Multi nutrient enriched diets restore cerebral

perfusion and protect against neurodegeneration in a

mouse model for Alzheimer’s disease

Valerio Zerbi1,2, D. Jansen1, X. Fang1, M. Wiesmann1, M. Mutsaers1, P.J. Dederen1,

I.Arnoldussen1, A. Veltien2, S. Van Asten2, A. Heerschap2 and A.J. Kiliaan1

1 Dept. Anatomy, Donders Centre for Neuroscience, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands 2 Dept. Radiology, Radboud University Nijmegen Medical Centre, Nijmegen, the Netherlands

Alzheimer’s Disease (AD)

Introduction

Clinical phenotype: gradual episodic memory impairment

Neuropathological changes: • Presence of plaque and tangle pathology • Massive loss neuronal cells and synapses • Neurodegeneration / white matter pathology

AD affects more than 24 million people world wide Age is the major risk factor for AD: 47% of people older than 85 years affected 80 million affected in 2040

Genetics & risk factors

1. Sporadic AD (late onset)

Causes unclear

Risk factors

2. Familial AD (early onset)

Mutations in amyloid precursor

protein (APP), presenilin (PS)-1

or -2 genes

contribute to increased

Aβ production

De la Torre JC (2002), Stroke

Risk factors for Alzheimer’s disease

Ageing

Presence of APOEε4 allele

Hypertension

Congestive heart failure

Atrial fibrillation

Atherosclerosis

Smoking

High intake of saturated fat

Diabetes mellitus

Stroke

Sedentary lifestyle

Overweight

White matter lesions

Epidemiology (end of 20th century)

Introduction

Regulation of β-amyloid production / clearance

Microglial cells

Blood flow

β-amyloid γ-secretase

β-secretase

Physiological situation

Energy supply

Introduction

Regulation of β-amyloid production / clearance

Activated Microglial cells

Blood flow

β-amyloid •monomers •dimers •trimers γ-secretase

β-secretase

β-amyloid oligomers

β-amyloid plaques

• Abnormal cleavage of APP by γ- and β-secretase

• Decrease Aβ clearance by reduced cerebral blood flow

•Aβ oligomers and plaques

• Vascular amyloid deposition (CAA)

• Chronic inflammatory response

• Energy crisis

• Neurodegeneration

Energy supply

Introduction

Regulation of β-amyloid production / clearance

Activated Microglial cells

Blood flow

β-amyloid •monomers •dimers •trimers γ-secretase

β-secretase

β-amyloid oligomers

β-amyloid plaques

• Abnormal cleavage of APP by γ- and β-secretase

• Decrease Aβ clearance by reduced cerebral blood flow

•Aβ oligomers and plaques

• Vascular amyloid deposition (CAA)

• Chronic inflammatory response

• Energy crisis

• Neurodegeneration

Energy supply

Introduction

Prevention strategies

• Immunotherapy

• “classic” vascular risk factors (statins, aspirin, NSAIDs)

• Docosahexaenoic acid (DHA)

•*AD2000 collaborative group. Lancet Neurology 2008;7:41-49

•#Freund- levi et al arch neurol 2008

Introduction

Improving neuronal connectivity Improvement of neuronal membrane fluidity Improves vascular status and reduces atherosclerosis Decrease Aβ levels Beneficial effect in patients with mild AD #

Prevention vascular disease No beneficial effect on Alzheimer pathology

Targeting Aβ Still need to prove their efficacy on clinical trials

Prevention strategies

• Immunotherapy

• “classic” vascular risk factors (statins, aspirin, NSAIDs)

• Docosahexaenoic acid (DHA)

•*AD2000 collaborative group. Lancet Neurology 2008;7:41-49

•#Freund- levi et al arch neurol 2008

Introduction

Improving neuronal connectivity Improvement of neuronal membrane fluidity Improves vascular status and reduces atherosclerosis Decrease Aβ levels Beneficial effect in patients with mild AD #

Prevention vascular disease No beneficial effect on Alzheimer pathology

Targeting Aβ Still need to prove their efficacy on clinical trials

Prevention strategies

• Immunotherapy

• “classic” vascular risk factors (statins, aspirin, NSAIDs)

• Docosahexaenoic acid (DHA)

•*AD2000 collaborative group. Lancet Neurology 2008;7:41-49

•#Freund- levi et al arch neurol 2008

Introduction

Improving neuronal connectivity Improvement of neuronal membrane fluidity Improves vascular status and reduces atherosclerosis Decrease Aβ levels Beneficial effect in patients with mild AD #

Prevention vascular disease No beneficial effect on Alzheimer pathology

Targeting Aβ Still need to prove their efficacy on clinical trials

Aim of the study

“to investigate the effects of DHA enriched diet on the

pathophysiology of Alzheimer’s Disease”

Aim of the study

Animal model

• 12 months old male mice

– Transgenic strain resembling familial AD:

APP695swe/PS1∆E9 [APP/PS1] (Dr. D. Borchelt, Baltimore, MD, USA)

– C57BL/6J [wild type] control mice

Material and methods

Diets

Starting from 2 months of age…

(Control) 5% soy oil Standard control diet

(DHA+) DHA EPA UMP

Kamphuis PJ and Scheltens P (2010), Wurtman RJ (2008)

Material and methods

(Fortasyn) DHA EPA UMP Choline Phospholipids B-vitamins Anti-oxidants

Delatour et al., In Vivo Imaging Biomarkers in Mouse Models of Alzheimer’s Disease: Are We Lost in Translation or Breaking Through? J Alzh dis 2010

Material and methods

1. 1H MR Spectroscopy

1H Magnetic Resonance Spectroscopy

Metabolites of interest:

• N-Acetylaspartate (NAA):

neuronal marker,

reflects neuronal dysfunction

• Myo-inositol (mI):

glial marker,

associated with inflammation

Methods – MRS

Results 1H MRS

Results – MRS

2. Cerebral blood flow

(X + CBF) – X = CBF

Slice selective inversion: (X+CBF)

Global inversion: (X)

Global slab

Label slab

90 180

IR

S-G Kim et al, MRM 37:425-435 (1997) KK Kwong et al, MRM 34:878-887 (1995)

FAIR (Flowsensitive alternating inversion recovery)

180

Imaging slice

Methods – Cerebral blood flow

T1-fitting selective / global

T1 selective

T1 global

TI (inversion time)

≈ CBF

Methods – Cerebral blood flow

Results – Cerebral Blood Flow

Results – Cerebral blood flow

3. Diffusion Tensor Imaging

Diffusion MR imaging

• Theoretical result

• Signal attenuation

• Loss of phase coherence

Methods – Diffusion Tensor imaging

Dr 62

Dt

rrrr

DtrtrP

4

)()(exp

)4(

1)0,|,( 00

30

2

0 exp3

xyM M G D

Brownian motion

Anisotropy

• Directionally dependent

• Restriction in diffusion direction – Because of axonal membrane

• Water molecules diffuse approximately 8 µm

in ~40 ms diffusion time.

Methods – Diffusion Tensor imaging

Diffusion tensor imaging

zzzyzx

yzyyyx

xzxyxx

DDD

DDD

DDD

D

http://www.cs.utah.edu/~gk/papers/tvcg00/img144.png http://www.ajnr.org/content/23/5/803/F6.large.jpg

• Needs 6 measurements with non co-linear directions

Methods – Diffusion Tensor imaging

• Axial diffusivity (λ1)

• Radial diffusivity (RD) [(λ2+ λ3)/2]

• Mean diffusivity (MD) • Fractional anisotropy (FA)

2

3

2

2

2

1

2

3

2

2

2

1

2

3

Methods – Diffusion Tensor imaging

Fractional anisotropy (FA, p < 0.05)

Change in APP compared to WT

corpus callosum ventricles optic tract hippocampus

FA ↓↓ ↓ ↓ -

Results – Diffusion Tensor imaging

Radial diffusivity (RD, p < 0.05)

Change in APP compared to WT

corpus callosum ventricles optic tract hippocampus

RD ↓ ↑↑ ↑ ↑

Results – Diffusion Tensor imaging

Summary

MR

Technique Hypothesis

Control

diet

DHA+

diet

Fortasyn

diet

1H MRS metabolic

alterations

FAIR - ASL Cerebral perfusion

DT-MRI

White matter

degeneration and

neuronal loss

Summary

Acknowledgements

Radboud Univ Nijmegen Medical Centre, Nijmegen, the Netherlands (RUNMC) Anatomy Amanda Kiliaan Diane Jansen Carola Janssen Maartje Mutsaers Jos Dederen Ilse Arnoldussen Xiaotian Fang Maximilian Wiesmann Michiel Kleinnijenhuis

Radiology Arend Heerschap Andor Veltien Sjaak Van Asten Alan Wright

EU consortium 7th framework LipidiDiet • Univ of Saarland (USAAR), Germany Hartmann & Fassbender • Univ of Kuopio (KU), Finland Tanila & Soininen • Univ of Szeged (USZEG), Hungary

Penke • Tel Aviv University (UTA), Israel Michaelson •Göteborgs Universitet (GU), Sweden Skoog & Gustafson • Danone Research B.V., the Netherlands Broersen •Karolinska Institutet (KAU), Sweden Wahlund •Institute of Physiology (ASCR), Czech Republic Dolezal •VU University Medical Centre (VUMC), the Netherlands Scheltens •University of Bonn (UKB), Germany Lütjohann

Support: European Community’s Seventh Framework Programme (FP7/2007-2013) Under grant agreement no 202167

Acknowledgements

Questions