2. What are Prions?
Prion :Proteinaceous infection partical.
Prions are unprecedented infectious
pathogens that cause a group of invariably
fatal neurodegenerative diseases
mediated by an entirely novel mechanism.
Infectious PrPsc protein induces a
conformational change on the normal PrPc
protein.
3. Cont.......
Infectious protein: causes prion diseases.
Discovered by Prusiner in 1982 in
Scrapie(neurological disease of sheep).
Prusiner won the nobel price in physiology
or medicine in 1997.
Misfolded/abnormally folded proteins in
humans/animals are pathogenic whereas
normally folded proteins are non
pathogenic.
4. Differences from bacteria and viruses
Prions do not have Nucleic acid and do
not have DNA or RNA.
They are extremely resistant to heat and
chemicals.
Prions are very difficult to decompose
biologically; they survive in soil for many
years.
Prions are 100 times smaller than the
smallest virus.
5. History of Prions:
1730s – Earliest written record of scrapie in
english sheep; already prevalent in central
Europe.
1950s – high level of kuru appear among the
fore people of new guinea.
1960s – scientists experimentally transmit Kuru
and CJD in chimpanzees, demonstrating the
transmissible nature of these diseases.
1967 - Griffith proposed that proteins could be
infectious pathogens and postulated their
involvement in scrapie, a universally fatal
transmissible spongiform encephalopathy in
goats and sheep.
6. Cont……
1980s – 60 people die from CJD after being
infected by contaminated surgical instruments.80
people die after receiving prion infected growth
hormone injections.
1982 – Dr.Stanley Prusiner coin the term “Prion”.
Highly purified PrP-res is shown to be infectious.
1985 – scientists identify the PrP gene and
discover that uninfected people produce the
normal form of the PrP protein.
1986 – by the year 2000,180000 cattles were
infected. To stop this 1000s of cattle were killed.
7. Two forms of Prions
The prions are in two forms;
1) PrP-sen
2) PrP-res
The first type “sen” stands for Sensitive (i.e) proteins are
sensitive to break down by Protease enzyme.
PrP-sen is produced by normal healthy cell. It is mainly
present in neurons in the brain.
It is commonly called as PrPc(C-Cellular).
The second type “res” stands for Resistant (i.e) proteins
are resistant to break down by protease enzyme.
PrP-res is disease causing form.
It is commonly called as PrPsc(Sc- Scrapie)
8. Structure of Prions
Structure of PrPc :
The prion protein gene, PRNP, encodes a 253
amino acid (aa) precursor protein with an
endoplasmic reticulum (ER)-targeting sequence
for translocation into the secretory route.
During transit through the ER and Golgi
apparatus, PrPc is modified by two complex
asparagine linked sugar moieties, a disulfide
bond, and a C-terminal
glycosylphosphatidylinositol (GPI) anchor,
localizing the protein to glycolipid-enriched
membrane domains.
9. Structure by NMR
NMR studies have shown that the N-
terminal half of the protein, of about 100 aa,
is unstructured, whereas the C-terminal half
is a well-structured globular domain
containing three α-helices and two short,
antiparallel β-sheets.
GPI-anchored PrPc at the cell surface, is
ideally placed for moving between
membrane domains and for interacting with
transmembrane signaling complexes.
10. Cont….
The primary sequence of murine PrPc is
protein of about 253 amino acids long
before post translational modification.
Signal sequence in the amino- and
carboxy- terminal ends are removed post
translationally, resulting in a mature
length of 208 amino acids.
Circular dichorism shows that PrPc has
43% alpha helical and 3% beta sheets.
11. Cont.....
Structure of PrPsc :
In the aberrant protein some these helices are
stretched out into flat structure called beta
strands.
PrPsc has the same amino acid sequence as the
normal protein; that is, their primary structures
are identical but its secondary structure is
dominated by beta conformation.
PrPsc with much hydrophobic aminoacyl side
chain exposed to solvent.
Circular dichorism shows that PrPSc has 43%
beta sheet and 30% alpha helix.
12.
13. Structure of Prnp gene and its location.
Structure :
The Prnp gene contains either three (in rat, mouse,bovine, sheep) or
two exons (in hamster, humans, tamar ,wallaby), of which a single
exon codes for PrPc protein.
Control of Prnp gene expression has been attributed to sequences
within the 5-flanking region, within the first intron, and to 3-untranslated
sequences.
Location :
PRNP is the human gene encoding
for the major prion protein PrP
also known as CD230 (cluster of differentiation).
It is located on the short arm of
chromosome 20 between the end of the
arm and position 13,from base pair
4,686,350 to 4,701,590.
14.
15. Comparision
PrPc PrPSc
Cellular
Globular protein
Composed of alpha helix.
Protease Sensitive.
Not infectious.
Easily soluble.
Monomeric
Consist of asparagine at
position 129.
Gene PRNP(short arm of
chromosome 20).
Scrapie.
Fibrous protein.
Composed of beta sheets.
Protease resistant.
Infectious.
Insoluble.
Multimeric.
Consist of valine at position
129.
Reproduce by binding to
Prpc
16.
17. Functions of prion protein
Knockout mouse model:
With the exception of 3 PrP knockout mouse
models, in which ectopic expression in the central
nervous system of the PrP paralogue Doppel leads
to loss of Purkinje cells in the cerebellum.
The first two Prnp KO strains, Zurich I (ZrchI) and
Edinburgh (Edbg), showed no developmental or
other phenotypic disturbances .
Further investigations of Prnp-ablated mice have
revealed subtle phenotypic changes, indicating
possible functions for PrPc . The majority of these
are related to the central nervous system (CNS),
where PrPc is abundant.
19. PrP regulates NMDAR(Nmethyl-
d-aspartate receptor)
Glutamate release from the presynaptic terminal,
NMDARs are activated on the postsynaptic
terminal, leading to calcium entry.
Via a series of molecular mechanisms, NO and
copper ions are released in the synaptic cleft.
Released Cu2+ ions are rapidly bound by copper-
binding proteins including PrPC, which is highly
expressed in both presynaptic and postsynaptic
terminals. PrPC has high affinity for both Cu2+
and Cu+ and it may reside in lipid raft domains,
which also contain NMDAR.
20. Cont……
Synaptic NO can react with extracellular cysteine
thiols of NMDAR subunits GluN1 and GluN2A,
leading to cysteineS-nitrosylation.
The S-nitrosylation inhibits NMDAR activation by
closing the channel.
The chemical reaction between NO and
cysteine thiol requires the presence of an
electron acceptor such as Cu2+.
According to this model, PrPC positions Cu2+
ions that support the reaction of NO with thiols,
leading to the S-nitrosylation of GluN1 and
GluN2A, thus inhibiting NMDAR.
21.
22. Conversion of PrPc to PrPsc
Conformational conversion of a protein from an
α- helix to a β- strand is usually associated with a
major change in the tertiary structure,which may
alter its physiological function.
This conversion result in loss of function of PrPc .
The accumulation of PrPSc is linked to apoptotic cell
death in animal models and in humans.
Recombinant PrPc converted into a cross -sheet
amyloid induce prion diseses in hamsters.
23.
24. Replication of Prions
1)Heterodimer model.
2)Fibril model.
1) Heterodimer model:
* This model assumes that a
single PrPSc molecule binds to a single PrPc
molecule and catalyses its conversion into PrPSc.
* The two PrPSc molecules
then come apart and can go on to convert more
PrPc.
25.
26. Cont…..
2) Fibril model:
* This model assumes that PrPSc exists only as
fibrils, and that fibril ends bind Prpc and convert it into
PrPSc.
* Then the quantity of prions would increase
linearly, forming ever longer fibrils.
* But the exponential growth of both PrPSc
and of the quantity of infectious particles is observed
during prion disease.
* This can be explained by taking into
account fibril breakage.
* Exponential growth rate resulting from the
combination of fibril growth and fibril breakage.
28. Neuroimmune crosstalk
PrPc and the immune system:
1)PrPc in blood immune cells.
2)PrPc in neuroinflammation.
3)Poteolytic processing of PrPc :
signalling by PrPc fragments.
29. PrPc in blood immune cells.
PrPC is expressed in immune-privileged stem-cell
niches of the hematopoietic bone marrow and has
been shown to be important for stem-cell renewal
under stressful conditions.
High levels of PrPC are maintained in mononuclear
cell precursors, but it is downregulated during
maturation of granulocytic and erythroid cell lines.
Cell-surface levels of PrPC are promptly
upregulated when T cells are activated.
Genetic removal of Prnp or pharmacological
blocking of PrPC impaired T-cell proliferation .
Removal of PrPC resulted in murine T cells being
skewed towards proinflammatory phenotypes.
30. Cont……
Another cell type with high PrPC expression is
Mast cells .
PrPC was not found to be obligatory for mast cell
differentiation, it was rapidly shed from the cell
surface upon activation, such as during mast
cell-dependent allergic inflammation.
A sub-type of mast cells resides on the brain
side of the blood-brain barrier and
communicates with neurons, astrocytes, and
microglia.
Thus, upon activation, mast cells act as first
responders to initiate,amplify, and prolong
immune and nervous responses.
31. Cont…
Another key element of immune responses
and neuroimmune crosstalk is tissue
invasion by activated blood leukocytes.
PrPC modulates leukocyte extravasation
including into the CNS .
PrPC can influence leukocyte migration is
through interaction with adhesion molecules
like β1 integrin, which is a co-receptor for
PrPC.
32. PrPc in neuroinflammation
At the cellular level, neuroimmune crosstalk is
maintained through an integrative network of
neurons, microglia, astrocytes, and infiltrating
immune cells, such as T cells.
All the cellular participants of neuroimmune
crosstalk express PrPC .
Acute neuroinflammation is activation of resident
microglia, which participate in the phagocytosis
of microbes or debris, as well as release of
cytokines.
Astrocytes also play significant roles in brain
inflammation, producing both pro- and anti-
inflammatory chemokines.
33. Cont……
Astrocyte PrPC seems to be important for the
survival and differentiation of both astrocytes
and neurons.
Astrocytes overexpressing PrPC showed higher
levels of GFAP, a general marker of astrocyte
activation and neuroinflammation.
PrPC also protected astrocytes from oxidative
stress , which could be important during
inflammation caused by infarctions.
The concept that PrPC protects against
neuroinflammation, probably by modulating the
effects of cytokines and other inflammatory
molecules, and thereby limiting tissue damage.
34. Proteolytic processing of PrPC:
signaling by PrPC fragments
Mature PrPC can be subject to proteolytic
processing . Soluble full-length PrPC can be
released from the cell membrane through the
action of ADAM10.
Mature full-length PrPC is attached to the cell
membrane through its GPI-anchor.
Ligands can bind PrPC which probably is
associated with transmembrane co-receptors
to initiate signaling into the cell.
Shedding of PrPC can be performed in close
proximity to the GPI-anchor releasing soluble
PrPC into the extracellular space .
35. Cont……
The N-terminal domain (N1 fragment) can be
released by α-cleavage leaving membrane
bound C1(mC1)attached to the cell surface.
Finally, the C1 fragment can be shed form the
cell surface in a soluble form sC1.
The released PrPC fragments can probably
mediate both intercellular communication
(paracrine) and autocrine signaling.
This demonstrates that secreted PrPC can be
amyloidogenic and potentially harmful,
suggesting that shedding of full length PrPC,
atleast in the brain, must be a tightly controlled
process.
36.
37.
38. Prion disease(TSE)
Transmissible, progressive and invariably fatal neurodegenerative
conditions associated with misfolding and aggregation of a host-encoded
cellular prion protein.
Also known as transmissible spongiform encephalopathy.
Large numbers of cells die off.
↓
Cysts form in the brain.
↓
Brain - spongy appearance.
↓
Prions aggregate on the membrane of the Neurons.
↓
Large plaques that are toxic to brain tissue.
↓
SPONGIFORM ENCEPHALOPATHY
39.
40. Different prion disease affect different
regions of the brain.
Cerebral cortex: The symptoms
include loss of memory and
mental acurity, also visual
imparement (CJD).
Thalamus : Fatal Familial
Insomnia(FFI) affects thalamus.
Cerebellum :Loss the control of
body movements and difficulties
to walk(Kuru,GSS).
Brain stem : In the mad cow disease
the brain stem is affected.
43. Prion diseases
Animal diseases
Scrapie (sheep and goats)
Transmissible mink encephalopathy
Wasting disease of deer and elk
Bovine spongiform encephalopathy.
Transmissible spongiform encephalopathy of captive wild
ruminants.
Feline spongiform encephalopathy.
Human diseases
Kuru
Sporadic CJD
Familial CJD
Iatrogenic CJD
Variant CJD
44. Animal diseases:
Scrapie disease
Scrapie was the first prion disease to be
identified and has been recognised by
shepherds for over 200 years.
Symptoms:
The disease is characterised by scraping of
fleece,stumbling, and behavioural changes and
has a progressive course, leading to death in
3–6 months.
Post-mortem examination shows changes only
in the CNS with neuronal loss, gliosis, and
vacuolisation of neural cells.
45. Transmissible mink encephalopathy
First reported in Wisconsin, USA, most
outbreaks have been traced to mink feed
suppliers with the assumption that scrapie
infected sheep were included in the feed.
46. Chronic wasting disease of cervids
Chronic wasting disease of deer (cervids) was
first found in the 1960s among captive mule
deer in a wildlife research facility in Colorado,
USA.
Symptoms:
Animals became emaciated,and developed
behavioural changes, unsteadiness, and
excessive salivation.
Death occurred within weeks to months, and
pathological examination of brains showed
widespread spongiform changes in grey
matter.
47. Bovine spongiform encephalopathy
In 1985, the first cases of BSE were observed
in the UK.
Also known as “Mad cow disease”.
Symptoms:
Cows with BSE may show nervousness or
aggressive behaviour, difficulty with
coordination, trouble standing up, decreased
milk production, and weight loss.
The disease is fatal, with death usually
occurring 2 weeks to 6 months after symptoms
start.
48. Human diseases:
Kuru
Kuru was described in 1957 in a remote area of New Guinea.
Symptoms:
Symptoms of more common neurological disorders such
as Parkinson’s disease or stroke may resemble kuru symptoms.
Three stages of symptoms:
In the first stage, a person with kuru exhibits some loss of bodily
control. They may have difficulty balancing and maintaining posture.
In the second stage, or sedentary stage, the person is unable to
walk. Body tremors and significant involuntary jerks and movements
begin to occur.
In the third stage, the person is usually bedridden and incontinent.
They lose the ability to speak. They may also exhibit dementia or
behavior changes, causing them to seem unconcerned about their
health.
Starvation and malnutrition usually set in at the third stage, due to the
difficulty of eating and swallowing. These secondary symptoms can
lead to death within a year. Most people end up dying from pneumonia.
49. Sporadic CJD
Sporadic CJD occurs throughout the world without
overall geographic or seasonal clustering.
Symptoms:
The initial symptoms fatigue,disordered sleep, and
decreased appetite; behavioural or cognitive changes.
The final focal signs such as visual loss, cerebellar
ataxia, aphasia, or motor deficits.
The disease progresses rapidly with prominent
cognitive decline and the development of myoclonus,
particularly startle sensitive myoclonus.
The median time to death from onset is only 5 months.
50. Familial diseases
Familial CJD cases show autosomal dominant
inheritance of mutations in PRNP.
Symptoms:
Progressive insomnia, autonomic dysfunction
and dementia.
Polysomnography shows little sleep, loss of
sleep spindles, and near absence of rapid-eye-
movement sleep.
The neuropathological changes are localised
largely to neuronal loss in the thalamus
particularly the anterior ventral and mediodorsal
nuclei, and the olivary nuclei of the brainstem
and there is little vacuolisation.
51. Iatrogenic CJD
Transmission of CJD among people has
occurred with corneal transplants, dural
grafts, injections of hormones extracted
from human pituitary glands, and
contaminated neurosurgical instruments.
52. Variant CJD
In 1994, a new form of human spongiform
encephalopathy emerged in the UK.
Symptoms:
Symptoms include psychiatric problems,
behavioral changes, and painful
sensations.
55. Prions in plants
A protein in a thale cress (Arabidopsis) behaves like a prion when
it is expressed in yeast.
This research was led by Susan Lindquist ,a biologist at the
Whitehead Institute for Biomedical Research in Cambridge.
In plants, the protein is called Luminidependens(LD) and it is
normally involved in responding to daylight and controlling
flowering time.
When a part of the LD gene is inserted into yeast, it produces a
protein that does not fold up normally, and which spreads this
misfolded state to proteins around it in a domino effect that causes
aggregates or clumps.
Later generations of yeast cells inherit the effect:their versions of
the protein also misfold.
Lindquist has shown that prion proteins can provide evolutionary
advantages for some living oganisms-such as ,in yeast, surviving
harsh environment.
56. Cont……
• In 2015, researchers at The
University of Texas Health science
Centre at Houston found that plants
can be a vector for prions.
• When researchers fed hamsters grass
that grew on ground where a deer
that died with chronic wasting
disorder was burried, the hamsters
became ill with CWD, suggesting that
prions can bind to plants, which then
take them up into the leaf and stem
structure where they can eaten by
herbivores, thus completing the cycle