Human Prion diseases

1. Speaker : Swarnendu Pal

2. o Introduction
o Discovery
o The PrP Prion Diseases
o The Prion Protein
o Molecular Pathogenesis
o Human Prion Diseases
o Diagnosis
o Risk of Prion Disease To Health Care Givers
o Receiving a sample
o Sterilization
o Treatment
o Our Interest

3. Introduction
Prion diseases encompass a group of
neurodegenerative disorders with a problem in the
metabolism of Prion protein affecting the nervous
sytem in both humans and animal .
The term prion comes from a combination of
‘protein’ and ‘infection’ to distinguish it from
other conventional infectious diseases.

5. Clinical Vignette
 A 50 year old female housewife presented with altered
sensorium .No H/O trauma.
 Symptoms of akinetic state, mutism, rapid progressive
dementia, and myoclonic jerks with duration of three
months.
 Mixed diet habits with meat consumption.
 No surgical procedure history in past.
 No family history.
 O/E, drowsy with flexor posturing of upper limbs on painful
stimulus. No signs of decerebrate and decorticate rigidity.
Rest unremarkable..

6. Clinical vignette ....
(MRI) of the brain revealed mild cerebral atrophy,
increase in signal intensity in putamen, caudate
nucleus, and cerebral cortex (cortical ribboning).
Cerebro spinal fluid (CSF) analysis showed elevated
protein of 45 mg/dl, normal glucose, and no
pleocytosis.
An EEG obtained one week later revealed bilateral
synchronous periodic epileptiform discharges
Gadgil NM, Chaudhari CS, Gohil SD, Kalgutkar AD. Creutzfeldt-Jacob disease: An autopsy case
report in tertiary care hospital. Indian J Pathol Microbiol 2012;55:97-9.

8. What’s so unique about Prion disease
?
Prions are the only known transmissible pathogens that
are devoid of nucleic acids.
Prion diseases may manifest as infectious, genetic, and
sporadic disorders

9. What’s so unique about Prion
disease ?
Prion diseases result from the accumulation of PrPsc
the conformation of which differs substantially from
that of its precursor, PrPc.
Distinct strains of prions exhibit different biologic
properties, which are epigenetically inherited.

10. Discovery
 During the 1960s, radiation biologist Tikvah
Alper and mathematician John Stanley Griffith
developed the hypothesis that some
transmissible spongiform encephalopathies are
caused by an infectious agent consisting solely
of proteins and resisted ionizing radiation.

11. Discovery contd...
 In 1982, Stanley B.Prusiner of the
University of California, San
Francisco announced that his team
had purified the hypothetical
infectious prion.
 Prusiner won the Nobel Prize in
1997 for his research into prions.

12. THE PrP PRION DISEASES
Disease in human
 Kuru
 iCJD
 vCJD
 fCJD
 sCJD
 GSS
 FFI
 sFI
Disease in animal
 Scrapie(M/C)
 BSE
 TME
 CWD
 FSE
 Exotic ungulate
encephalopathy

13. The Prion Protein
 PrP - major component of prions. PrP is a normal cell
membrane protein expressed at a particularly high level in
nerve cells.
 PrP exists in 2 isoforms:
 (PrPc) - rich in α helix and little β structure
 (PrPSC) also designated PrPres(protease resistant) - less α
helix and a high amount of β structure.

14. The Prion Protein contd...
Both isoforms encoded by the same cellular gene
PRNP, located on short arm of human chromosome 20
Identical molecular weights of 33-35 kDa
Same amino acid sequence,but differ in their three
dimensional, folded structure.

15. The Prion Protein contd...
 PrPc is a glycoprotein anchored to the external surface of
neurones by a glycolipid moiety.
 Function
 Unknown.
 May play a role in cell adhesion,
 Transmembrane signalling or may be some form of
receptor.
 PrPsc is post-translationally modified isoform of the host
PrPc

17. Conversion of PrPc to PrPSC
 The two mechanisms believed to give rise to PrPsc are:
 1. Post-transcriptional modification leading to an
alteration in the conformation of the normal prion
protein
 2. Mutations in the prion gene (PRNP) resulting in the
translation of a protein with sequence abnormalities that
predispose to the spontaneous adoption of an abnormal
conformation."

18. Prion-induced neuronal damage--the
mechanisms of neuronal destruction

19. Prion-induced neuronal damage--the
mechanisms of neuronal destruction
 Cell death in prion disease occurs by apoptosis.
 Apoptosis of neuronal cells can also be induced in vitro by
exposure to PrPSC
 PrPSC causes microglial activation.
 Activated microglial cells  cytokines and reactive oxygen species
 Disturbances in cerebral copper metabolism and antioxidative
defense mechanisms
 loss of function of PrPC may contribute to neuronal cell death
Curr Top Microbiol Immunol. 2001;253:203-17. Prion-induced neuronal damage--the mechanisms of neuronal
destruction in the subacute spongiform encephalopathies. Giese A1, Kretzschmar HA

20. Affected areas of Brain

21. Etiology
Genetic CJD
Fatal familial insomnia
Gerstmann-Sträussler-
Scheinker
Kuru
Iatrogenic CJD
Variant CJD

22. Molecular Pathogenesis
 Sporadic
 Somatic mutation of the open reading frame of the PrP
gene
 The activation energy barrier theory by Dobson
 PrPsc may be present at low levels in some normal
cells.
Altered metabolic states, the cellular mechanisms for
clearing PrPSC compromised,  rate of PrPSC formation
exceed the capacity of the cell to clear it.

23. Molecular Pathogenesis
contd..
 Inherited prion diseases
 dominantly inherited caused by mutations in the
PRNP.
 Misense mutations and expansion in the octapeptide
repeat region of the gene cause familial prion
diseases.

24. Molecular Pathogenesis
contd..
 Infectious prion diseases
 Iatrogenic CJD - Accidental transmission during
corneal transplantation, contaminated(EEG)
electrode implantation & surgical procedures Dura
Mater Grafts
 Kuru- (means shivering and trembling) due to
ritualistic cannibalism.

25. Molecular Pathogenesis
contd..
 AMYLOID IN PRION: Amyloid plaques found in prion
disease . Amyloidosis is found in 10% cases of CJD,70%
case of Kuru and in all cases of GSS.

26. Sporadic CJD
 Sporadic forms of prion disease comprise most cases.
Worldwide incidence of approximately 1 case/1 million
population
 Patients as young as 17 years and as old as 84 years
have been recorded.
 Progressive disease, death within a year of onset

27. CJD - Clinical features
Nonspecific prodromal symptoms –
fatigue
sleep disturbance,
weight Loss,
headache,
anxiety，
vertigo, malaise, and ill-defined pain.
Deficits in higher cortical function.
Psychiatric symptoms – depression, psychosis, visual
hallucinations

28. CJD - Clinical features
 Cerebellar deficits followed by progressive
dementia.
 Visual problems
 Extrapyramidal dysfunction –rigidity, masklike facies,
myoclonic jerks
 Seizures

30. Radiological investigations
 CT Scan - normal or
show cortical atrophy.
 MRI and FLAIR
sequences and DWMRI
show increased
intensity in the basal
ganglia and
“cortical ribboning”

31. EEG
60% of individuals
show the typical
pattern
Periodic sharp wave
complexes (PSWCs)

32. Laboratory Analysis
Cerebrospinal fluid (CSF) analysis-
 Normal.
 Elevated levels of protein 14-3-3 (not specific for
diagnosis).
 Neurone specific enolase in CSF may also be increased.
Detection of PrPsc- IHC and western blotting have
been used to detect PrPSC in brain tissue.
IHC either fixed or unfixed tissue.
Western Blot– only on unfixed tissue (Gold Standard)

33. IHC

34. IHC

35. Laboratory Analysis
 PrP gene analysis
This can be performed on DNA extracted from blood
leucocytes antemortem as well as from frozen brain
tissue.

36. Gross appearance
 Macroscopically normal.
 Some or severe atrophy
with a reduction in brain
volume.
 Marked ventricular
enlargement
 Meninges and blood
vessels appear normal

38. Few Terms explained
 Neuropil - Nuclei of both neurones and neuroglia are
surrounded by a dense network of branching cytoplasmic
processes, axons and dendrites- seen as fibrillar material on
HE stain
 Neuroglia- 4 types
 Oligodendrocytes- forms myelin sheath in CNS(equivalent
to schwann cells)
 Astrocytes- Pack the interstices between neurones. Provide
mechanical support, exchange of metabolites & part of
BBB.

39. Few Terms explained
• Microglia - CNS representative of Monocyte-macrophage
system. Role in defence and immunology.
• Ependymal cells – Specialised epithelium which lines the
ventricles and spinal canal.
 Gliosis- Gliosis is reaction of the CNS to injury of the brain
or spinal cord
(a) increased number of astrocytes, or
(b) increased number and length of process or increased
cytoplasmic synthesis of GFAP representing a
gemistocytic astrocyte.

40. Neurones
Oligodendrocyte
Astrocyte

42. H/P features of CJD
 Vacuolation of the neuropil
 Reactive gliosis
 Few vacuoles in the neuropil are
glassy or lightly eosinophilic.
 Microglial cells are increased in
number
 Negligible perivascular
inflammatory infiltrates.

43. H/P features of CJD
 PAS-positive plaques of
‘spiked ball’ type (‘kuru’
plaques).
 Mild spongiform change
present in the molecular
layer.

44. Points to consider...
Lack of identification of diagnostic histologic changes
from a single biopsy site does not rule out CJD
Different subtypes of the disease exist, variable with
the mutation in the prion protein,
Considerable H/P variation can be seen from gyrus to
gyrus in CJD and at different stages of the disease
Diffuse cortical vacuolization—is a common finding in
advanced Alzheimer disease

45. Variant CJD/ “Mad Cow Disease”
 In the mid-1990s ‘‘new-variant’’ CJD (vCJD)
emerged in Great Britain and France, originally
called ‘‘mad cow disease’’.
 linked to the eating of meat from cows with BSE
 Patients are young

46. Variant CJD/ “Mad Cow Disease”
 Clinical Features
 behavioural changes,
 ataxia and
 peripheral sensory disturbances rather than change in
mentation
 Time between onset and death range from 7.5 - 22.5
months

47. H/P features
Cerebral and cerebellar
cortex striking stellate,
‘‘florid’’ plaques
composed of prion
protein,
coarse vacuolization (fine
vacuolation of CJD)
Marked gliosis

48.  Iatrogenic CJD
 Progressive cerebellar syndrome in a recipient of
human cadaveric-derived pituitary hormone; or
 sporadic CJD with a recognized exposure risk, e.g.,
antecedent neurosurgery with dura mater
implantation.
 Familial CJD
 Definite or probable CJD plus
 definite or probable CJD in a first degree relative;
and/or Neuropsychiatric disorder plus
 disease-specific PrP gene mutation.

49. Clinical Course
 I.P - 1.5-2 years. In other cases I.P of up to 40
years.
 Most patients with CJD live 6-12 months after the
onset of clinical signs and symptoms,
Some live for up to 5 years.

50. Differential Diagnosis
Alzheimer’s Disease
Hashimoto's encephalopathy
AIDS dementia complex
Diffuse intracranial tumors

51. Feature Prion Disease Alzheimer's disease
Onset Any age Mid-to-Iate life
Progression 6-8 months 8-12 years
Signs Dementia, ataxia,
myoclonus
Dementia
Chromosomal loci 20 21, 14, 19
Gene with mutation PRNP APP
Vulnerable cells Many regions of the brain Cortical, hippocampus,
cholinergic basal
forebrain
neurones
Cytoskeletal pathology Rare NF Tangles, neurites
and neuropil threads
Amyloid PrPsc Ab
Cell Death Variable Severe

52. Fatal Familial Insomnia
 Thalamus most affected part in FFI.
 Symptoms:
 Sleep-wake cycle disturbance,
 progressive insomnia,
 complex hallucinations, depression
 stupor, coma.
Mild atrophy of the frontal cortex and moderate
atrophy of the thalamus
H/P : Neuronal loss & reactive gliosis. No spongiform
pathology.

53. GSS
 Gerstmann, Straussler, Scheinker in 1936.
 One of the rarest disease entity.
 Three dominant disorders (not a single disease)
•Ataxic GSS,
•Telencephalic GSS,
•GSS with neuro fibrillary tangles.
 Symptoms-
• Difficulty in walking,
• unsteadiness,
• leg pain,
• paraesthesia,
• mental deterioration.

55. Making a diagnosis
DEFINITIVE DIAGNOSIS
requires satisfaction of any one of the following
criteria:
Finding of “spiked ball”-type PrP amyloid plaques
Demonstration of protease-resistant PrP in biopsy
material by specific immunohistochemical or
immunoblot methods
Detection of a PrP gene mutation that is recognized
as pathogenic

56. Making a diagnosis
 PROBABLE
Rapidly progressive dementia; and at least two out of
the following four clinical features:
Myoclonus
Visual or cerebellar signs
Pyramidal/extrapyramidal signs
Akinetic mutism
AND

57. Making a diagnosis
 Positive result on at least one of the following
laboratory tests:
 a typical EEG (periodic sharp wave complexes) during an
illness of any duration; and/or
 a positive 14-3-3 cerebrospinal fluid (CSF) assay in
patients with a disease duration of less than 2 years
 MRI high signal abnormalities in caudate nucleus and/or
putamen on (DWI) or (FLAIR)
 AND without routine investigations indicating an
alternative diagnosis.

58. Workup of a suspected case of Prion
disease
 Physical exam and patient history –
actual date of onset,
patient’s ability to complete activities of daily living helps
quantify Ds
disease progression,
F/H of neurodegenerative ds,
Medical and travel history
 Blood work –
CBC,
chemistry panel (including Ca, Mg and P),
 LFTs
thyroid function,
anti-thyroglobulin and anti-thyroperoxidase antibodies
HIV,
B12, homocysteine
Paraneoplastic antibodies.

59. Workup of a suspected case of
Prion disease
Urine analysis - 24-hour urine for heavy metal screen
Imaging-
 MRI
 CT Scan- chest, abdomen and pelvis - rule out malignancy and
paraneoplastic disorders.
EEG- Periodic Sharp Wave Complexes (PSWCs) about
once every second
Brain Biopsy-

61. Receiving a sample of a suspected
case
 Biopsy material from patients suspected to have CJD
require cautious handling
 Formalinized and paraffin-embedded archival
specimens retain transmissibility.
 Commonly used - Formic acid immersion following
initial formalinization.

62. Tissue Preparation
 Histology technicians wear gloves, apron, laboratory coat, and
face protection
 Adequate fixation f/b post-fixation in 96% absolute formic acid
for 30 minutes, followed by 45 hours in fresh 10% formalin.
 Liquid waste to be collected in waste bottle containing 600 ml 6N
NaOH
 In addition to gray matter, white matter and meninges should be
sampled
 Gloves, embedding moulds, and all handling materials are
disposed in regulated medical waste.
 Tissue cassettes are processed manually

63. Tissue Preparation
The knife stage is wiped with 2N NaOH, & the knife
used is discarded immediately.
Slides labeled with “CJD Precaution”
Staining:
 Reagents prepared disposable specimen cups
 After placing the cover slip on, slides are
decontaminated by soaking them for 1 hour in
2N NaOH

64. Sterilization
 Prions are extremely resistant to common
inactivation procedures
 Autoclaving at 1340 C for 5h or treatment with 2N
NaOH for several hours is recommended

65. Care of CJD Patients
 Risk of inoculation by aerosols - very small
 Procedures should be performed in certified
biosafety cabinets
 EEG,EMG needles should not be reused.
 Private room nursing care not required.
 Wastes handled accordingly.
 Pathologists or other morgue employees should not resist
performing autopsies on patients whose clinical diagnosis
was CJD.

66. Risk to Health Care Providers
 High Infectivity- Brain, Spinal cord, Eye
 Low Infectivity- CSF, Kidney, Lymph nodes/spleen
 No detectable infectivity – Tears, Nasal mucous, urine,
faeces, blood

67. Prevention and Therapeutics
 No known effective therapy
 Phenothiazines and Acridines inhibit PrPSC formation
in cultured cells
 Anti-PrP Abs have been shown to eliminate PrPSC
from cultured cells
 Several drugs, including Pentosan Polysulfate as
well as Porphyrin and Phenylhydrazine derivatives
tried.

68. Take Home Message
 Prion diseases are a group of neurodegenerative
disorders with a problem in the metabolism of Prion
protein.
 Alzheimers disease is a close differential diagnosis.
 Suspected Prion Disease specimens and Laboratory
samples require careful handling and should be
according to protocol.

69. Our Interest
Describe the pathogenesis and pathology of prions
disease. April 2008 Paper 4
Prions disease. April 2001,2002 Paper 2

70. References
 Harrisons Principles of Internal Medicine - 19th Edn
 Sternberg’s Diagnostic Surgical Pathology - 5th Edn
 Rosai & Ackermans Surgical Pathology – 10th Edn
 CDC's Diagnostic Criteria for Creutzfeldt-Jakob Disease (CJD), 2010
Global Surveillance, diagnosis, and Therapy of Human Transmissible spongiform Encephalopathies:
Report of WHO consultation, February 9-11, 1998, Geneva, Switzerland; and b) Zerr, I, Kallenberg K,
Summers DM, et al. Brain 2009, 132; 2659-2668.
 Human prion diseases - RAJEEV THAKUR, YASMEEN MARBANIANG VINCENT,
SUJATA CHATURVEDI THE NATIONAL MEDICAL JOURNAL OF INDIA VOL. IS, NO.
6,2002
 Gadgil NM, Chaudhari CS, Gohil SD, Kalgutkar AD. Creutzfeldt-Jacob disease:
An autopsy case report in tertiary care hospital. Indian J Pathol Microbiol
2012;55:97-9.
 Curr Top Microbiol Immunol. 2001;253:203-17. Prion-induced neuronal
damage--the mechanisms of neuronal destruction in the subacute spongiform
encephalopathies. Giese A1, Kretzschmar HA
 CDC guidelines for handling tissue specimens.

71. THANK YOU