Creutzfeldt-Jakob’s disease or Prion Disease or Mad Cow disease

1. Creutzfeldt-Jakob’s disease or Prion Disease or Mad Cow disease It belongs to a group of neurodegenerative diseases called Transmissible Spongiform Encephalopathy (TSE). The infectious agents responsible for TSE are Prions, PrPc converted into PrPsc. They are generally hard to eliminate, and are extremely resistant to: Heat Radiation Disinfection Protein digestion (degradation)

2. Creutzfeldt-Jakob’s Disease (Mad cow disease or Prion disease)

3. Severe Brain Atrophy in CDJ’s patient www.scienceclarified.com

4. KURU: laughing death -Papua New Guinea (1957) http://www.biologie.uni-duesseldorf.de KURU and prion’s disease: similarities in the symptoms and in cerebellar ataxia

5. Models for conformational conversion of PrPc into PrPsc

6. Tissues in which PrPsc accumulates !!! vCJD is of oral origin, and PrPsc could take years before converting PrPc Role of oral mucosas in first accumulating then spreading PrPsc

7. Symptoms in Creutzfeldt-Jakob’s Disease -It could take years (decades) before a carrier of prion disease will become fully symptomatic. -Symptoms are characterized by cognitive decline, which may be fulminant and progress to akinetic mutism within few weeks. -Cerebellar signs are evident (balance and coordination dysfunction -ataxia, changes in gait, rigid posture, and seizures). mood swings depression anxiety memory lapses social withdrawal clumsiness or lack of coordination insomnia

8. As the disease rapidly progresses, patients with all forms of CJD generally experience: * visual deterioration and eventual blindness * dementia * involuntary muscle contractions * muscle paralysis * slurred speech * difficulty swallowing * incontinence * coma

9. Cow affected by Bovine Spongiform Encephalitis www.jonbarron.org

10. Diagnosis: -Electroencephalography -MRI “Probable” CJD is based on the clinical symptoms. -Correct and definite diagnosis only post-mortem, performing immunohistochemical analysis of brain sections. -Biopsy of the tonsils and, in 30% of the cases, of skeletal muscles can confirm CDJ. The target of the analysis is the presence of the scrapie, protease-K resistant form of Prion Protein (PrPsc). This form is transported in the hematopoietic system, tends to accumulate in the lymphatic system, is enriched in the B-lymphocytes, and is present also in the mucosa olfactoria.

11. Absence of Protease-K-digested PrP in CJD used for diagnosis CJD Control Prion protein

12. Spongiform (intracellular vacuolation) change in the cortical gray matter of the brain, characteristic of TSEs and prions aggregates Walker et al.,

13. Features of TSEs and CJD *Neuronal death *Neuronal apoptosis *Astrogliosis (as a cause or a consequence of inflammation) *Protein misfolding and aggregation *Precipitation of aggregates (proteinaceous material) both at an intracellular and extracellular level (amyloid plaques)

14. Deposition of fibrillar proteinacious material in Creutzfeldt-Jakob’s disease (prion disease) Prion disease: Alteration in the prion protein lead to both intracellular and extracellular accumulation of amyloid aggregates, plaques, similar to those characteristic of AD, and positive to prion protein staining. Probably, replication and accumulation of the protease insensitive PrPsc results in fibril formation and plaque deposition. Alzheimer’s Creutzfeldt-Jakob’s Aguzzi A, Haass C. Science. 2003 Oct 31;302(5646):814-8. Review.

15. Epidemiology of Creutzfeldt-Jakob’s disease (CJD) CJD is, among the Transmissible Spongiform Encephalopathies, the most diffuse one. CJD can be classified as Sporadic sCJD: etiology not known, caused by both exogenous and endogenous factors, represents 85% of all the cases of CJD. In the United States, there are approximately 200 sporadic CJD cases per year. Familial fCJD: caused by mutations in the gene for PrP (prion protein). 15% of CJD cases are inherited. Iatrogenic iCJD: caused by the spreading of the infectious agent due to contaminated surgical tools, to the transplantation of tissues, or to the administration of pituitary hormones from deceased patients affected by the disease. 1% of CJD cases. Variant vCJD: caused by the transmission of Bovine Spongiform Encephalopathy (BSE) prion to humans (aka Mad Cow disease).

16. Incidence of BSE reported worldwide

17. Incidence of vCJD reported worldwide

18. The prion protein: functional domains and mutations causing inherited prion’s diseases

19. Functional domains of the prion protein OR: not required for PrPc function, it might influence the change of conformation in PrPsc, as OR KO mice do not propagate the disease. Protects from apoptosis. CC1: probably involved in protein internalization/trafficking. CC1 KO mice are viable and could develop the disease. CC2: might work in concert with HC region, as partial deletion of either or the other domain, or ablation of one domain and partial deletion of the other accelerate the pathology in mice. C-terminal: gene KO on H2, H3 or both domains leads to ataxia and neuron disease, BUT FAIL TO REPLICATE PRIONS. No transmission of disease from H2 KO and H3 KO to other animals. H2 and H3 might stabilize the conformation of the protein. C-terminal deletion prevents GPI anchoring of the protein, no development of the disease.

20. Prion Protein: domains and a- and b- helical structures www.chemsoc.org -PrPc contains 208 aminoacid residues and is abundantly expressed in neurons and glial cells -Signal peptide sequence -Octarepeats followed by a short Hydrophobic/toxic structure -The C-terminal portion of the protein is a globular structure that contains 3 a-helical domain and 2 b-helical domains. This domain folds rapidly and is extremely stable

21. Amyloid plaques in TSE Kuru disease GSS disease Gerstmann-Straussler-Sheinker disease Kuru disease

22. Aguzzi et al., Nat Rev Mol Cell Biol. 2007 Jul;8(7):552-61

23. Physiologic role of PrPc Caughey and Byron, 2006 Nature 443-19

24. Antiapoptotic function: PrPc KO mice are more susceptible to apoptosis. Following ischemic injury, PrPc KO mice have increased infarct volume and increased caspase 3 activation.

25. Infarcts are larger in PrPc KO mice after ischemic injury

26. Levels of activated caspase 3 are increased in PrPc KO mice after ischemic injury

27. PrPc protects against oxidative stress PrPc Ko mice are more susceptible to damage by H2O2 PrPc KO mice have reduced SOD activity Brain of PrPc KO mice has increased levels of oxidated proteins, lipids, DNA. PrPc also involved in maintaining mitochondrial integrity

28. Mitochondrial structure is disrupted in Prion’s infected hamsters Control Prion’s infected

29. PrPc is has a role in maintaining synaptic architecture and function PrPc localizes mainly at the synaptic terminal PrPc KO mice have impaired Glutamatergic and GABAergic transmission, as well as decreased LTP. An early pathologic change in prion’s disease is synaptic loss

30. PrP stains as a flocculate/amorphous form at a synaptic level

31. PrPc physiological functions

32. How does the prion form and how it propagates?

33. Nature of the prion The prion is the minimum required infectious agent able to convert normal cellular prion protein PrpC into the scrapie PrPsc. Is the prion a virus? NO, the prion is not a virus, as RNA and DNA material are totally absent in its composition, and the minimal molecular weight necessary for infectivity is ~2x105Da, so small to exclude the size of a virus. Is the prion proteinaceous material? YES

34. The “unfortunate” goal of CDJ disease is to convert normal cellular prion protein PrPC into the scrapie form PrPsc. This will result in 1-reduction of the form with a-sheet conformation, 2-accumulation of the form with b-sheet conformation, which will form aggregates and deposit both at an intracellular and extracellular level.

35. Mechanism of replication of PrPSc from PrPC Sakaguchi

36. Models for conformational conversion of PrPc into PrPsc

37. Different biochemical and structural properties between PrPC and PrPSc Sakaguchi

38. Prion’s disease and PrPc/PrPsc Loss of physiologic function or gain of toxic function?

39. PrPc KO mice are viable, develop normally and have no severe pathologies observed later in life. PrPc KO do not develop the disease as they do not propagate the formation of PrPsc Gain of toxic function rather than loss of physiological function

40. The “protein-only” hypothesis • -In vitro data suggest that prion infectivity is achieved also de novo in the test tube • -Propagation of conformationally changed yeast prions has been achieved • -In vivo, prion protein null mice (PrP-/- or PrP KO) are resistant to prion infection, do not propagate prion infectivity after exposure to PrPSc,suggesting that PrPC is necessary to propagate the infectivity caused by PrPSc. • Prion protein null mice do not propagate prion infection also when infected with infectious brain tissue. This implies that the protein PrPC ALONE (and not in combination with other cellular or putative viral factors) is sufficient to propagate the prion infectivity. • -Brain homogenates spiked with PrPSc and subjected to sonication and recovery amplify the infectious species which maintains infectivity when “transferred” to new tissue.

41. Fibrils of prion protein: self propagating mechanism? Prion Particles Prion Particles+yeast protein Prion Profile: Far left, infectious prion particles extracted from yeast cells. At right is an example of what yeast prions can do when mixed with an isolated yeast protein. These fibrils are essentially long series of prions all linked together after replicating many times in the presence of the protein. Soto and Castilla Nat Med. 2004 Jul;10 Suppl:S63-7. Review.

42. PMCA Protein Misfolding Cyclic Amplification

43. PMCA: protein misfolding cyclic amplification: a technique to amplify prions in vitro

44. Hypothetical model for PrPsc endocytosis

45. Prion proteins scrapie accumulation and propagation from normal cellular Prion protein (PrPC to PrPsc) Caughey and Byron, 2006 Nature 443-19

46. Post-translational modifications on Prion protein: Glycosylation and binding to metals Caughey and Byron, 2006 Nature 443-19

47. GPI anchor as a target for the treatment of CJD?

48. Inhibition of GPI-anchor could improve the internalization AND the degradation of PrPC, that in this way will not be converted into PrPSc at and intracellular level (PrPSc intracellular more toxic than extracellular) Caughey and Byron, 2006 Nature 443-19

49. PrPsc is toxic both when extracellular and intracellular MECHANISM?

50. PrPc and PrPsc form aggregates intracellularly, but only aggregates form PrPsc are toxic UPS impairment as a consequence of PrPsc toxicity