Title: Fatal Familial Insomnia: Pathogenesis caused by a mutation affecting the metabolism of the normal prion protein. By Sabrina T. Gillig BIO-475 Seminar Dr. Peter Lin
1Fatal Familial Insomnia Pathogenesis caused by a
mutation affecting the metabolism of the normal
prion protein.By Sabrina T. GilligBIO-475
SeminarDr. Peter Lin
2FATAL FAMILIAL INSOMNIAthe nightmare of those
who never sleep
- Dateline NBC. 2005. Fatal Insomnia Genetic
mutation inflicts rare disease through
generations. http//www.msnbc.msn.com/id/6822468/
. Accessed 11 APR 2006. CLICK ON THE LINK TO
WATCH THE VIDEO !
3What are Prions?
- Prions are the smallest infectious particles
known to date. They are made only of a protein. - Prions are abnormally folded proteins.
- Prions are the cause of transmissible spongiform
encephalopathies. - Prion diseases are fatal and untreatable.
Cann, 1997.
4More on prions
- The normal prion protein PrPc is found primarily
on the surface of neurons, and is likely to be a
synaptic protein with functional role in the
synaptic transmission. - Prion diseases exhibit an extended latency
period, spending this time performing
neuroinvasion.
5Prion diseases (Collinge, 2005)
6What is Fatal Familial Insomnia?
- FFI is an autosomal dominant inherited disease
cause by a mutation of the normal prion protein. - A mutation in codon 178 replaces asparagine (N)
for aspartic acid (D)? D178N - First symptoms to arise trouble sleeping,
difficulty concentrating , and personality
changes - These symptoms usually appear during midlife,
after appearance of the first symptoms death
follows usually with 18 months.
7The Thalamus (Wikipedia, 2006)
- The thalamus is the point where most signals from
the CNS pass to the cerebrum. - Severe loss of neurons in the thalamic nuclei,
and accumulation of amyloid plaques. - Sponge-like appearance under the microscope-TSE-
8Treatment
- There is no treatment for FFI.
- Two drugs (quinacrine and chlorpromazine) were
being tested, but the individuals in the clinical
trials worsened.
9Based on
Petersen, R.B., P. Parchi, S.L. Richardson, C.B.
Urig, and P. Gambetti. 1996. Effect of the D178N
mutation and the codon 129 polymorphism on the
metabolism of the prion protein. The Journal of
Biological Chemistry 271 12661-12668.
10Metabolism of the mutated prion protein
- Codon 129 Polymorphism
- PRNP encodes for methionine or valine.
- CJD and FFI similarities
- The normal haplotype is designated 178D, the
haplotype in FFI is designated D178N.
11- The metabolic and mutational events that lead to
the syndrome will be examined further along this
presentation.
12Expression, Localization and metabolism of the
PrP in humans
- PrPc is a glycoprotein attached to the cell
membrane . - During the process of translocation in the ER,
PrPc continues its folding process. - Further folding occurs in the Golgi apparatus
13N-Linked Glycosylation
-
- Is common to all eukaryotic cells. It is
imperative for proper folding of the protein.
14The secretory Pathway ( Schuldiner et al., 2005)
15Schematic Representation of The Prion Protein
-The human PrP. Polymorphic sites 129 and 178 are
shown. GPI (ground positioning indicator) anchors
the prion protein to the cell membrane.
16Three forms of PrPc
- Called GLYCOFORMS and differ in the level of
glycosylation (addition of sugar units). - 1) Unglycosylated
- 2)monoglycosylated
- 3)diglycosylated
17Cell lines and their different genotypes
18Glycosylation differences among mutant and normal
cells on the cell surface
- The cells were treated with PI-PCL.
- Scientists were able to study the prion protein
itself and quantify solely the amount of PrP
present on the cell surface.
M mature I Intermediate U Unglycosylated
19Results
- 3 different glycoforms were observed
- Only 1/3 the amount of PrPm was released from the
cell surface of mutant cells when compared to the
control.
20Glycosylation differences on the cell
surface-Normal vs. Mutant-
- Enzyme N-Glycosidase F was used to remove the
sugar groups.
21Results
- The unglycosylated form is underrepresented on
the cell surface.
22Efficiency of the PI-PCL on the mutant PrP
- To prove that the mutant prion protein is
efficiently unanchored by the PI-PCL treated (),
and untreated (-) cells were labeled with biotin.
As seen in this figure, PrPm is powerfully cut
by PI-PCL treatment
23Scientists concluded that
- The three PrP forms differ in the level of
glycosylation? In mutant cells the unglycosylated
form is virtually inexistent.
24Prion Protein representation inside the cell and
on the cell surface
- Scientists had to quantitate the amount of PrP
present inside and on the cell surface. - INTRACELLULARLY
- Retention of the prion protein
- EXTRACELLULARLY
- Amount present on the cell surface
25Graphical Representation of unglycosylated PrP
intracellularly and on the cell surface.
26What does this mean?
- INTRACELLULARLY At zero time PrP is still in the
ER-Golgi complex - EXTRACELLULARLY The mutant PrP is inadequately
transported during the secretory pathway
27How does the stability of PrPm affects its
transport to the cell surface?
- Glycosylation was prevented using the antibiotic
tunicamycin. - -Tunicamycin treated ()
- Untreated (-)
28Cells treated with Tunicamycin
29Results
- The unglycosylated form of the D178N PrPm
- is degraded inside the cell, while the
normal PrPc necessitates glycosylation to reach
the cell surface. - When glycosylation is prevented, the PrPm hardly
arrives to the cell surface, and untraceable
after synthesis.
30PrP Degradation
- Experimental
- In order to find out whether PrPm is degraded
when kept in the ER-Golgi compartment scientists
used brefeldin A (which blocks transport of
glycosylated proteins from the ER to the Golgi
complex).
31Effects of transport block in the degradation of
the PrP
- Scientists wanted to find out where the PrPm is
digested. - The PrPc showed no major changes with time
- PrPm amounts decreased
32Results
- Normal cells Brefeldin A
- All three glycoforms were observed
- Mutant cells Brefeldin A
- Mutant cells exhibited degradation or change to
the glycosylated form - ?More unglycosylated PrPm reaches the cell
surface when valine is present in codon 129.
33Results (continued)
- Degradation of the mutant prion protein does not
occur in the Golgi compartment, but in the
endosomal-lysosomal system, which contains highly
acidic enzymes.
34D178N mutant cells lack PrPres
- Normal and mutant cells were tested for
proteinase K-resistant PrP. - Mutant cells lack PrPres which provides
resistance to powerful denaturing conditions.
35Underrepresentation in the brain of the PrPm
- Western Blot was used to determine whether the
unglycosylated form of the mutant prion protein
is decreased in FFI patients. - Portions of normal and diseased brain were
examined.
36Presence of unglycosylated PrP in the brain of
FFI patients
- Scientists examined the PrPm present in a FFI
patient, in a section of the brain which does not
have PrPres. All N-linked sugars in the FFI
patient sample were removed by the enzyme PNGase,
to study the original glycoforms.
37Results
- In the mutant gray matter the unglycosylated form
is present at only about 1/3 when compared to the
normal samples.
38What appears to be the cause of disease in FFI?
- FFI appears to result only from the degradation
of the unglycosylated form before it reaches the
cell surface.
39Conclusion
- Pathogenesis in FFI and other prion diseases is
believed to be caused by a change in the shape of
the normal protein. - It is imperative to continue research, since in
other neurodegenerative diseases (e.g.
Alzheimer's) a misfolded protein could also be
the cause. - A detailed analysis of the different factors,
mechanisms and disease expression may be critical
in the even of an epidemic (Mad Cow disease in
the mid 1990s).
40Conclusion
- Even though FFI and other prion diseases are rare
and sporadic, science should always try to stay a
step aheadfor the sake of all humanity.
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