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what are prion diseases?

fatal neurodegenarative diseases in animals and humans


name a few prion diseases in animals?

1. Sheep - Scrapie
2. Cattle - Bovine spongiform encephalopathy (BSE)
3. Mink - Transmissible mink encephalopathy (TME)
4. Mule, deer, elk - Chronic wasting disease (CWD)
5. Cats - Feline spongiform encephalopathy (FSE)
6. Greater kudu, nyala, onyx - Exotic ungulate encephalopathy (EUE)


name a prion diseases in human for
1. sporadic
2. genetic
3. acquired

1. sporadic - CJD (Creutzfeldt-Jakob disease)
2. familial - familial CJD, GSS, FFI (fatal familial insomnia)
3. acquired - vCJD (variant CJD), iatrogenic CJD, Kuru


what are the THREE main characteristics of PD?*

1. vacuolation
2. amyloid deposition
3. gliosis


what are the clinical signs of scrapie sickness?
1. appearance
2. muscular tone
3. behaviour
4. changes in movement

1. general appearance
- ruffled fur, arched back
- lacerations on neck or back
- loss of appetite
- decrease (or increase) in weight
- shivering
- rigid or straight tail
2. muscular tone - involuntary muscular spasms/jerks
3. stereotypical behaviour
- characteristic scratching
- gnawing or chewing of paw
- biting and aggressive behaviour
4. changes in movement
- slow onset or no movement
- hyperactivity
- uncoordinated hind leg movements
- paraplegia of hind limbs


what are the clinical signs of CJD

1. cognitive decline and dementia - short term memory impairment, poor concentration, expressive/receptive dysphasia, temporo-spatial disorientation and confusion
2. behavioural disturbances - agitation restless, irritability, aggressive
3. cerebellar and movement defects - coordination problems, myoclonus, pain or weakness in limbs, brush reflexes, immobility, mutism unresponsiveness
4. abnormal MRI patterns - cortical atrophy, ischemic lesions, signals from basal ganglia/thalamus
5. abnormal EEG patterns - biphasic/triphasic waves associated with front-temporal regions in spCJD


abnormal EEG is seen and not seen in which prion diseases of humans?

seen in sporadic cases like spCJD and not seen acquired cases like variant CJD, kuru, iatrogenic CJD


what are the characteristics of EEG patterns in spCJD?

1. 1-2 cycles/sec in triphasic periodic sharp wave complexes
2. identifies ~60% cases neuropathological confirmed as CJD
3. the EEG patterns disappear characteristic with progressive clinical duration of disease


what are the FIVE pathological hallmarks (microscopic) of prions disease?*

1. spongiform encephalopathy (appearance of vacuoles) (also seen AD, dementia with frontal type, dementia in MN disease, diffuse levy body disease)
2. PrPsc deposition
3. neuronal loss (ALS)
4. astrocytosis (ALS, TBI)
5. microglial reactivity (ALS, TBI)
6. amyloid deposition? (AD)


what are the various ways of PrPsc deposition ?

1. diffuse, punctate deposits and
2. plaque formation


what are the two different hypothesis for the cause of prions disease?

1. spongiform encephalopathy is due to an unusual pathogen associated with PrPsc
2. PrPsc itself is both, pathogenic and transmissible agent


what is the evidence for the involvement of virus as pathogen in prions disease?*

1. classical infectious course (spreads from the periphery to brain in the incubation period)
2. existence of strains
3. species barrier - humans developed from eating sheep but not beef so could be the virus


what are SEVEN the factors the PD infectious agent is resistant to?*

4. 70% ALCOHOL


what are the FIVE methods that can lead to partial destruction of the infectious agent in PD?

1. autoclaving (121 degreeC for 1 hour)
2. 10% hypochlorite
3. 2M NaoH
4. Formic acid
5. guanidine thiocyanate


what are prions?

prion is proposed to denote a small proteinaceous infectious particle


what are the forms of prion?

1. scrapie associated form (PrPsc/PrPd)
2. Normal cellular form (PrPc)


compare the structures of PrPc and PrPsc on the basis of
1. content
2. % of alpha helix and beta sheet
3. expression and metabolism
4. solubility in non-denaturing detergents
5. digestion by proteases

1. content
PrPc - alpha alpha-helical content
PrPsc - high-beta sheet content
2. % of content
PrPc - 42% alpha helix + 3% beta sheet
PrPsc - 43% beta sheet + 30% alpha helix
3. expression + metabolism
PrPc - normal glycoprotein expressed on the cell surface + high levels in the neurone synaptic terminals
PrPsc - abnormal metabolism resulting in aggregates of fibrillar and plaque deposits of PrPsc (27-30KD isoform)
4. solubility in non-denaturing proteases
PrPc - soluble
PrPsc - insoluble
5. digestion by proteases
PrPc - readily digested
PrPsc - partially digested


despite the differences between PrPc and PrPsc structures, what's the similarity?

they have similar primary structure consisting of 209 A.A but differ in secondary structure


how does PrPc convert into PrPsc?

PrPc converts to PrPsc in post translational modification and co-regulators protein x


where does PrPsc attach to on PrPc after protein x interaction?

attaches on the n-terminal of PrPc


what is thought to be the function of prions in the CNS and where is it prevalent?*

- prevalent in the neurones and particularly synapses
its functions are
1. modulates synaptic plasticity
2. modulates NT release/activity
3. protective against oxidative stress (regulate SOD-1/ Bcl-2?)
4. cell adhesion (since it is a cell surface protein)
5. sleep regulation
6. circadian rhythm
7. BACE1 inhibitor


what is the role of energy barrier and PrPsc monomers in the transformation of PrPc to PrPsc?

1. high energy barrier - required for the spontaneous conversion of PrPc to PrPsc
2. PrPsc monomers - the interaction between PrPc and PrPsc leads to the unfolding of PrPc and refolds into 2 monomers of PrPsc and these monomers then aggregate to form plaques


what prevents the immediate conversation version of PrPc into PrPsc?

prevented by high energy barrier


which is more important for the transmission of the disease?
1. monomer
2. aggregates

monomer is more essential for the transmission


what is the prion propagation mechanism?

1. large alpha-helical PrPc proceeds via an unfolded state (denatured PrP) to re-fold into a large beta-sheet form B-PrP
2. B-PrP is prone to aggregation in physiological salt concentrations forming an aggregate termed as PrP seed since it may be critical for the prion propagation
3. unfolded PrP (denatured PrP) or B-PrP monomers are recruited which combines with PrPsc seed to form PrPsc, this is a thermodynamically irreversible process by intermolecular interactions


what is the structure of PrPc?

- consists of a N and C terminal
- along with three alpha helix (alpha helix 1,2 and3)
- the alpha helix 2 and 3 are connected via a single disulphide bond
- and a GPI anchor which attaches the protein to the outer surface of the cell membrane


which part of the PrPc is essential for prion toxicity/clinical manifestation of PD?

the GPI anchor mediating the anchoring of PrPc to the cell membrane therefore, the GPI anchor form of PRPc is essential for clinical manifestation of prion disease


what is the evidence for PrPc being the signalling molecule in the prion disease and PrPsc for the plaques?

- GPI negative transgenic mice expressing monomeric soluble secreted form of PrP do not develop clinical prion disease when infected with PrPsc
- however, the brains of these mice are packed with prion plaques
- Thus indicating, the removal of GPI anchor abolishes the susceptibility to clinical disease while maintaining the replication of aberrant/abnormal prion protein
- clinical disease refers the clinical symptoms seen in terms cognitive, behavioural and motor deficits are absent despite the neuropathological findings being intact (plaques)


how can we identify the prevalence of pion protein isoforms?

western immunoblot analysis of WT and infected brain samples with the application of proteinase K


what are the three isoforms of prion protein and how do they differ from each other?

1. PrPc (209 A.A)
2. PrPsc (209 A.A)
3. PrP27-30 (~142 A.A)
they differ in their primary structure as PrPc and PrPsc have 209 A.A (from codon 23-231) while PrP27-30 has ~142 A.A (from codon. 94-231)


where is the PRNP gene located?*

on short arm of
1. chromosome 20 - humans
2. chromsome 12 - mice


what are the mutations found in the PRNP gene?

1. point mutation codon 129 methionine to valine
2. stop codon - tyrosine changes to stop codon 145
3. additional mutation - proline glycine, octapeptide repeat region inserted


what is the evidence for the central role of PrPc or PRNP gene?*

1. incidence is controlled by the PRNP gene
2. transmission is controlled by the PRNP gene


what are the factors supporting the evidence for incidence being controlled by PRNP gene?*

1. inherited forms of CJD and GSS are caused by PrP mutations with variable penetrance and pathogenic features
2. incidence of natural scrapie in sheep is controlled by PrP genotype
3. risk of sporadic CJD is controlled by PrP genotype (80% due to MM homozygous gene)


what are the factors supporting the evidence for transmission being controlled by PRNP gene?*

1. the species barrier which makes disease transmission b/w species difficult in controlled by PrP gene
2. mice lacking PrP genes are resistant to transmission of mouse scrapie
3. PrP is the major gene that controls mouse adapted scrapie incubation period


what are the three proposed routes for transmission of prion to the CNS?

1. via B cells and vagus nerve
2. via M cells and enteric nerves
3. via infected B cell or monocyte neuroinvasion


what are the three essential requirements for transmission of prions?

1. host expression of PrPc (PrPc knock out mice do not develop prions disease)
2. material from an infected animal (brain>spleen>blood - infection rate)
3. an appropriate route (intracerebral/intravenous> intraperitoneal>intraocular/oral)


what are the four barriers to the infection of prions?

1. genetic susceptibility - codon 129 (met-val) have less susceptibility
2. species specific co-regulatory factors of host (protein X)
3. strain specific - strain of infective agent (PrPsc)
4. host phenotype state of PrPc


how are the strains differentiated once inoculated into animals?

1. length of incubation
2. lesion profile


what are the diagnosis available for prions disease?

1. tonsil biopsy - vCJD samples show heavy PrP deposits
2. appendix biopsy
3. preclinical (subclinical) blood test - screening of blood with antibodies to PrPsc + plasminogen bead capture system and PMCA (protein misfolding cyclic amplification)


what are the treatments for prions disease under clinical trials?

1. pentosan polysulphate
2. quinacrine
3. doxycycline


why is CJD currently an incurable disease?

antiviral, antibiotic, anti-malarial, steroid hormones, cytokines and growth factors have proven unsuccessful


what are the current treatment options available for the prion disease symptoms?

1. pain - opiates and analgesics
2. myoclonus - clonazepam or sodium valproate
3. immunotherapy - PRN100 (given for the first time in clinic)


what are the proposed therapeutic interventions?

1. keep the alpha sheet structure
2. knock out of the gene responsible for the production of cellular prion protein - anti gene therapy


How are prions important in Alzheimer's disease (AD)?

1. PrPsc increases abeta production (abeta 1-42 instead abeta 1-40)
2. PrPc interacts directly with BACE1
3. PrPc is reduced in late onset AD but not familial AD – decrease in PrP with age
4. PrPc mediates impairment of synaptic plasticity by abeta oligomers - Anti PrP antibodies prevent abeta oligomer binding interaction


how does BSE cause variant CJD?*

1. same prion stain causes vCJD and BSE
2. similarities in patterns of PrP deposition, glycoform ratios and fragments sizes in BSE and vCJD inoculated mice
- human prion protein= dissimilar to sheep prion but similar to that of cow and cow's is similar to that of sheep which lets the conversion easier
- which is why humans don’t develop PD from sheep meat but do get it from eating beef
. Scrapie – BSE – vCJD


what is protein X?

In the post translation modification a co-regulator called protein X converts PrPc into PrPsc


what is the role of protein X in prions disease?

1. Species specific molecule that reduces the energy barrier required for PrPc to unfold and refold into more of beta sheet structure
2. It helps the PrPc and PrPsc to directly interact with each other
- reduces energy barrier for the conversion


focal spongiform is seen in which other disorders apart from prions disease?*

1. AD
2. Pick's disease
3. dementia of frontal lobe type
4. dementia in motor neuron disease
5. diffuse lewy body disease
6. Grey matter related - oedema, metabolic encephalopathies, neuronal stage disorders
7. white matter related - oedema, metabolic encephalopathies, ischaemia, canavan's disease


what could be resulting in focal spongiform in disease instead of the actual pathology?

tissue fixation and processing artefacts


how do microglia, astrocytes and neuron result in status spongiosis?

1. microglia -> activation -> release of inflammatory mediators, ROS, ECM degrading enzymes -> neurotoxicity + phagocytosis -> status spongiosis
2. astrocyte -> activation ->release of inflammatory mediators, glutamate toxicity, ECM degrading enzymes -> neurotoxicity + astrocytosis + fibrillary gliosis -> status spongiosis
3. neuron -> degradation -> apoptosis -> neuronal loss + spongiform vacuolation -> status spongiosis


the prion protein was first originated from which medium?

scrappie associated fibrils (SAFs)


Cu2+ binds to the which region human PrP structure?



what does the human PrP primary structure consist of?

1. signal peptide (1-23)
2. octapeptide (51-96)
3. conserved hydrophobic region (B1+A1+B2)
4. disulphide bridge - N-glycosylation site (140-214; A2+A3)
5. GPI anchor (258)


what are the three codon 129 polymorphisms?

1. MM - methionine homozygous (vCJD>sdCJD>normal population)
2. MV - heterozygous (normal population>sdCJD)
3. VV - valine homozygous (normal population>sdCJD)


where is pentosan polysupphate derived from and what properties does it have?

- derived from beechwood
- properties - anti-thrombotic and anti-inflammatory


what are the advantages and disdvantages of pentosan polysuphate?

1. relieves bladder pain and discomfort in interstitial cystitis
2. inhibits neurological signs in scrapie infected mice
3. PPS reduced PrP staining in periphery
1. PPS does not cross the brain barrier
2. its clinical relevance is uncertain
3. no effect on neuropathology of brain


what property does quinacrine have?



when is doxycycline effective?

when administered in the earliest stages of disease


why species barrier poses a problem for transmission of prion disease?

1. longer incubation period - as transmission of prion from one species to another is characterised by prolonged incubation period (due to species barrier) of months-years as compared to transmission were hosts pieces is unchanged
2. species specific differences in genome composition


what is the difference between human PrP and mouse PrP?

human PrP differs at 28 of 254 position from mouse PrP - species barrier for the transmission of infection


state three ways to overcome the human-mice species barrier for transmission of prion disease?

1. transgenic mice - expressing chimeric human/mouse PrP transgene with PrP differ only in 9 residues = lower incubation period
2. transgenic mice exclusively expressing HuPRNP (HuPRNP+/+ Prnp0/0)


what are the advantages if using transgenic mouse models to study prions disease?*

1. PrnP0/0 (knock out) mice do not express PrPc and do not develop prion disease
2. introduction of neurograpft from a WT mouse into CNS and then innoculation of prion infected brain homogenates result in scrapie pathology only in the neurograft area
3. In PrPSc-infected mice, depleting endogenous, neuronal PrPc using the Cre-Lox system prevents neuronal loss, reverses spongiosis and recovers the behavioural and cognitive abilites of the mice


on what basis are the CJD types classified?

according to the ratio of mono, di- and tri-glycosylated forms and electrophoretic mobilities of PrPSc


what are the four types of sporadic CJD?

1. Met/Met 1
2. Met/Met 2
3. Met/Val 2
4. Val/Val 2


out of the all the sporadic types which one has the shortest incubation period?

Met/Met 1 - 2-14 months