Lecture 21 Prions

Kuru

•Presented with prolonged headache and minor loss of coordination

•Symptoms rapidly progressed and by 4 months victims unable to walk requiring constant care for washing and feeding

•Speech became disturbed and completely disappeared

•Victims lost ability to swallow

•Death occurred within 1 year of onset of symptoms

Possibilites of Kuru

•Infectious agent?
>Disease occurred in clusters of cases
>Victims failed to show classic signs of infectious disease (no fever, no increased blood lymphocyte count)
>No inflammatory response in brain tissues
>Tissue culture and animal (mice, rats, guinea pigs) inoculations negative

•Environmental factors?
>Metallic poisons, Smoke from cooking fires, Hot stones used for cooking food, Food and water, Straw used to construct huts + Soil not involved

•Heredity?
>Disease developed within families
>Outsiders never developed Kuru

•Mass hysteria?
>Not a disease of old age or senility
>Genuine neurologic signs cannot be mimicked by hysteria
>Neither a psychotic disease nor hysterical disease

•Cannibalism
>Practice not unusual to New Guinea that would provide an avenue for   passage of infectious agent
>Fore tribe ate tissues (especially the brain) of close relatives as expression of sorrow, respect, and love for deceased
>A religious ceremony
>But no infectious agent could be detected in laboratory studies

What were the abnormalities of the Kuru brain, and what was closely associated?

1. Loss of neuron (spongiform encephalopathy)

2. Activation of astrocytes (react to foreign entities)

3. No infectious sign

4. No inflammation

Creutzfeldt-Jakob disease

Creutzfeldt-Jakob disease

•Rapidly progressive global dementia, myoclonus, and marked progressive motor dysfunction (tremors)

•Normal blood, cerebrospinal fluid, CT, and MRI findings

•Death occurs within 1 year of onset of symptoms

•Definitive diagnosis depends on brain pathology at time of biopsy/autopsy

•Termed spongiform encephalopathy due to gaping holes where neurons had once been

•Sporadic, Familial, Iatrogenic form for regular CJD, and variant form (vCJD)

Why are adult men not affected by Kuru, and only Fore tribe?

Only women and children of the Fore tribe ate the brains of deceased relatives

Men + older boys lived away from women/children, didn’t share meat with the women. Women raised pigs but gave them to the men, survived on vegetables, insects, frogs, and small rodents (little protein)

When did Kuru appear? Why is this significant?

In 1900 in Northern Fore territory, then down to Central/South Fore by 1930s. Fore women began eating brains of deceased disguised under religious practice due to lack of protein.

Women cooked infectious brains, but also through cuts, opens sores, scratches, mouth mucus membranes. Small kids would get pieces of brain during playtime.

Kuru’s impact on medicine, neurology, infectious diseases

•Opened new area of slow virus diseases

•Opened new area of transmissible spongiform encephalopathies

•Lead to identification of new infectious agent, the prion

Slow virus diseases

Years may separate the time of initial contact with infectious agent and appearance of clinical disease

>Conventional agents (viruses)

>Unconventional agents (prions)

Measles and Subacute Sclerosing Panencephalitis (SSPE)

Conventional, viral agent

•Usually presents clinically 6 - 10 years after primary measles virus infection

•Occurs in immunologically normal persons who have had measles at younger than 2 years of age (50%) or prior to 4 years of age (80%)

•A slowly progressive measles virus infection of the brain that is poorly controlled by immune responses

•Pathology is significant for inclusion bodies / inflammation

•Recognized in 1 : 1,000,000 cases of primary measles virus infection

Transmissible Spongiform Encephalopathies

rare, but always fatal, slow neurodegenerative diseases caused by an    unconventional infectious agent(s)

•Dementia and/or ataxia; loss of brain function; death

•Prominent astrocytosis and neuronal loss

•Lack of inflammation!

•Possible appearance of amyloid-like plaques or fibrils

•Etiologic agent traced to prion (small proteinaceous infectious particle), a protease-resistant protein (PrP)

Unconventional Agents

Animals

•Scrapie in sheep

•Transmissible mink encephalopathy

•Bovine spongiform encephalopathy (mad cow disease)

Humans

•Creutzfeldt-Jakob disease

•Variant Creutzfeldt-Jakob disease (mad cow disease)

•[Kuru]

Scrapie agent

passed through filters that do not transmit bacteria but transmit viruses

agent infectivity not completely eliminated by ultraviolet light, nucleases, standard fixation of tissue samples, and many kinds of sterilization procedures including standard autoclaving → self- replication protein, no nucleic acid

PrP

proteinase-resistant protein → infectious protein

•Smaller in size than conventional viruses

•Remarkable hydrophobic properties (loves binding to membranes)

•Resistant to ionizing radiation & UV light

•Resistant to formaldehyde, alcohols, nonionic detergents, proteinase K

•Resistant to conventional autoclaving (15 - 30 min at 121 C)

•Resistant to nucleases including DNase

Sequencing of PrP followed by cloning its gene = PrP is a protein found in normal hosts including humans that would explain lack of immune response (immunologic tolerance)

•Entire ORF resides in single exon, so no RNA splicing

•A 33-35 kd protein encoded by the PRNP human gene found on  chromosome 20

•An analogous gene identified in mice, other mammals, birds, and fungi 

Protein found attached to membrane rafts and found on cell surface

•PrP mRNA is constitutively expressed in brains of healthy animals including humans
>Highest levels of PrP mRNA found in neurons
>PrP expressed on surface of neurons 

•PrP also expressed in many non-neuronal cells that include T-lymphocytes and B-lymphocytes

PrP^C

cellular isoform of the prion protein [good form]

PrP^{SC or CJD}

abnormal pathogenic isoform of prion protein that causes illness [bad form]

PrP^C vs. PrP^SC

Both encoded by same gene and have same amino acid comp. (immunological tolerance and therefore no inflammation)

•PrP^C is sensitive to proteinase K, whereas PrP^SC is resistant

•PrP^C is soluble in nondenaturing detergents, whereas PrP^SC  insoluble

•PrP^SC (but not PrP^C) aggregates and forms amyloid plaques in brains with TSE

•PrP^C has an α-helix content of 42% and little β-sheet, whereas PrP^SC has an α-helix content of ~30% and 45% β-sheet content
>Explains differences in sensitivities to proteinase K / detergents
>Explains ability of PrP^SC to aggregate and form amyloid plaques

Kuru meaning

‘trembling from fear and cold’

Prion hypothesis

after infection with the PrP^SC isoform of the PrP protein, the PrP^SC isoform causes the PrP^C isoform to be converted into the PrP^SC isoform

PrP summary

•PrP is a normal cellular protein (PrPc)

  > Protease sensitive

  > Alpha helix conformation

•PrPc is converted to abnormal form (PrPsc)

  > Protease resistant

  > Beta helix conformation → abnormal beta-sheets of protein → amyloid plaques (TSE)

•PrPsc can induce other PrPc to become PrPsc

Sporadic form

•Accounts for ~85% of all cases of CJD

•Bulk of cases occur between ages 50 and 79
>By age 80 years, incidence drops
>Suggests an exposure to infectious agent at specific age followed by very long incubation period

•Presents as a presenile dementia characterized by rapidly progressive mental deterioration, myoclonic jerking, deterioration of motor functions, ataxia, and death within one year of onset of symptoms

Familial form

•Accounts for 5% to 15% of all cases of CJD

•A genetic disorder due to point mutations / insertions in the PRNP gene found on short arm of chromosome 20

•Onset takes place at a younger age when compared with sporadic CJD

•Disease progression characterized by progressive insomnia, autonomic dysfunction, endocrine changes, decline in motor / cognitive functions, and death within one year of onset of symptoms

Iatrogenic Form

•Accounts for ~1% of all cases of CJD

•Medical transmission of infectious agent
>Corneal transplantation
>Electrodes used in neurosurgical procedures
>Human growth hormone
>Human gonadotropin
>Human cadaveric dura mater in neurosurgical procedures

•Median incubation period of 13 years

•Disease progression similar to sporadic

•Kuru, an epidemic of iatrogenic form of CJD

Variant form (Mad Cow Disease)

•First recognized in 1996 in Great Britain as a form of CJD whose clinical picture differs greatly from that of sporadic CJD

•Cases occur in younger age group (mean age = 26 years)

•Early stages of illness present with psychiatric symptoms (depression, withdrawal, anxiety, and irritability)

•Sensory symptoms develop (limb pain, numbness, cold feelings) that progress to ataxia, dementia, and death usually 13 months after onset of symptoms

 •Origin unclear
>Transmission of infectious agent responsible for bovine spongiform encephalopathy that was recognized in Great Britain in 1986?
>Identified in patients who received blood transfusions from donors who later died of vCJD

Prion Inactivation [BONUS, will ask for 4/5 for 8 pts]

•Incineration

•Treatment with mercaptoethanol, SDS (breaks sulfide bond → change structure, no more beta sheets)

•Treatment with sodium hypochlorite (20,000 ppm)

•Immersion for 60 min in 2N sodium hydroxide

•Autoclaving for 90 min at 132-136 C (versus 15-30 min at 121 C)

Gloves must be worn when handling biopsy/autopsy tissues even after fixation