Fatal Familial Insomnia: Pathogenesis caused by a mutation affecting the metabolism of the normal pr

1
Fatal Familial Insomnia Pathogenesis caused by a
mutation affecting the metabolism of the normal
prion protein.By Sabrina T. GilligBIO-475
SeminarDr. Peter Lin
2
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.
3
What 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.
4
More 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.

5
Prion diseases (Collinge, 2005)
6
What 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 for
  aspartic acid.
• 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.

7
The 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.
• When brain tissue is examined under the
  microscope, numerous tiny holes are visible,
  giving it a sponge-like appearance- From these
  observation the name TSE arose.

8
Treatment

• There is no treatment for FFI.
• Two drugs (quinacrine and chlorpromazine) were
  being tested, but the individuals in the clinical
  trials worsened.

9
Metabolism of the mutated prion protein

• The polymorphism in codon 129 is exclusive to
  humans. The two common forms of PRPN are the
  major determinants in the phenotypic expression
  of TSEs.
• PRNP encodes for methionine (sulfur-containing
  aminoacid) or valine (essential aminoacid for
  growth).
• CJD and FFI both present a mutation in codon 178,
  but codon 129 is the one that determines the
  phenotype.
• The normal non-mutant haplotype is designated
  178D, the haplotype in FFI is designated D178N.
• The metabolic and mutational events that lead to
  the syndrome will be examined further along this
  presentation.

10
Expression, Localization and metabolism of the
PrP in humans

• PrPc is a glycoprotein attached to the cell
  membrane .
• During the process of translocation
  (rearrangement occurring when a piece of one
  chromosome is broken off and joined to another
  chromosome ) in the ER PrPc continues its folding
  process.
• Further folding occurs in the Golgi apparatus

11
The secretory Pathway ( Schuldiner et al., 2005)
12
Three forms of PrPc

• Called GLYCOFORMS and differ in the level of
  glycosylation (addition of sugar units).
• 1) Unglycosylated
• 2)monoglycosylated
• 3)diglycosylated

13
-Purpose-Comparison among normal and mutant
cells in the metabolism of the prion protein

• Cells from the FFI haplotype only released 1/3
  the amount of PrP to the cell surface when
  compared to normal cells.
• Confirms that the three PrP forms differ in the
  level of glycosylation? In mutant cells the
  unglycosylated form is virtually inexistent.

14
Transport in the secretory pathway

• Distinct amounts of glycosylation in normal and
  mutant forms indicate that the mutant PrP is not
  adequately transported during the secretory
  pathway.

15
Stability and transport of the prion protein

• Experimental
• By using the antibiotic tunicamycin (which
  prevents glycosylation in newly synthesized
  proteins), effect of the glycosylation in the
  transport of the PrPm was evaluated.

16
Results

• 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.

17
PrP 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).

18
Results

• 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.

19
Results (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.

20
D178N 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.

21
Underrepresentation 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.

22
Results

• Normal gray matter presented the three previously
  discussed glycoforms which transfer as a single
  unit after deglycosylation.
• In the mutant gray matter the unglycosylated form
  is present at only about 1/3 when compared to the
  normal samples.

23
Conclusion

• 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).

24
Conclusion

• 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.

25
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