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