Lysophosphatidic Acid (LPA) Signaling in Post-Hemorrhagic Hydrocephalus

Lysophosphatidic acid (LPA) signaling in post-hemorrhagic hydrocephalus

Background Lysophospholipid signaling Lysophosphatidic acid (LPA) Sphingosine 1-phosphate (S1P) Prenatal cerebral cortical development

LPA mechanisms in the prenatal cerebral cortex: Normal effects Under hypoxia

Jerold Chun, M.D., Ph.D. LPA signaling and hydrocephlaus Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute Jerold Chun, MD, Ph.D., [email protected] Google or Yahoo: Chun Lab

Acknowledgements Fetal Hydrocephalus: Fetal Brain Hypoxia: Yun C. Yung Keira J. Herr Tetsuji Mutoh Deron Herr Mu-en Lin Chang-Wook Lee Kyoko Noguchi Kyoko Noguchi

Rich Rivera

Ji Woong Choi HA Fellow! Marcy Kingsbury Guillermo Estivill Torrus Background: Fernando Rodriguez de Fonseca Rocky Contos Nobuyuki Fukushima Peppi Prasit (Amira Pharmaceuticals) Adrienne Dubin Chang-Wook Lee Supported by the NIH (NIMH, NINDS, NIDA, Jonathan Hecht Tetsuji Mutoh NICHD, NIDCD) Isao Ishii Joshua Weiner Fellowship support: NSF, NIH, ASTAR, Hydrocephalus Jerold Chun, MD, Ph.D., [email protected] Association, Novartis, Pfizer, Amira

Over 50% of the brain’s dry weight is lipid

Phospholipids are t he most abundant brain lipid and were first discovered in the brain

Jerold Chun, MD, Ph.D., [email protected] Phospholipids make-up the lipid membrane bilayer

Jerold Chun, MD, Ph.D., [email protected] Lysophospholipids Lysophosphatidic Acid (LPA) Sphingosine 1-phosphate (S1P)

Jerold Chun, MD, Ph.D., [email protected] Trends Mol. Med. 12, 65 (2006) Possible LP mechanisms

Calcium chelator

Ionophore

Membrane disruptor

Second messenger

Intracellular receptor

Extracellular receptor

Jerold Chun, MD, Ph.D., [email protected] Identification of a first LP receptor, a GPCR for LPA

Ventricular zone cells Cell lines

Degenerat e PCR For GPCRS Followed by In sit u hybridizat ion

Neurit e Present in serum retraction Heat stable Morphogenic Ventricular zone expression Proliferative

LPA1 “ VZG-1” Homologous to CB1

J Cell Bio 135:1071 (1996)

Jerold Chun, MD, Ph.D., [email protected] Lysophospholipid (LPA and S1P) G protein-coupled receptors (GPCRs)

~40% of human medicines t arget Jerold Chun, MD, Ph.D., [email protected] GPCRs Lysophospholipid (LP) Receptors

Receptor (gene*) Agonist G proteins Gene structure G , G , G LPA1 (LPAR1) LPA i/o 12/13 q LPA2 (LPAR2) LPA Gi/o, G12/13, Gq

LPA3 (LPAR2) LPA Gi/o, Gq

S1P1 (S1PR1) S1P>SPC Gi/o

S1P3 (S1PR3) S1P>SPC Gi/o, G12/13, Gq, Gs

S1P2 (S1PR2) S1P>SPC Gi/o, G12/13, Gq, Gs

S1P5 (S1PR5) S1P>SPC Gi/o, G12/13

S1P4 (S1PR4) S1P>SPC Gi/o, G12/13, Gs

LPA4 (LPAR4) LPA G12/13, Gq /i?, Gs LPA5 (LPAR5) LPA G12/13, Gq, Gs? LPA G LPA6 (LPAR6) s? Ann. Rev. Biochem. 73, 321 (2004) 30% 40% 50% 60% JBC 279, 20555 (2004) Approximate amino acid identity Ann. Rev. Pharm. Tox. 50,157 (2010) * HUGO & MGI Jerold Chun, MD, Ph.D., [email protected] Pharmacol. Rev. 62, 579 (2010) gene names

Lysophospholipid (S1P) receptors as bona fide drug targets

Fingolimod targets S1P receptors and is efficacious in MS

FDA approved (9/2010) as a first oral MS treatment (Gilenya, Novartis)

Acts on both immune and central nervous systems (astrocytes)

Jerold Chun, MD, Ph.D., [email protected] Choi et al., Ann. Rev. Pharm. Toxicol. 2010 Jerold Chun, MD, Ph.D., [email protected] Mutoh et al., Brit. J. Pharmacol. (2012)

Lipid (LPA) signaling in prenatal brain development and related disorders

Background Lysophospholipid signaling Lysophosphatidic acid (LPA) Sphingosine 1-phosphate (S1P) Prenatal cerebral cortical development

LPA mechanisms in the prenatal cerebral cortex: Normal effects Under hypoxia

Prenatal cerebral cortical development

Jerold Chun, MD, Ph.D., [email protected] Cortical Plate (CP)

Intermediate Zone (IZ)

Ventricular Zone (VZ) (Neural Progenitor Cells (NPCs) Ventricle (V) (Cerebral Spinal Fluid (CSF))

Embryonic neural tube

Jerold Chun, MD, Ph.D., [email protected] Postmitotic neurons

Neural Progenit or Cells (NPCs)

NPCs in t he VZ

Jerold Chun, MD, Ph.D., [email protected] Mitotic cells (phosphoH3 positive)

Lipid (LPA) signaling in prenatal brain development and related disorders

Background Lysophospholipid signaling Lysophosphatidic acid (LPA) Sphingosine 1-phosphate (S1P) Prenatal cerebral cortical development

LPA mechanisms in the prenatal cerebral cortex: Normal effects Under hypoxia

LPA1 gene expression in the ventricular zone of the embryonic cerebral cortex

Choroid Plexus

Jerold Chun, MD, Ph.D., [email protected] LPA receptor subtypes modulate calcium signaling in neural progenitor cells 90 -1 -2 -1,2,3 Nestin+ 80 70 21 0.25 60 50 40 6 30 4 20 10

Percent responsive Percent 3 0 WT A1 -/- A2 -/- A1; A2; A3 -/-; -/-; -/- Ratio (340/380) Ratio Nestin TuJ1

DAPI

0 100 200 300 400 500 600 700 800 900 1000 1100 50 mm J. Neurosci. 30, 7300-7309 (2010) Jerold Chun, MD, Ph.D.,Time [email protected] (sec) LPA signaling in the fetal cerebral cortex • Morphological changes, NPCs, young neurons (process retraction) • Cell migration effects • Increased chloride and non-selective cation conductances (whole- cell patch clamp) • Increased calcium signaling • Modest proliferative effects • Anti-apoptotic effects • Cytoskeletal remodeling, adhesive changes • Loss-of-function (KO): mild, strain-dependent effects on cerebral cortical organization from single receptor deletion • Gain-of-function (increased receptor activity by LPA exposure): a model for developmental CNS diseases?

Jerold Chun, MD, Ph.D., [email protected]

Gain-of-function cerebral explants to assess global, “in situ” effects, wildtype vs. nulls

Jerold Chun, MD, Ph.D., [email protected] LPA produces cortical folding and increases cortical thickness

Nat. Neurosci. 6, 1292 (2003) Trends in Neuroscience 27, 362 (2004)

Jerold Chun, MD, Ph.D., [email protected] LPA produces cortical folding and increases cortical thickness

Nat. Neurosci. 6, 1292 (2003) Trends in Neuroscience 27, 362 (2004)

Jerold Chun, MD, Ph.D., [email protected] LPA increases mitotic progenitor/stem cells: accelerated or premature cell cycle exit

Jerold Chun, MD, Ph.D., [email protected] LPA increases the number of postmitotic neurons and alters their positions

Jerold Chun, MD, Ph.D., [email protected] (-/-) (-/-) No effect in lpa1 lpa2 : LPA receptors are required

Jerold Chun, MD, Ph.D., [email protected] Lipid (LPA) signaling in prenatal brain development and related disorders

Background Lysophospholipid signaling Lysophosphatidic acid (LPA) Sphingosine 1-phosphate (S1P) Prenatal cerebral cortical development

LPA mechanisms in the prenatal cerebral cortex: Normal effects Under hypoxia In Fetal Hydrocephlaus Jerold Chun, M.D., Ph.D. Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute Jerold Chun, MD, Ph.D., [email protected] Google or Yahoo: Chun Lab

Hypoxia induces displacement of mitotic

NPCs in the embryonic brain via LPA1 Genetic approach: LPA1 null mice a a b e

c d

Jerold Chun, MD, Ph.D., [email protected] Model Normoxia Hypoxia

108 Jerold Chun, MD, Ph.D., [email protected] PNAS , 15444 (2011)

Lipid (LPA) signaling in prenatal brain development and related disorders

Background Lysophospholipid signaling Lysophosphatidic acid (LPA) Sphingosine 1-phosphate (S1P) Prenatal cerebral cortical development

LPA mechanisms in the prenatal cerebral cortex: Normal effects Under hypoxia

Jerold Chun, M.D., Ph.D. LPA and Fetal Hydrocephlaus Department of Molecular Biology Dorris Neuroscience Center The Scripps Research Institute Jerold Chun, MD, Ph.D., [email protected] Google or Yahoo: Chun Lab

Major epidemiological signal: Fetal bleeding results in the increased incidence of FH

LPA is found in many normal and pathological body fluids, and can be locally produced.

A prominent source of LPA and/or its precursors is blood, particularly plasma and serum.

In vivo levels of LPA produced during hemorrhage

can reach 15,000X the apparent KD of LPA1.

Jerold Chun, MD, Ph.D., [email protected] LPA1 gene expression in the ventricular zone of the embryonic cerebral cortex

Choroid Plexus

Intraventricular bleeding often precedes

hydrocephalus formation, and LPA can reach high concentrations during hemorrhage Jerold Chun, MD, Ph.D., [email protected] LPA signaling may init iat e fet al hydrocephalus

Yung et al. Sci Transl Med (2011) 3:99ra87

Jerold Chun, MD, Ph.D., [email protected] In vivo injection of serum, plasma or LPA

produces hydrocephalus phenotype

Jerold Chun, MD, Ph.D., [email protected] Science Transl Med (2011) 3:99ra87 Jerold Chun, MD, Ph.D., [email protected] In vivo injection of LPA disrupts cell adhesion and alters cell fates of NPCs resulting in loss of ciliated ependymal cells

Jerold Chun, MD, Ph.D., [email protected] Prevention of FH and comorbid

sequelae in LPA1-null animals

Jerold Chun, MD, Ph.D., [email protected] Pharmacological prevention of LPA-induced FH

Jerold Chun, MD, Ph.D., [email protected] LPA signaling Alterations via Hypoxia & Bleeding

Ot her CNS disorders linked To bleeding and/ or hypoxia: LPA-signaling "hypomorphs?"

Jerold Chun, MD, Ph.D., [email protected] Summary • LPA receptor signaling functions in normal cerebral cortical development, and its dysregulation profoundly alters the brain.

• Alterations in LPA receptor signaling – as occurs with hemorrhage, hypoxia or infection may contribute to one or more forms of hydrocephalus and may be THERAPEUTICALLY TRACTABLE

– Hypoxic insults (LPA1) – Hydrocephalus (LPA1 & ? LPA Receptors?) – LPC and other lipids? – Other CNS disorders ? • Autism: bleeding and hypoxic insult, increased cortical cell number and brain mass • Schizophrenia: bleeding and hypoxic insult, ependymal disruption

Jerold Chun, MD, Ph.D., [email protected]