Frontiers in : Translational research

Carlos A. Pardo, MD Johns Hopkins University School of Baltimore, Maryland [email protected]

1 FRONTIERS IN NEUROLOGY: TRANSLATIONAL RESEARCH

Looking for answers: From the bench to clinical pracce And from clinical pracce to the bench and back to clinic.

JHU/Neuroimmunopath/CA Pardo 2 Froners in neurology: Translaonal research Answering crical quesons about pathogenesis of neurological disease • Are neuroinflammatory changes involved in pathogenesis of ausm? • Why ausm? • Why inflammaon? • How to approach the quesons and design an expreimental approach to answer the quesons

JHU/Neuroimmunopath/CA Pardo 3 •Between 1 in 80 and 1 in 240 with an average of 1 in 110 children in the USA have an ASD. •ASDs occur in all racial, ethnic, and socioeconomic groups, yet are on average 4 to 5 mes more likely to occur in boys than in girls. •If 4 million children are born in the United States every year, approximately 36,500 children will eventually be diagnosed with an ASD. •Assuming the prevalence rate has been constant over the past two decades, we can esmate that about 730,000 individuals between the ages of 0 to 21 have an ASD. •Studies in Asia, Europe and North America have idenfied individuals with an ASD with an approximate prevalence of 0.6% to over 1%.

www.cdc.gov/ncbddd/ausm/data.html#prevalence

•Among idencal twins, if one child has an ASD, then the other will be affected about 60-96% of the me. In non-idencal twins, if one child has an ASD, then the other is affected about 0-24% of the me.

•Parents who have a child with an ASD have a 2%–8% chance of having a second child who is also affected.

It is esmated that about 10% of children with an ASD have an idenfiable genec, neurologic or metabolic disorder, such as fragile X or Down syndrome.

•5% of people with an ASD are affected by fragile X and 10% to 15% of those with fragile X show ausc traits. 1-4% of people with ASD also have tuberous sclerosis.

www.cdc.gov/ncbddd/ausm/data.html#prevalence Johns Hopkins Phipps Psychiatric Clinic Leo Kanner, Autistic disturbances of Affective contact, Nervous Child 1943, 2:217-250 Eologic Model of ASD Including Gene- Environment Interacon

Genecally Suscepble Host

Environmental Trigger? Abnormal Immune Response Autism Spectrum Disorder Genec Complexity in ASD

People with ausm harbor more copy number variants (CNVs) — deleons or duplicaons of large chunks of DNA — compared with controls, but only in the protein-coding regions of the genome

Pinto et. Al. Nature : 466: 368–372, 2010 8 ACC OFC OFC OFC OFC PPC

FG

Cerebellum Amygdala

Social Impairment Communicaon Deficits Repeve Behavior OFCIFG OrbitofrontalInferior frontal cortex (Broca ’s) OFC Orbitofrontal cortex ACCSTS AnteriorSuperior cingulate temporal cortex sulcus ACC Anterior cingulate cortex FGSMA Fusiform Supplementary gyrus motor area BG Basal ganglia STSBG Superior Basal ganglia temporal sulcus Th Thalamus ASN Amygdala Substantia nigra IFGTh Inferior Thalamus frontal gyrus PPCPN Posterior Pontine nuclei parietal cerebellum cortex

Adapted from Amaral D et al. of Autism. Trends in 31:137, 2008 9 Funcon, Behavior & Cognion

CNS Acvaon

Immune Neurological synapse System

Stereotyped Behaviors Regulaon

10 &

Autism is a brain disorder in which there is : • Abnormal cortical organization and brain growth • Disproportionate increase of white matter (e.g. frontal lobe)

Filipek et al. 1991 • Decrease cerebellar volume Piven et al.1995,1996 Courchesne E et al. 2001-2004 (vermis?) Sparks et al. 2002 Aylward et al 2002 Herbert et al 2003-2005 Hardan A, 2006 Others… 11 ASD as a neuropathological problem

• Unorganized cerebral cortex – Increase neuronal “packing” – Reduction in cortical minicolumns • Cerebellar atrophy – Purkinje cell reduction • Cytoarchitectural changes in subcortical structures • Increase in frontal lobe and white matter

(Kemper and Bauman 1994, 1998) (Bailey et al, 1998) (Casanova 2001, 2002) (Courchesne et al 2002; Herbert M. 2004) (Wegiel J et al 2010)

(Wegiel J et al 2010) Molecular Neuropathology in ASD: Clues in pathogenesis

Two observations in the neuropathology of ASD: 1-Excess , white matter and enlarged frontal lobe What is abnormal in the white matter?

Schumann, C. M. et al. J. Neurosci. 2010; 30:4419-4427

2-Neuroinflammatory changes in the brain of ASD patients What are the triggering factors in in the brain of ASD patients ? 13 Queson 1: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Adapve Immunity Innate Immunity Lymphocyte and & astroglia Anbody producon acvaon

? ? ?

14 IMMUNE & ENVIRONMENTAL FACTORS IN PATHOGENESIS OF ASD

Neurotoxins Infecons Maternal Immunity Maternal Infecon Host immunity Stress

CRITICAL PATHOGENIC PERIOD ADAPTATION PERIOD

Intra-uterine Brain Development Postnatal brain Brain Brain Genec Influences development maturaon adaptaon 1st 2nd 3rd First year Childhood Adulthood trimester trimester trimester

Developmental Immune Factors Neuroimmune Factors • Radial & Microglia Modeling Function • Neuronal-glial interactions • Developmental / Pathways • Cytokine networks • Developmental TLRs and MMPs function • Cytokine/neuroglial & • Developmental complement function interaction Adaptive Developmental Synaptic Plasticity Synaptic Plasticity

NEUROBIOLOGICAL TRAJECTORIES NEUROBEHAVIORAL Neuronal migraon, corcal & neuronal TRAJECTORIES network organizaon Language & Communicaon Sociability Behavior

The Neuroimmune System

Central Innate immunity Astroglia Microglia Microglia & astroglia

BBB BBB Perivascular Blood Brain Barrier Adapve immunity Specific Modulation of immune responses and anbody responses Lymphocyte/monocyte trafficking

16 Microglia Astroglia

11 cases of ausm (age 5-44) Assessment of Neuroglial 12 controls reactions: Quantification Brain regions: of microglia and astroglia Frontal (MFG), cingulate (ACG), and activation cerebellum 17 Innate Immunity: Neuroglial acvaon in ausm

Astroglia activation

Microglia activation

Vargas DL et al. Ann Neurol 2005 18 Microglia cell density increases in the cerebral cortex in ASD

19 Morgan JT et al. Biological 15;68(4):368-76, 2010 Astroglial reacon as an indicator acvaon of innate immunity in ausm

GFAP

Expression of the astroglial protein GFAP as an indicator of astroglial activation

20 Vargas DL et al. Ann Neurol 2005 Immune CNS Environment Environment Neurotransmission pathways Pro-inflammatory Neurotoxicity Tissue repair pathways inflammatory An- funcon Leukocyte trafficking Interneuron migraon

Neuromodulaon

Queson 1: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Adapve Immunity Innate Immunity Lymphocyte and Microglia & astroglia Anbody producon acvaon

? ? ?

22 Ausm: Profiles of Cytokine/ Chemokine in the Brain

Vargas DL et al. Ann Neurol23 2005 Brain Cytokine-Chemokine-Growth Factors in ASD

IGF-1 IL-6 IGFBP-1

CCL-2 TGF-β1 (MCP-1) IL-10 MCP-3 MCP-2

MIG Osteoprotegerin

Based on Vargas DL et al. Ann Neurol 2005 24 Brain Cytokine-Chemokine-Growth Factors in ASD CCL2 (MCP-1) TGF-β1 CCL-2 (MCP-1) IGFBP-1 IL-6 TGF-β1 IGFBP-1 IGFBP-1

TGF-β1

Based on Vargas DL et al. Ann Neurol 2005 25

Queson 2: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD during childhood?

Adapve Immunity Innate Immunity Lymphocyte and Microglia & astroglia Anbody producon acvaon

?

28 QUESTIONS?

•Do peripheral immune responses influence the clinical phenotype in ASD?

•Does the profile of peripheral immune responses reflects changes within the CNS?

•Does the profile of peripheral immune markers parallels the changes in the CFS?

29 Ausm Intramural Program S. Swedo, S. Spence, A. Thurm & others

• Prospecve study of paents with ASD – Non-regressive vs. “regressive” – Clinical, behavioral and cognive assessment – Neuroimaging – Blood/CSF studies for evaluaon of immune acvaon

JHU/Neuroimmunopath/CA Pardo 30 HOW TO STUDY NEURAL AND IMMUNE INTERACTIONS IN ASD? BRAIN IMMUNITY

Cellular Immune Responses

Cytokines/chemokines networks

Immune & Neurotrophic growth factors CSF BLOOD

31 EGF

CXCL8 TGFα

CCL2 CX3CL sCD40L Flt3L

NIMH Study of Immunological Factors in Autism: Profile of cytokines and chemokines in serum & CSF

SUBJ37 SUBJ36 SUBJ35 SUBJ39 SUBJ44 SUBJ46 SUBJ47 SUBJ51 SUBJ42 SUBJ41 SUBJ50 SUBJ45 SUBJ49 SUBJ43 SUBJ40 SUBJ38 SUBJ30 SUBJ29 SUBJ27 SUBJ25 SUBJ65 SUBJ57 SUBJ59 SUBJ66 SUBJ63 SUBJ54 SUBJ64 SUBJ56 SUBJ55 SUBJ52 SUBJ24 SUBJ48 SUBJ14 SUBJ5 SUBJ18 SUBJ9 SUBJ16 SUBJ7 SUBJ17 SUBJ3 SUBJ11 SUBJ8 SUBJ15 SUBJ13 SUBJ23 SUBJ33 SUBJ19 SUBJ31 SUBJ22 SUBJ26 SUBJ21 SUBJ28 SUBJ20 SUBJ53 SUBJ32 SUBJ34 SUBJ58 SUBJ60 SUBJ61 SUBJ62 SUBJ67 SUBJ12 SUBJ6 SUBJ1 SUBJ4 SUBJ2 SUBJ10 IL8 FIt3L GRO MDC sCD40L VEGF EGF TGFa GMCSF 5 FGF2 GCSF IFNa2 IL1a MCP3 IL15 TNFa MIP1b MIP1a IL9 0 MCP1 IP10 Fractalkine IL10 IL12p40 IL5 IL1RA IL7 IL12p70 IL4 −5 IL3 sIL2Ra IFNy TNFb Eotaxin IL6 IL13 IL2 IL1b IL17

AgeSexRank2=1

AgeSexRank2=0

AgeRank2=1

AgeRank2=0

Sex=M

Sex=F

Profile of cytokines & chemokines in CSF

What is the significance of monocyte-linked chemokines expression in CSF? • CCL2 (MCP1), CX3CL (Fractalkine) and FLT3L are “monocyte” linked cytokines/chemokines

• Fractalkine is the ligand of CX3CR, a receptor critical for microglial activation.

• Fractalkine is expressed in neurons and its production increased in periods of neuronal “distress”

• Interaction between CX3CL and CX3CR play roles in neuronal-neuroglial interactions critical for CNS homeostasis

JHU/Neuroimmunopath/CA Pardo 37 Queson: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Are we able to treat microglia “acvaon”?

Adaptive Immunity Innate Immunity Lymphocyte and Microglia & astroglia Antibody production activation

? ? ?

38 How to modify microglial acvaon? • Minocycline hydrochloride, is a broad spectrum tetracycline antibiotic.

• Bacteriostatic antibiotic, classified as a long- acting type. long half-life : serum levels 2-4 times that of the simple water-soluble tetracyclines (150 mg giving 16 times the activity levels compared to 250 mg of tetracycline at 24–48 hours).

• Minocycline is the most lipid-soluble of the tetracycline-class antibiotics, greatest penetration into the prostate and brain, but also the greatest amount of (CNS)-related side effects. JHH/Neuroimmunopath/ JHU/Neuroimmunopath/CA Pardo Pardo39 Minocycline in neurological disorders

Neuroprotective and anti-human immunodeficiency virus activity of minocycline . Zink MC, et al. JAMA. 2005 Apr 27;293(16):2003-11.

Minocycline attenuates HIV infection and reactivation by suppressing cellular activation in human CD4+ T cells. Szeto GL, et al J Infect Dis. 2010 Apr 15;201(8):1132-40

Minocycline slows disease progression in a mouse model of amyotrophic lateral sclerosis. Kriz J, Nguyen MD, Julien JP. Neurobiol Dis. 2002 Aug;10(3):268-78.

Placebo- controlled phase I/II studies of minocycline in amyotrophic lateral sclerosis . Gordon PH, et al. Neurology. 2004 ;62(10):1845-7.

Efficacy of minocycline in patients with amyotrophic lateral sclerosis: a phase JHH/Neuroimmunopath/ III randomised trial. JHU/Neuroimmunopath/CA Pardo Pardo40 Gordon PH, and the Western ALS Study Group. Lancet Neurol. 2007 (12):1045-53..

Minocycline in ASD: Study Design

Minocycline 1.4 mg/kg

ASD paents with developmental regression

Post-Tx Pre-Tx 6 months

Neurobehavioral testing

Blood/CSF: cytokines, chemokines, , microbial translocation

JHU/Neuroimmunopath/CA Pardo JHH/Neuroimmunopath/ JHU/Neuroimmunopath/CA Pardo Pardo42 JHH/Neuroimmunopath/ JHU/Neuroimmunopath/CA Pardo Pardo43 JHH/Neuroimmunopath/ JHU/Neuroimmunopath/CA Pardo Pardo44

Minocycline study in ASD: Conclusions q No significant clinical behavioral effects were seen in this small group of children with autism in response to minocycline treatment. q Frequent GI symptoms were observed as side effect of the use of minocycline and 2 of 11 patients presented urinary problems (UTI and hematuria). q Changes in the pre-/post-treatment profiles of the proform of BDNF in CSF and blood, HGF in CSF and IL8 in serum, suggest that minocycline may have effects in the CNS by modulating the production of neurotropic growth factors. q At present, there is no evidence that minocycline is useful for modifying the behavioral or cognitive status of patients with autism. q Higher doses may be required for achieving biological effects on neuroinflammation or neuroprotection but the increased risk for adverse effects and toxicity may limit the use of this medication in children with autism.

JHU/Neuroimmunopath/CA Pardo 46 Johns Hopkins/ Arun Azhagiri Acknowledgments… Sharifia Wills

Humera Khan Norman Haughey Li-Chin Lee Diana L. Vargas David Wheeler Kennedy Krieger Instute Andrew W. Zimmerman Becky Landa

Rebecca NIH-NIMH Landa Peter Emch Fund Sue Swedo for Ausm Research Sarah Spence Bart McLean Fund for Audrey Thurm Research 47