Is the infectious prion protein PrPsc present in blood of variant CreutzfeldtJakob Disease patients

1
Is the infectious prion protein PrPsc present in
blood of variant Creutzfeldt-Jakob Disease
patients?

• Riet De Smet

2
Introduction

• Prions and related diseases
• Structure of prions
• vCJD
• Aim of the project

3
Prions and related diseases

• Conversion of the normal prion protein PrPc
  (cellular) into the infectious form PrPsc
  (scrapie) leads to TSE (Transmissible Spongioform
  Encephalophaties)
• TSEs are fatal, neurodegenerative disorders with
  a very long incubation period followed by a rapid
  neurological dysfunction and specific lesions of
  the CNS. These lesions cause the characteristic
  spongiform aspect of brain tissues
• Prion diseases may occur as genetic (PRNP gene),
  infectious or sporadic disorders all of which
  involve modification of the cellular prion
  protein PrPc

4
Prions and related diseases
Abbreviations used CJD, Creutzfeldt-Jakob
disease GSS, Gerstmann-Sträussler-Scheinker
disease FFI, fatal familial insomnia FSI, fatal
sporadic insomnia BSE, bovine spongiform
encephalopathy TME, transmissible mink
encephalopathy CWD, chronic wasting disease
FSE, feline spongiform encephalopathy HGH, human
growth hormone MBM, meat and bone meal.
5
Structure of prions

• PrPc is a glycosylphosphatidylinositol-anchored
  membrane protein that is expressed at significant
  levels in normal neurons, glial cells and
  lymphoid cells
• PrPc and PrPsc have the same primary amino acid
  sequence
• Secondary structure differs drastically
• PrPc is rich in a-helices but poor in ß-sheets
• PrPsc is rich in ß-sheets

6
Structure of prions
Models of PrPc and PrPsc deduced from Syrian
hamster recombinant PrP 90-231. Helix A appears
to be converted into a ß-sheet along with some
other variable regions. Helix B and C belong the
stable core of PrP and are stabilized by a
disulfide bridge between Cys179 and Cys214. This
core contains 2 asparagine-linked
oligosaccharides.
7
Structure of prions

• Their different secondary structure impart very
  different physicochemical properties

8
Structure of prions

• PrPc is protease sensitive while PrPsc is only
  partially protease sensitive
• Product of treatment of PrPsc with protease K is
  the N-terminally truncated form, PrP 27-30

Diagram of SHaPrP (Syrian hamster) which consists
of 254 amino acids
9
vCJD

• Case in which prion diseases cross the species
  barrier
• Consumption of BSE infected meat causes vCJD in
  humans
• 145 cases of vCJD in the UK and some cases in
  France and Italy
• Epidemical models predict at least several
  hundred cases as the ultimate extent of the
  disease. But predictions are uncertain because
  the long incubation time of the disease and lack
  of knowledge of ways of transmission
• The increasing amount of patients raises concern
  about transmission of the disease through blood

10
Aim of the project

• Prion diseases generally spread in the
  lymphoreticular system before crossing the
  blood-brain barrier
• This means an infectious form is already present
  in blood
• Up till now PrPsc hasnt been detected yet in
  human blood
• So, either the concentration in blood is too low
  or either the infectious form is a structural
  intermediate between PrPc and PrPsc with
  different physicochemical properties
• In this project we try to increase the
  concentration of potential PrPsc in vitro so that
  it can be detected in a Western blot

11
Experimental plan

• Blood collection and separation in components
• Amplification (PMCA)
• Electrophoresis and Western blot
• What if the results are negative?

12
Blood collection and separation in components

• Blood samples from vCJD patients are taken
• Anticoagulant is added
• Separation in components by centrifugation
• Components are platelet-containing plasma, buffy
  coat (contains WBCs), RBCs
• For details Cervenakova et al., 2003

13
Amplification (PMCA)

• PMCA Protein-Misfolding Cyclic Amplification
• Method aggregates formed when PrPsc is incubated
  with PrPc are disrupted by sonication to
  generate multiple smaller units for the continued
  formation of PrPsc (Gabriela et al., 2001)

14
(No Transcript)
15
Amplification (PMCA)

• PrPc from brain homogenate of healthy
  transgenetic mice (Tg(HuPrP) PrnP0/0)
• Homogenisation of brains a homogenator containing
  PBS buffer with detergents (0.5 Triton-X-100,
  0.05 SDS) and protease inhibitor
• 5 samples
• Brain homogenate
• Brain homogenate incubated with all blood
  components
• Brain homogenate incubated with plasma
• Brain homogenate incubated with WBCs
• Brain homogenate incubated with RBCs
• PMCA is applied to samples 2-5
• Samples are collected of 5, 10, 20 and 40 cycles

16
Electrophoresis and Western blot

• Samples 2-5 are digested with protease K and
  reaction is stopped with 50 mM phenyl-methyl
  sulfonyl fluoride
• SDS-PAGE of samples
• Transfer of proteins to nitrocellulose membrane
  for Western blot
• Nitrocellulose membrane is blocked with 5
  non-fat milk and incubated with mAB 6H4
  (Prionics, Zürich)
• Immunodetection is accomplished by incubation
  with goat anti-mouse secondary antibody labelled
  with HRP and ECL chemoluminescence Kit (Amersham)

17
Electrophoresis and Western blot

• PrPsc is cleaved at the N-terminal by protease K.
  This causes a reduction in molecular mass from 35
  till 27-30 kDa

Western blot of BSE animal (Schaller et al.,
1999). The weak signal at 31 kDa represents an
internal marker. On the right, undigested
full-length PrP from a normal brain homogenate is
shown
18
What if the results are negative?

• Infectious PrP is probably present in blood as a
  structural intermediate between PrPc and PrPsc
• Further research can than be focused on
  determining the ß-sheet content of PrP present in
  blood
• the infrared spectrum and far-UV CD spectrum of
  recombinant ß-PrP is shown to differ from the one
  of recombinant a-PrP (Kazlauskaite et al., 2003)
• So, ß-sheet content can be determined using a
  spectropolarimeter and infrared spectrometer

19
Relevance

• Early diagnostics of vCJD
• If PrPsc is present in blood, transmission of
  vCJD via blood or blood products is very probable

20
References

• Cervenakova, L., Yakovleva, O., Mc Kenzie, C. et
  al., 2003. Similar levels of infectivity in the
  blood of mice infected with human vCJD and GSS
  strains of transmissable spongiform
  encephalopathy. Transfusion complications 43,
  1687-1694.
• Gabriela, P., Saborio, B.P., Soto, C., 2001.
  Sensitive detection of pathological prion protein
  by cyclic amplification of protein misfolding.
  Nature 411, 810-813.
• Kazlauskaite, J., Sanghera, N., Sylvester, I. et
  al., 2003. Structural changes in lipid membranes
  leading to aggregation and fibrillization.
  Biochemistry 42, 3295-3304.
• Mc Kinley, M.P., Meyer, R.K., Kenaga, L., et al.,
  1991. Scrapie prion rod formation in vitro
  requires both detergent extraction and limited
  proteolysis. J. Virol. 65, 1340-1351.
• Pan, K.-M., Baldwin, M., Nguyen, J., Gasset, M.
  et al., 1993. Conversion of a-helices into
  ß-sheets features in the formation of the scrapie
  prion proteins. Proc. Natl. Acad. Sci. USA 90,
  10962-10966.
• Prusiner, S.B., 1991. Molecular biology of prion
  diseases. Science 252, 1515-1522.
• Prusiner, S.B., 1997. Nobel lecture.
• Schaller, O., Fatzer, R., Stack, M. et al., 1999.
  Validation of a Western immunoblotting procedure
  for bovine PrP detection and its use as a rapid
  surveillance method for the diagnosis of bovine
  spongiform encephalopathy (BSE). Acta Neuropathol
  98, 437-443.
• Sparkes, R.S., Simon, M., Cohn, V.H., et al.,
  1986. Assignment of the human and mouse prion
  protein genes to homologous chromosomes. Proc.
  Natl. Acad. Acad. Sci. USA 83, 7358-7362.
• United Kingdom Department of health Monthly
  Creutzfeldt-Jakob Disease statistics web site.
  Http//www.doh.gov.uk/cjd/stats
• Valleron, A.J., Boelle, P.Y., Will, R., Cesbron,
  J.Y., 2001. Estimation of epidemic size and
  incubation time based on age characteristics of v
  CJD in the United Kingdom. Science 294,
  1726-1728.
• Will, R.G., Zeidler, M., Stewart, G.E., et al.,
  2000. Diagnosis of new-variant Creutzfeldt-Jakob
  disease. Ann. Neurol. 47, 575-582.