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 !