Yijun Pan BPharm Hons Ph D candidate The

Yijun Pan, BPharm (Hons) Ph. D candidate The involvement of fatty acidbinding protein 5 in the bloodbrain barrier transport of docosahexaenoic acid www. pharm. monash. edu. au/mips

Docosahexaenoic acid (DHA) • Essential for brain function

Docosahexaenoic acid (DHA) • Essential for brain function • The brain has a limited ability to synthesise DHA de novo • Plasma derived DHA needs to traverse the blood-brain barrier (BBB)

Docosahexaenoic acid (DHA) • Essential for brain function • The brain has a limited ability to synthesise DHA de novo • Plasma derived DHA needs to traverse the blood-brain barrier (BBB) • Diet is the main source of DHA • Regular fish consumption is beneficial to cognitive function – DHA is one of the key components (epidemiological studies)

Docosahexaenoic acid (DHA) • Essential for brain function • The brain has a limited ability to synthesise DHA de novo • Plasma derived DHA needs to traverse the blood-brain barrier (BBB) • Diet is the main source of DHA • Regular fish consumption is beneficial to cognitive function – DHA is one of the key components (epidemiological studies) • Brain DHA level decreases in many neurodegenerative diseases, e. g. Alzheimer’s disease (AD)

Alzheimer’s disease (AD) Beta amyloid accumulation CNS inflammation Alzheimer’s disease Hyperphosphorylated tau protein Neuronal cell death

DHA relieves AD pathology in mouse models of AD Beta amyloid accumulation CNS inflammation DHA Alzheimer’s disease Hyperphosphorylated tau protein Neuronal cell death

DHA relieves AD pathology in mouse models of AD Beta amyloid accumulation CNS inflammation DHA Alzheimer’s disease Hyperphosphorylated tau protein DHA Neuronal cell death

DHA relieves AD pathology in mouse models of AD Beta amyloid accumulation CNS inflammation DHA Alzheimer’s disease Hyperphosphorylated tau protein DHA Neuronal cell death

Why are DHA levels lower in AD brains? 8 months old **p < 0. 01 Mean ± SEM, n = 8

DHA transport across the blood-brain barrier (BBB) BLOOD brain microvascular endothelial cell BRAIN PARENCHYMA

DHA transport across the BBB BLOOD Paracellular Transcellular Tight junctions BRAIN PARENCHYMA

DHA transport across the BBB DHA Paracellular BLOOD Transcellular Tight junctions Aqueous intracellular environment BRAIN PARENCHYMA

DHA transport across the BBB DHA Paracellular Tight junctions BLOOD Transcellular Carrier protein Aqueous intracellular environment BRAIN PARENCHYMA

DHA transport across the BBB DHA Paracellular Tight junctions BLOOD Transcellular Carrier protein Aqueous intracellular environment BRAIN PARENCHYMA

Fatty acid-binding protein (FABP) • FABP has 9 isoforms • FABP 2 assists lipophilic drug transport in the small intestine (Velkov 2004) – Binds to lipophilic drugs – Assists lipophilic drug transport across the aqueous intracellular environment in the enterocyte

Expression and function of FABP at BBB • Is FABP expressed at the BBB? • Does FABP bind to DHA? • Is FABP involved in BBB transport of DHA? • Is FABP expression altered in AD?

Expression and function of FABP at BBB • Is FABP expressed at the BBB? • Does FABP bind to DHA? • Is FABP involved in BBB transport of DHA? • Is FABP expression altered in AD?

FABP 5 is expressed at the human BBB • FABP 3, 4, and 5 m. RNA detected in immortalized human brain microvascular endothelial cells (h. CMEC/D 3 cells)

FABP 5 is expressed at the human BBB • FABP 3, 4, and 5 m. RNA detected in immortalized human brain microvascular endothelial cells (h. CMEC/D 3 cells) • FABP 5 protein is expressed (Western blot) β-actin FABP 5

FABP 5 is expressed at the mouse BBB • Brain microvessels were isolated fresh from mice FABP 5

Expression and function of FABP 5 at BBB • Is FABP 5 expressed at the BBB? • Does FABP 5 bind to DHA? • Is FABP 5 involved in BBB transport of DHA? • Is FABP 5 expression altered in AD?

Synthesis and purification of h. FABP 5 Ladder (k. Da) Pure h. FABP 5

h. FABP 5 binds to DHA with n. M affinity Isothermal titration calorimetry Negative control (no DHA) Kd = 155 ± 8 n. M (mean ± SEM, n=15)

Expression and function of FABP 5 at BBB • Is FABP 5 expressed at the BBB? • Does FABP 5 bind to DHA? • Is FABP 5 involved in BBB transport of DHA? • Is FABP 5 expression altered in AD?

FABP 5 is down-regulated with si. RNA transfection • In vitro: 14 C-DHA cellular uptake in h. CMEC/D 3 cells with FABP 5 m. RNA silencing m. RNA ****p < 0. 0001 Mean ± SEM, n = 4

FABP 5 is down-regulated with si. RNA transfection • In vitro: 14 C-DHA cellular uptake in h. CMEC/D 3 cells with FABP 5 m. RNA silencing m. RNA ****p < 0. 0001 Mean ± SEM, n = 4 protein **p < 0. 01 Mean ± SEM, n = 3

FABP 5 si. RNA decreases 14 C-DHA uptake in h. CMEC/D 3 • In vitro - 14 C-DHA cellular uptake in h. CMEC/D 3 cells with FABP 5 m. RNA silencing ****p < 0. 0001 Mean ± SEM, n = 12

14 C-DHA brain transport is reduced in FABP 5 -/- mice • In vivo - BBB transport of 14 C-DHA in FABP 5 -/- mice (**p<0. 01) Mean ± SEM, n = 5 -6

Expression and function of FABP 5 at BBB • Is FABP 5 expressed at the BBB? • Does FABP 5 bind to DHA? • Is FABP 5 involved in BBB transport of DHA? • Is FABP 5 expression altered in AD?

FABP 5 protein expression reduces in APP/PS 1 mice Mean ± SEM, N = 4 ** p<0. 001

Summary • FABP 5 is expressed at both the human and mouse BBB • FABP 5 binds DHA in the n. M range • FABP 5 involved in BBB DHA transport in vitro and in vivo • FABP 5 expression at the BBB is reduced in AD, which is associated with reduced BBB transport of DHA

Acknowledgements • Monash University – Dr. Joseph Nicolazzo – Prof. Chris Porter – A/Prof. Martin Scanlon • Yamaguchi University – Prof. Yuji Owada • Financial support – Mason Foundation – Victorian government – Faculty of Pharmacy and Pharmaceutical Sciences, Monash University