A human iPSC platform for studying pathogenic protein aggregation in neurodegeneration Eve Corrie1, Anna-Lena Zepernick1, Matthieu Trigano1, and Emma V. Jones1 1Medicines Discovery Catapult, Block 35, Alderley Park, Cheshire, SK10 4ZF, UK
Pathogenic misfolded protein
Introduction
Protein misfolding, phosphorylation, and aggregation are central features of many neurodegenerative diseases These pathogenic proteins can seed further misfolding and propagate pathology across anatomically connected brain regions Native protein These processes contribute to cellular dysfunction and death, and are increasingly being targeted by novel therapeutics in the drug discovery landscape MDC has developed human in vitro systems to study the pathology of α-synuclein and tau in iPSC-derived cortical neurons in 96- and 384-well format Native protein using high content imaging assays and analysis pipelines
Live imaging of tau aggregation using lentiviral systems
0N4R-EGFP tau 2 weeks post-PFF No PFF control
• Wildtype neurons were transduced with a lentivirus to express a full-length 4R tau isoform with a familial frontotemporal dementia P301L mutation labelled with EGFP • Neurons were then exposed to recombinant tau preformed fibrils (PFFs) and imaged
3μg/ml K18 (P301L) PFFs
30μg/ml 2N4R PFFs
800
200
0
K18 FRET biosensor system 2 weeks post-PFF No PFF control
3μg/ml K18 (P301L) PFFs
Area of NFRET signal of K18 (P301L) fragments
30μg/ml 2N4R PFFs
10000
0 days 3 days
8000
7 days
6000
• Neurons were transduced with lentiviruses 4000 to express 4R microtubule binding domain 2000 (K18) fragments with P301L mutation 0 labelled with EYFP or Cerulean • When aggregation occurs, the fluorophores become close enough for FRET to occur • Increasing over time, PFF exposure induced areas of bright EGFP-positive aggregates of full-length tau, and • The transduced neurons were then aggregation of the K18 fragment FRET pair as seen by positive normalised FRET (NFRET) signal exposed to tau PFFs and FRET imaging • K18 PFFs with P301L mutation induced aggregation more readily than full length 2N4R PFFs in both systems performed
Phosphorylation and aggregation of native α-synuclein
14 days
WT/WT
pS129 α-synuclein NeuN
SNCA A53T/A53T
2 weeks
3 weeks
20
20
%pS129 positive
2 weeks post-PFF
15 10
8.8
5 0.9
1.6
1.5
0
15 10 5
2.8
2.2
0.7
PRKN R275W/R275W
SNCA A53T/A53T
WT/WT
0
-PFFs
• Isogenic control and early onset Parkinson’s disease SNCA A53T mutation neurons were exposed to α-synuclein PFFs with A53T mutation 3 weeks • Neurons were fixed and labelled for α-synuclein post-PFF phosphorylated at serine 129 (pS129) • PFF treated neurons show Lewy body-like perinuclear aggregates of pS129 α-synuclein
%pS129 positive
16.9
PRKN R275W/R275W
pS129 α-synuclein β-tubulin
Immunocytochemistry of phospho-S129 α-synuclein
SNCA A53T/A53T
NFRET
14 days
WT/WT
FRET
7 days
0. 3μ g/ m 1 μ l K1 - PF F g/ 8 m (P s 3μ l K1 301 g/ L) 8 m (P lK 3 18 01L ) (P 30 1μ 1L g/ m ) 3μ l 2N g/ 4R m l2 10 N μg /m 4R l 30 μg 2 N4 /m R l2 N 4R
K18 (P301L) Cerulean
3 days
400
Overexpressed K18 tau fragment FRET biosensor system K18 (P301L) EYFP
0 days
600
-PFFs
0N4R (P301L) Tau-EGFP MAP2
Area of aggregated GFP-0N4R (P301L) Tau
NFRET area (μm2/well)
Overexpressed full-length 0N4R-EGFP tau
Hyperphosphorylated protein aggregate
0. Aggregated tau area (μm2/well) 3μ g/ m -P 1μ l K FF 1 g/ 8 m (P s l 3 K 3μ 18 01L g/ ) m ( l K P30 1L 18 ) (P 30 1μ 1L g/ m ) 3μ l 2N g/ 4R m l 10 μg 2 N4 /m R 30 l2 N μg /m 4R l2 N 4R
• • • •
Dysfunction, toxicity and cell death
+PFFs
+PFFs
• A53T mutation increases the proportion of pS129 positive neurons after PFF exposure
Inter-neuronal seeding of α-synuclein pathology in microfluidics devices
15 10
8.9
5 0
High content imaging of human iPSC-derived neurons provides a platform to model the pathological seeded aggregation and phosphorylation of neurodegenerative proteins like tau and α-synuclein The ability to induce and detect disease-relevant protein aggregation using human-relevant assays early in the discovery pipeline makes this system a valuable tool for therapeutic developers targeting protein misfolding, phosphorylation, and propagation In vitro assays quantifying tau and α-synuclein aggregation following disease-relevant PFF exposure enable sensitive, reproducible measurement of seeded pathology and support evaluation of mechanism-modifying interventions
SNCA A53T/A53T
WT/WT
A53T/A53T
Conclusions
•
17.5
0
Live neuron dye
•
20
Donor
1.1
SNCA A53T/A53T
pS129 α-synuclein NeuN
Projection of neurites through microchannels at 2 weeks
•
Immunocytochemistry of phospho-S129 α-synuclein
WT/WT
WT/WT
Acceptor chamber – seeded PFFs
%pS129 positive
• Isogenic control or SNCA A53T “Donor” “Acceptor” mutation neurons were plated in both chambers of a microfluidics device then cultured to allow the neurons to connect via the microchannels • PFFs were added to the donor eNuvio Omega4 device chamber only before fixing and labelling for pS129 α-synuclein • pS129 positive neurons were detected in donor chambers due to directly applied PFFs and acceptor chambers due to seeded PFFs, though automated analysis failed to detect the small proportion of positive wildtype neurons in acceptor chambers
Donor chamber – PFFs directly applied
Acceptor
• A53T mutation increases pS129 pathology in both donor and acceptor chambers