Research Trends in Understanding How Maternal Stress and Health Interacts with Prenatal Brain Development Hanna Stevens, M.D., Ph.D

Research trends in understanding how maternal stress and health interacts with prenatal brain development Hanna Stevens, M.D., Ph.D. Department of Psychiatry Iowa Neuroscience Institute Carver College of Medicine University of Iowa Disclosures

• Advisory board – Klingenstein Third Generation Foundation • Research funding (past year): – Roy J Carver Trust – Nellie Ball Trust – Chromadex – Environmental Health Sciences Research Center (NIEHS) – Hypertension Research Center

Important Questions

• What happens during prenatal brain development? • Does maternal prenatal stress and health matter for brain development that occurs in utero? • How can we think about buffering brain development in utero? – What molecular mechanisms are involved? Prenatal Brain Development Andersen (Neurosci and Biobehav Rev; 2003) General Principles of Development Patterning the Nervous System: regions defined by unique combinations of factors Patterning and Regional Differences • Different regions become defined by these early patterning signals to be the place where complex sensory, motor, emotional, executive and integrated processing occurs. Development of the Nervous System Sanes, Reh and Harris, Copyright 2006. Expanding Cell number

Vaccarino et al. J. Autism Dev. Dis., Vol 39, 2009 Human Brain Expansion

Dorus et al. Cell, Vol 119, 2004 Processes for Cortex Formation • Neuronal Migration: – newer cells migrate along processes of older, parent cell

Marin et al (CSH Perspectives; 2010) Childhood psychiatric disorders and GABAergic neurons of the Caudate nucleus • The caudate is >95% inhibitory GABAergic neurons of multiple types – Decreased caudate GABAergic interneurons in Tourette Syndrome patients – Overgrowth of GABAergic progenitors in macrocephalic Autism

García-Montes et al 2012; Health García-Montes et al 2012; Health

Andersen (Neurosci and Biobehav Rev; 2003) Maternal Health and Stress Factors • Anxiety and mood disorders are common and can disrupt adaptive physiology & health behaviors • The demands of pregnancy are high and can be difficult to meet • Psychological and biological problems that arise or continue in pregnancy are highly interactive • Medication treatment during pregnancy should be personalized, but access to care impedes this process

Christian 2012 Modeling Maternal Health and Stress Factors

Bale 2015 Prenatal Stress and Later Brain Function

Prenatal stress and stress-related states are associated with: •Neonatal outcomes: –Preterm birth and low birth weight •Childhood outcomes: –Cognitive developmental delay –Increased behavioral & stress reactivity –Altered cortical development •Childhood diagnosis: –Autism Spectrum Disorder risk –Tourette Syndrome symptomatology Bergman et al., 2007 –Mood, anxiety, and ADHD symptoms

Buss et al., 2009 Model System of Prenatal Stress

Pregnant Dam

Embyonic Day 0 (E0)

E12 Electrophysiological Studies showing reduced hippocampal LTP Postnatal Day 0

Repetitive Restraint Stress Decreased dopamine transporter 45 min of physical restraint in ventral telencephalon Three times daily Days E12-E19 Serotonin receptor and ligand changes in prefrontal cortex

Elevated Plus: increased anxiety HPA axis dysregulation

Forced Swim: increased immobility

REM sleep alterations Increased sensitivity to drugs of abuse Changes in Caudate GABAergic

Neuron populations StephanieParker AbbottLussier Neurobiological differences • Increased production and populations of GABAergic neurons in caudate: Only in Males

Caudate GABAergic neurons

Lussier et al in preparation Childhood Psychiatric Disorders and the Caudate Nucleus • Autism, ADHD, and Tourette syndrome affect the same brain functions: – Repetitive behavior and procedural learning – Disruptions of motor regulation (hyperactivity, impulsivity) • These brain functions depend on the caudate nucleus, a subcortical region in the forebrain that contributes to cognitive and motor behaviors Anatomography • MRI changes in caudate – Activation during motor tasks, habits, and procedural learning – Volume changes in patients with schizophrenia, autism, Tourette syndrome, and ADHD

Persistence in Caudate GABAergic

Neuron populations Stephanie Lussier Adult GABAergic differences * * • Increased Caudate

GABAergic numbers: Only NS PS NS PS NS PS NS PS in Males P24 P150 P24 P150

Rotarod: Motor learning Behavioral differences • Learning impairments and motor hypoactivity: Only in Males

Lussier et al in preparation Moderators of Prenatal Stress: Sex Differences Interaction of Sex with Prenatal Stress in effects on Gene Expression in GABAergic Progenitors at Embryonic Day 13 0.0014 0.0012 Ankrd17 0.001 0.0008 0.0006 * Upregulated in Males 0.0004 0.0002 • Cellular Proliferation/Stem Cell Genes 0 Sox2; Ankrd17; PCNA; Smc3; Cdc6; Cdk2; 25 Pcna 20 p=0.1 Cenph; Kif18a; Kif4; Mtbp; Nusap1; Rad21 15 10 • Mitochondrial Genes 5 Aifm1; Atp5a1; Cox7b; Hadhb; Idh2; Ndufa5; 0 0.6 Ndufb4; Ndufb5; Ndufs4; Pmpcb; Sdh 0.5 Sox2 0.4 Upregulated in Females 0.3 * 0.2 • Neural Differentiation Genes 0.1 0 Adora1; Alk; Diap1; Epb4.1l1; Hcn3; Hnmt; NS NS PS PS Kcnc4; Klhl14; Map3k12; Mapt; Nrxn1; Rac3; F M F M Rgs7; Sort1; Stx1a; Stxbp1 Jake Michaelson Lab Collaboration Fcon|Fps|Mcon|Mps Male susceptibility to Stress Factors in vitro Neurosphere preparation from embryonic mouse brain • Neuronal proliferation of male and female neural stem cells • The inflammatory stress mediator, interleukin-6 (IL6), increases neurosphere growth only in males, not females

Interleukin-6 blockade during stress blocks proliferation increase

5 4 3 2 1 Relative 48 Hr 48 Hr Relative 0 Neurosphere growth growth Neurosphere Control IL6 Control IL6

Edenia Menezes; Jessica Armer, Stevens Lab 2018 Moderators of Prenatal Stress: Sex Differences • Stress differential impacts on E13 Placenta IGF-1 Expression males and females may 3.5 * 3 diverge in the placenta and its 2.5 2 1.5 support for growth 1 0.5 0 NS PS NS PS Males Females

E13 Placenta Endogenous Antioxidant Glutathione Peroxidase

0.25 * 0.2 0.15 0.1 0.05 0 NS PS NS PS Females Males Dimasuay et al 2016 Prenatal Stress and Mechanisms

Mechanisms & Developmental Stage = potential targets for treatment and prevention

GeneDisRegulationGene What mechanisms at the molecular DisRegulationGeneDisRegulat ionGeneDisRegulationGeneDi and cellular level may be responsible EmbryonicsRegulationGeneDisRegulatio Brain Development for: nGeneDisRegulationGeneDis RegulationGeneDisRegulatio nGeneDisRegulationGene Changes in the brain during Prenatal Stress? GeneDisRegulationGene AbnormalDisRegulationGeneDisRegulat Childhood, AdolescentionGeneDisRegulationGeneDi and Adult What could define maternal stress sRegulationGeneDisRegulatio nGeneDisRegulationGeneDisBrain that is important for these effects? StructureRegulationGeneDisRegulatio and Function nGeneDisRegulationGene Consequences for mature brain?

Cortical Inhibitory Neuron Development

• GABAergic interneurons are born in the ganglionic eminence (at embryonic day 10 to 11 in mice- at about 4 weeks gestation in humans) • Cortical interneurons migrate tangentially into the cortical plate, then migrate/integrate radially into the cortical layers

Stevens et al 2013; PNEC Embryonic Day 14: GABA Cell Tangential Migration

Altered distribution of inhibitory cells at Embryonic Day 14

Rostral Caudal

Non- stressed

Prenatally Non stressed Stressed GAD67GFP Prenatally Stressed Stevens et al 2013; PNEC Postnatal Trajectories of GABA Populations and Inhibited Behavior

Lussier and Stevens 2016 Maternal Stress Factors and the Embryonic Brain

Pregnant Dam

Embyonic Day 0 (E0)

E12

E14 Collection for Migration Assay Repetitive • Corticosterone • IL1beta • IL6

injection compared to oil or PBS injection Three times daily Days E12-E13

Results: Migration Delay with cytokines but not glucocoticoids

Jada Bittle and Banu Gumusoglu * p=.004 1 * p=.030 No delay 0.9 * p<.001 ????? 0.8

0.7

0.6

0.5

distance 0.4

0.3

0.2

0.1 GPM as a % of total cortical plate plate cortical as a %total GPM of

0 NS PS NS/Saline NS/IL-6 NS/anti IL-6 PS/anti IL-6 NS/IL-1beta NS/Cort

 IL-6 but no other mediator alone can reproduce the effect of prenatal stress on inhibitory neuron migration

 Blocking IL-6 during prenatal stress does not rescue migration delay in embryonic brain Mechanisms of Prenatal Stress

Oxidative stress and the mitochondrial function that regulates it may be critical for effects on migrating cells

Ant1 mutants have disrupted mitochondrial ADP/ATP flux and abnormal build up of pro-oxidants. Inhibitory neuron migration is also disrupted

Barry et al 2006

Lin-Hendel et al 2016 Oxidative Stress and Neuronal migration • Collaboration with Mike Dailey’s lab to do live imaging of neuronal migration in mouse embryonic cortical slices to directly test mediators

Jada Bittle Oxidative state: Disrupted migration

Velocity Angle Deviation 80 100

70 80 60 50 60

per hour 40

30 n = 4 40 n = 4

μm n = 4 20 20 10 n = 4

0 deviation in degrees Average 0 Control H2O2 Control H2O2 Oxidative Stress and Neuronal Migration

• There was a significant effect of the antioxidant, N- acetyl cysteine (NAC), on Inhibitory Neuron migration Jada Bittle

Inhibitory Neuron Migration at E13 100 * 80

Cortex 40

Percent Migration into into Migration Percent 0 NS PS NS NAC PS NAC Prenatal toxic exposure and GABAergic migration

• Insecticides are commonly used to reduce the risk of mosquito born diseases and agricultural pests – This is becoming more common during pregnancy to protect against the neurodevelopmental risks of mosquito born diseases – The CDC specifically recommends two insecticides for pregnant women: DEET and permethrin – Evidence is lacking on neurodevelopmental impacts of permethrin/cypermethrin Prenatal toxic exposure and GABAergic migration

% Migration Prenatal Stress and Mechanisms

Mechanisms & Developmental Stage = potential targets for treatment and prevention

Preventive Measures

1940’s-1960’s Folic Acid and nucleic acid synthesis 1980’s Dietary Crider et al 2011 Folate Supplements and Neural Late 1980’s Tube Defects and 1990’s Folic Acid RCTs ↓ NTDs by Early 1990’s: CDC 70-100% recommends Supplementation for women Mandatory Fortification by 2000

Brumbach et al 2017 Acknowledgments

Pattern Trust

• Nellie Ball Trust • Nat’l Center for Advancing Translational Science (Yale Center for Clinical Investigation) • APIRE/Wyeth Pharmaceuticals • NARSAD YI Award: Dr. Mortimer D. Sackler Developmental Psychobiology Research Program

Glial Development

• Begins embryonically following the production of neurons • Different types (astrocytes or oligodendrocytes) develop from different regions • Continues throughout life, particularly in response to damage Development of the Nervous System and injury Sanes, Reh and Harris, Copyright 2006. Programmed Cell Death

In worms, all neurons have a name and their eventual function/fate is known

Death of some cells appears necessary for appropriate fate specification of other cells

www.wormatlas.org Neuronal Signaling

Even as neurons and their branches are being produced, they begin sending nonspecific signals

These internally-generated, initial signals play a role in neuronal development