Post on 14-Apr-2017
Pizza ClubZoé Hanss
28.09.2016
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
Misconformed protein Fragmentation
Propagation
Nucleus Aggregates
New nucleus
Native Protein
Limited step
Spontaneous aggregation
Aggr
egat
es n
umbe
r
Temps
Prion mechanism
Latency
Nucleus formation
Misconformed protein Fragmentation
Propagation
Nucleus Aggregates
New nucleus
Native Protein
Limited step
Spontaneous aggregation
Aggr
egat
es n
umbe
r
Time
Exogenous nucleus
Decreased latency
Exogenous nucleus
Prion mechanism
Parkinson’s disease Multiple system atrophyAlzehimer’s disease
Prion Disease
AβAmyloïdes plaques
TauTangles
PrionPrPsc aggregates
α-synucleinLewy Body
α-synucleinGlial cytoplasmic inclusions
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 ?
Previous experiment (2013)
2 MSA casesTgM83+/-α-syn A53T
120 days Progressive CNS dysfunctionPhosphorylated 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
Aggr
egat
es n
umbe
r
TimeDecreased latency
Exogenous nucleus
Brain homogenates
Mouse model prone to form α-syn aggregates
Characterisation of samples1) Neuropathological characteristic of patients samples
SN depigmentation
Phosphorylated α-syn staining
GCILB
By ELISA
Characterisation of samples2) Total α-synuclein amount
FC: Frontal cortex SN: Substancia nigraBG: Basal gangliaP: Pons
MSA/PD : higher amounts of phosphorylated α-syn than controls
Characterisation of samples3) Level of insoluble α-synuclein
Experiment 1 :
Transmission of MSA prions to cultured HEK cells
HEK cells expressing full length α-synuclein containing the A53T mutation fused to yellowfluorescent protein : exposed for 4 days to precipitated sample from brains
4 days
Sodium phosphotunstic acid (PTA)
Precipitation of α-syn ImagingConfocal fluorescence microscopy
A53T mut
YFP
- 1 control brain- 6 PD brains- 14 MSA brains
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
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
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
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
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
Shorter incubation time with MSA inoculated mouse brain
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice
TgM83+/-
MSA
TgM83+/+
Primary transmission Secondary transmission
TgM83+/- TgM83+/-
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice
Same incubation time: replication of MSA prions at same level
Experiment 3 :
Serial transmission of MSA prions to TgM83+/- mice
TgM83+/-
WT
Tg(SNCA) Snca0/0
Experiment 4 :Role of endogenous α-syn
A53T mutation facilitate prion replication
TgM83+/-
WT
Tg(SNCA) Snca0/0
Experiment 4 :Role of endogenous α-syn
IC
IM
Intraglossaly
Incubation time
133 ± 6 days
136 ± 6 days
> 220 days
MSA
Experiment 4 :Role of inoculation routes
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
Thank you for your attention !