease is also transmitted thru oxytocin injections which is given to increase milk yeild ,these injections are derived from Animals

CHRONIC WASTING DISEASE (CWD)

• Occur in the deer family of mule deer and elk.

• Prion proteins accumulate in the brain of affected individuals, causing neural degeneration and inevitably, death.

• Only place where the disease is known to occur is in northeastern Colorado and southeastern Wyoming.

In humans :• Creutzfeldt-Jakob disease (CJD) and its

varieties• Gerstmann-Sträussler-Scheinker syndrome

(GSS) • Fatal familial insomnia (fFI) • Sporadic fatal insomnia (sFI) • Kuru• Alpers syndrome

KURU

• Found in geographically isolated tribes in the Fore highlands of New Guinea. The disease is caused in Cerebellum

• Established that ingesting brain tissue of dead relatives for religious reasons was likely to be the route of transmission.

CANNIBALISM• It was usually found in women and children since they were

the ones eating the brains by grinding up the brain into a pale grey soup, heated and ate it.

• It shows slow onset and is manifested by cerebellar signs and shaking ataxia. Death occurs with 18 to 24 months.

• Since the discovery of the relationship between cannibalism and kuru, the practice of cannibalism had been stopped and the incidence of the disease has abated.

CREUTZFELD-JACOB DISEASE (CJD)

• Cerebral cortex is infected• An uncommon, sporadic, degenerative disease in

humans.• Occurs during middle decades of life.• The course is sub acute , ranging from 3 to 6

months in duration.• Manifested by dementia accompanied by motor

signs and blindness.

26

vCJD, in the USA • A girl lived in Florida, USA.

• She was born in England (1979).

• Moved to the United States in 1992.

• Contracted the disease when she lived in England. • November 2001, began to forget things and lose her temper, her hand began to

shake pretty rapidly and stumble walking .

• Took to England in early 2003, where she was diagnosed with probable vCJD. • Received experimental treatment with quinacrine for 3 months but not improve

conditions.

• All treatments failed, she died in June 2004 (25 years)

GERSTMANN-STRAUSSLER-SCHEINKER SYNDROME

• Abbreviated as GSS (Cerebellum)• Characterised by cerebellar ataxia and

concomitant motor problems.• Disease course lasts several years to death

FATAL FAMILIAL INSOMNIA (THALAMUS)

• An autosomal dominant disorder characterized by degeneration of the thalamus and progressive insomnia.

• That means both sexes are affected and there are no carriers.

• Caused by mutation in the prion that is associated with with brain tissue and if individual inherits the mutant gene, that individual will at some point suffer the disease.

“FFI”• Symptoms are vegetative state of sleep, inability to

produce tears or feel pain as well as poor reflexes and dementia.

• The disease also results in the formation of amyloid plaques. This is the build up of a waxy substances made of proteins associated with polysaccharides.

DIAGNOSIS1 . EEG : Two cycles per second triphasic sharp-wave discharges. 2. CSF : Presence of 14-3-3 protein, 3. Histopathology : Biopsy or Autopsy (confirmatory diagnosis): The

typical neuropathology consists of a microscopic picture of spongiform changes, gliosis, and neuronal loss in the absence of inflammatory reaction. Amyloid plaques demonstrated in >>10% of patients.

4. PrPsc : The presence of PrPsc in biopsy or autopsy of brain samples can be demonstrated by immunodiagnostic tests, such as:

a) immunohistochemical staining. b) Histoblot c) Western blot techniques d) Conformation-dependent immunoassay(CDI)5. Genetic test : Sequencing the PRNP gene

Prion diseases damage the brain

PREVENTION AND THERAPEUTICS

• There is no known effective therapy for preventing or treating prion diseases.

• The finding that phenothiazines and acridines inhibit PrPSc formation in cultured cells led to clinical studies of quinacrine in patients.

• Although quinacrine seems to slow the rate of decline

in some CJD patients, no cure of the disease has been observed.

DECONTAMINATION OF CJD PRIONS

• Prions are extremely resistant to common inactivation procedures, and there is some disagreement about the optimal conditions for sterilization.

• Autoclaving at 132°C for 5 h or treatment with 2 N NaOH for several hours is recommended for sterilization of prions.

• The term "sterilization" implies complete destruction of prions; any residual infectivity can be hazardous.

STATISTICS• The incidence of sporadic CJD is about 1 per

million per year.

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

• It is estimated that 1 in 10,000 people are infected with CJD at the time of death.

DO NOT UNDERESTIMATE!

• These figures from previous page are likely to be underestimated since prion diseases may be misdiagnosed as other neurological disorders.

• The diseases are characterized by loss of motor control, dementia, paralysis wasting and eventually death, typically following pneumonia.

THANK YOU !