Pizza club Zoé 28.09.16

1. Pizza Club
Zoé Hanss
28.09.2016

2. What are prions ?
• Kuru, Creutzfeldt–Jakob
disease, Scrapie…
→ Caused by a new pathogenic
element
• Non viral or bacteriologic
• Resistant to standard
decontamination
• Transmission process
unclear
β-sheetsα-helix
Native
protein
Alternative
shape
SOLUBLE Aggregates
PrP PrPSc

3. Misconformed
protein Fragmentation
Propagation
Nucleus Aggregates
New
nucleus
Native
Protein
Limited
step
Spontaneous aggregation
Aggregatesnumber
Temps
Prion mechanism
Latency
Nucleus formation

4. Misconformed
protein Fragmentation
Propagation
Nucleus Aggregates
New
nucleus
Native
Protein
Limited
step
Spontaneous aggregation
Aggregatesnumber
Time
Exogenous
nucleus
Decreased latency
Exogenous nucleus
Prion mechanism

5. Parkinson’s disease Multiple system atrophyAlzehimer’s disease
Prion Disease
Aβ
Amyloïdes plaques
Tau
Tangles
Prion
PrPsc aggregates
α-synuclein
Lewy Body
α-synuclein
Glial cytoplasmic inclusions

6. Multiple System Atrophy (MSA)
• Sporadic, adult-onset, neurodegenerative disorder
• Incidence 3/100 000 (>50 Yo)
• Progressive loss of autonomic nervous system function
• Signs of parkinsonism
• Glial cytoplasmic inclusions (GCI) of filaments of α-synuclein
Prion behaviour ?

7. Previous experiment (2013)
2 MSA cases
TgM83+/-
α-syn A53T
120 days Progressive CNS dysfunction
Phosphorylated α–syn in neurons
Are these 2 cases real “transmission” ?
2) Transmission to TgM83+/- :1) Transmission to HEK cells
- 1 control brain
- 6 PD brains
- 14 MSA brains
Spontaneous aggregation
Aggregatesnumber
TimeDecreased latency
Exogenous nucleus
Brain homogenates
Mouse model prone to form α-syn aggregates

8. Characterisation of samples
1) Neuropathological characteristic of patients samples
SN depigmentation
Phosphorylated α-syn staining
GCILB

9. By ELISA
Characterisation of samples
2) Total α-synuclein amount

10. FC: Frontal cortex
SN: Substancia nigra
BG: Basal ganglia
P: Pons
MSA/PD : higher amounts of phosphorylated α-syn than controls
Characterisation of samples
3) Level of insoluble α-synuclein

11. Experiment 1 :
Transmission of MSA prions to cultured HEK cells
HEK cells expressing full length α-synuclein containing the A53T mutation fused to yellow
fluorescent protein : exposed for 4 days to precipitated sample from brains
4 days
Sodium phosphotunstic acid (PTA)
Precipitation of α-syn Imaging
Confocal fluorescence microscopy
A53T mut
YFP
- 1 control brain
- 6 PD brains
- 14 MSA brains

12. MSA samples induce
aggregate formation in HEK
cells
Experiment 1 :
Transmission of MSA prions to cultured HEK cells
No aggregates with
control brains
No aggregates with
PD brains
Aggregates with all
MSA brains

13. 30 µl of 1% homogenate
Right parietal lobe
2 month old
Checked for neurological
illness twice a week
Experiment 2 :
Transmission of MSA prions to TgM83+/- mice
Brain homogenates
- 1 control brain
- 6 PD brains
- 14 MSA brains

14. Inoculation with all MSA samples caused CNS dysfunction at 100-150 dpi: dysmetria and circling behavior
PD samples : no sign of neurological dysfunction at >360 dpi
Aggregates of phosphorylated a-syn
Astrocytic gliosis
Experiment 2 :
Transmission of MSA prions to TgM83+/- mice

15. Contralateral hemisphere
De novo formation of α-synuclein aggregates in contro-lateral
hemisphere of mouse brain
Experiment 2 :
Transmission of MSA prions to TgM83+/- mice

16. TgM83+/-
MSA
TgM83+/+
Primary transmission
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice
Mouse model prone to form α-syn aggregates
Mouse model spontaneously forming
α-syn aggregates

17. Shorter incubation time with MSA inoculated mouse brain
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice

18. TgM83+/-
MSA
TgM83+/+
Primary transmission Secondary transmission
TgM83+/- TgM83+/-
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice

19. Same incubation time: replication of MSA prions at same level
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice

20. TgM83+/-
WT
Tg(SNCA) Snca0/0
Experiment 4 :
Role of endogenous α-syn

21. A53T mutation facilitate prion replication
TgM83+/-
WT
Tg(SNCA) Snca0/0
Experiment 4 :
Role of endogenous α-syn

22. IC
IM
Intraglossaly
Incubation time
133 ± 6 days
136 ± 6 days
> 220 days
MSA
Experiment 4 :
Role of inoculation routes

23. Discussion
• All 14 human brain samples from MSA patients could be transmitted to
both HEK culture and Tg mice expressing α-syn A53T mutation
• At least 2 strains of α-synuclein prions : MSA and TgM83+/+
• MSA and PD prion strains are different
• Important to note that transmission of MSA prions requires A53T α-
synuclein mutation
• Health care issues

24. Thank you for your attention !