PERK Inhibitor GSK2606414 + GADD34 Mimetic Sephin1 Rescues eIF2alpha Stalling in ALS iPSC Motor Neurons

In_Vitro Score: 0.830 Price: $0.50 ALS / FTD iPSC-derived motor neurons from ALS patients (TDP-43 M337V, n=3 donors) + isogenic WT; G93A-SOD1 mice (secondary) Status: proposed

What This Experiment Tests

In_Vitro experiment designed to assess clinical efficacy targeting EIF2S1, EIF2AK3/PERK, PPP1R15B, EIF2B in iPSC-derived motor neurons from ALS patients (TDP-43 M337V, n=3 donors) + isogenic WT; G93A-SOD1 mice (secondary). Primary outcome: p-eIF2alpha/eIF2alpha ratio normalised to <=1.3x isogenic WT; TDP-43 nuclear:cytoplasmic ratio >=2.5

Description

Test whether combined PERK inhibition (GSK2606414) + GADD34 phosphatase mimetic (Sephin1) restores eIF2alpha-phosphorylation to physiological range, rescues stalled polysomes, and protects TDP-43-aggregating motor neurons in iPSC-derived cultures and G93A-SOD1 mice.

TARGET GENE

EIF2S1, EIF2AK3/PERK, PPP1R15B, EIF2B

MODEL SYSTEM

iPSC-derived motor neurons from ALS patients (TDP-43 M337V, n=3 donors) + isogenic WT; G93A-SOD1 mice (secondary)

ESTIMATED COST

$67,000

TIMELINE

16 months

PATHWAY

eIF2alpha phosphorylation -> stalled translational initiation -> stress granule formation -> TDP-43 mis-localisation

SOURCE

task:a989715e-c687-4558-91ca-74fce1474bd2

PRIMARY OUTCOME

p-eIF2alpha/eIF2alpha ratio normalised to <=1.3x isogenic WT; TDP-43 nuclear:cytoplasmic ratio >=2.5

Scoring Dimensions

Protocol

Differentiate iPSC-MNs (35-day protocol; ISL1+/HB9+ purity >=85%). 2. Treat at day 35 with GSK2606414 (200 nM) +/- Sephin1 (4 uM) for 72 h. 3. Endpoints: - Western blot: p-eIF2alpha(S51)/total eIF2alpha, ATF4, CHOP - Puromycin incorporation assay (SUnSET, 5-min pulse) for nascent translation - G3BP1/TDP-43 co-IF for stress granule count and TDP-43 nuclear:cyto ratio - LDH for cell viability; CellTiter-Glo for ATP. 4. In-vivo: G93A-SOD1 mice (70-100 days), GSK2606414 (50 mg/kg PO daily) + Sephin1 (4 mg/kg IP daily) x 30 days; hindlimb grip, rotarod, SMN IHC.

Expected Outcomes

Combined treatment will normalise p-eIF2alpha by >=50%, restore polysome/monosome ratio by >=30%, reduce TDP-43 cytoplasmic aggregates >=60%, and extend rotarod performance >=20%.

Success Criteria

Primary: p-eIF2alpha/eIF2alpha <=1.3x isogenic WT at 72 h (ANOVA p<0.01). Secondary: TDP-43 nuclear:cyto ratio >=2.5; LDH <=120% of WT vehicle.

Related Hypotheses (1)

H6: Aberrant eIF2α Phosphorylation Creates Stalled Translation State0.834

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