Based on…

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. Based 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.

3. FATAL 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 !

4. 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.

5. 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.

6. Prion diseases (Collinge, 2005)

7. 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 (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.

8. 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.

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

10. 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.

11. 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

12. The secretory Pathway ( Schuldiner et al., 2005)

13. N-Linked Glycosylation Is common to all eukaryotic cells. It is imperative for proper folding of the protein.

14. Schematic 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.

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

16. -Cell lines in this study were obtained from human neuroblastoma cells. These were inserted with the prion protein sequence either normal or mutant encoding for MET (methionine) or VAL (valine); in order to evaluate the outcome of the polymorphism on the metabolism of the PrP; these differences lead to two distinct phenotypes. Cell lines and their different genotypes

17. The cells were treated with PI-PCL (Phosphatidylinositol-specific phospholipase C). This enzyme was used to separate the PrP from the cell membrane. Scientists were able to study the prion protein itself by dissolving the anchor, and quantify solely the amount of PrP present on the cell surface. Glycosylation differences among mutant and normal cells on the cell surface M: mature I: Intermediate U: Unglycosylated

18. Carbohydrates in the samples were removed by the enzyme N-Glycosidase F. This helped the scientists observe exclusively the amount of PrP present in normal and mutant cell surface, according to their weight . Glycosylation differences on the cell surface-Normal vs. Mutant-

19. 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 Efficiency of the PI-PCL on the mutant PrP

20. The distinct amounts of glycosylation in the mutant and normal forms, indicate that the mutant PrP is inadequately transported during the secretory pathway; which is the method that the cells use to transport substances from the ER to the Golgi apparatus and the outside . In mutant cells, the absence of the unglycosylated forms further sustain these statements .

21. Scientists concluded that… • The three PrP forms differ in the level of glycosylation→ In mutant cells the unglycosylated form is virtually inexistent.

22. : In order to support the hypothesis that FFI results exclusively from the degradation of the unglycosylated form before it arrives at the cell surface; scientists had to quantitate the amount present inside and outside the cell. Once again PI-PLC was used to unanchor the prion protein. Scarcely noticeable quantities inside the cell give evidence that in the mutant forms, PrP is still inside the ER-Golgi complex. Unglycosylated PrP found in the cell and on the cell surface (Normal vs. Mutant)

23. Graphical Representation of unglycosylated PrP intracellularly and on the cell surface.

24. -By preventing glycosylation, scientists were able to demonstrate the hypothesis that the unglycosylated PrPm is degraded intracellularly and needs glycosylation to arrive at the cell surface. PrPc does not need glycosylation in order to reach the cell surface. (-) untreated cells; (+) tunicamycin treated. This experiment gives a powerful insight on the transport and stability characteristics . How does the stability of PrPm affects its transport to the cell surface?

25. Cells treated with Tunicamycin

26. 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.

27. 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).

28. By treating the cells with Brefeldin A transport of the glycosylated PrP from the ER through the Golgi complex was blocked; Scientists wanted to find out where the PrPm is degraded. Normal prion protein (WM or WV) were by far noticeable; on the other hand, the PrPm (178 MET and 178 VAL) show a decrease in the amount found between the 0-chase and the 2-hour chase when compared to the normal PrP; this indicates that PrPm is either degraded or glycosylated . Effects of transport block in the degradation of the PrP

29. 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.

30. 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.

31. 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.

32. The extreme vulnerability to these denaturing conditions, could leave the PrP biologically inactive, contributing to the pathogenesis in FFI

33. 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.

34. Scientists examined the PrPm present in a FFI patient, in a section of the brain which does not have PrPres. They wanted to prove that the U glycoform is unstable in FFI subjects. All N-linked sugars in the FFI patient sample were removed by the enzyme PNGase, to study the original glycoforms. Presence of unglycosylated PrP in the brain of FFI patients

35. 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.

36. 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 1990’s).

37. Conclusion • Even though FFI and other prion diseases are rare and sporadic, science should always try to stay a step ahead…for the sake of all humanity.

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