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Grant Proposal –Epigenetics: driver of amyloid pathology?
Julie Dela CruzSandro Da MesquitaXenos MasonJulie SavageJochen De Vry
Background
• Age-related increases in DNA methylation in humans
• AD patients show reduced DNA methylation• DNMTs/HDACs may be involved in cognitive
decline, changes in learning & memory• Parallels methylation patterns in AD mouse
models
Research questions
• Are epigenetic changes responsible for altered Aβ deposition?
Epigenetic changes
• Environmental influences: negative (chronic mild stress) AND positive (enriched environment) …
• … induce epigenetic changes• Do these epigenetic changes accelerate or
delay Amyloid deposition and memory performance?
• Wild-type and AD model
Aim 1 - Chronic Mild Stress (CMS)
• 3 weeks CMS (Cuadrado-Tejedor et al., J Alzheimer Dis. 2012) in young (3m) and old (12m), wild-type and APP/PS1 mice
• Examine epigenetic markers in different brain regions (hipp, EC, PFC, cerebellum) select brain region with biggest differences in epigenetic state
• Selected brain region: ChiP focus on genes involved in epigenetic regulation, validate with qPCR
• Examine identified gene(s) (up- or down-regulation) by overexpression or RNAi - functional assay check alterations in brain pathology and cognitive performance
Aim 1 - Enriched Environment (EE)
• 3 weeks EE (Fischer et al., Nature 2007) Increased Histone-tail acetylation
• Similar approach to CMS
Aim 2 – Epigenetic manipulation
• Focal up- or down-regulation of HDAC2 in selected brain region (Aim 1) mimic EE
• Electroporation HDAC2-siRNA-plasmid, HDAC2-plasmid, or scrambled plasmid
• After 3-4 weeks: memory performance, IHC (check pathology)
• Young (3m) and old (12m) – wild-type and APP/PS1
Aim 3 – Comparative human epigenetics
• Familial cases AD, sporadic AD, age-matched ctrls brain bank and/or collaboration
• IHC: HDACs, DNMTs, HATs• Specific brain region: FISH or RT-PCR for
candidate genes Aim 1
Timetable
8 mon
ths
12 m
onths
16 m
onth
s
20 m
onth
s
24 m
onths
28 m
onth
s
32 m
onth
s
Enriched Environment (Aim 1)
HDAC2 up/down regulation (Aim 2)
Start End
4 mon
ths
Chronic mild stress(Aim 1)
Human study (Aim 3)
Budget
• Aim 1: $65,000– Animals: EE and stress 2 x 2 x 2 x 12= 96 animals $15,000– CHiP, reagents,…: $50,000
• Aim 2: $38,000– Animals: $15,000– Consumables: $10,000– Stimulator: $13,000
• Aim 3: $50,000– Consumables: $50,000
• 2 PhD students = 6 years * $30,000 = $180,000
• Total: $333,000
Output
• 4 papers!!!
• Paper: EE• Paper: CMS• Paper: HDAC2 up/down regulation• Paper: human study