Frontiers in neurology: Translational research
Carlos A. Pardo, MD Johns Hopkins University School of Medicine Baltimore, Maryland [email protected]
1 FRONTIERS IN NEUROLOGY: TRANSLATIONAL RESEARCH
Looking for answers: From the bench to clinical prac ce And from clinical prac ce to the bench and back to clinic.
JHU/Neuroimmunopath/CA Pardo 2 Fron ers in neurology: Transla onal research Answering cri cal ques ons about pathogenesis of neurological disease • Are neuroinflammatory changes involved in pathogenesis of au sm? • Why au sm? • Why inflamma on? • How to approach the ques ons and design an expreimental approach to answer the ques ons
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 es mate that about 730,000 individuals between the ages of 0 to 21 have an ASD. •Studies in Asia, Europe and North America have iden fied individuals with an ASD with an approximate prevalence of 0.6% to over 1%.
www.cdc.gov/ncbddd/au sm/data.html#prevalence
•Among iden cal twins, if one child has an ASD, then the other will be affected about 60-96% of the me. In non-iden cal 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 es mated that about 10% of children with an ASD have an iden fiable gene c, 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 au s c traits. 1-4% of people with ASD also have tuberous sclerosis.
www.cdc.gov/ncbddd/au sm/data.html#prevalence Johns Hopkins Phipps Psychiatric Clinic Leo Kanner, Autistic disturbances of Affective contact, Nervous Child 1943, 2:217-250 E ologic Model of ASD Including Gene- Environment Interac on
Gene cally Suscep ble Host
Environmental Trigger? Abnormal Immune Response Autism Spectrum Disorder Gene c Complexity in ASD
People with au sm harbor more copy number variants (CNVs) — dele ons or duplica ons 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 Communica on Deficits Repe ve 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. Neuroanatomy of Autism. Trends in Neurosciences 31:137, 2008 9 Func on, Behavior & Cogni on
CNS Ac va on
Immune Neurological synapse System
Stereotyped Behaviors Regula on
10 Neuroimaging & Neuropathology
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 neurons, 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 neuroinflammation in the brain of ASD patients ? 13 Ques on 1: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Adap ve Immunity Innate Immunity Lymphocyte and Microglia & astroglia An body produc on ac va on
? ? ?
14 IMMUNE & ENVIRONMENTAL FACTORS IN PATHOGENESIS OF ASD
Neurotoxins Infec ons Maternal Immunity Neurotoxins Maternal Infec on Host immunity Stress
CRITICAL PATHOGENIC PERIOD ADAPTATION PERIOD
Intra-uterine Brain Development Postnatal brain Brain Brain Gene c Influences development matura on adapta on 1st 2nd 3rd First year Childhood Adulthood trimester trimester trimester
Developmental Immune Factors Neuroimmune Factors • Radial Glia & Microglia Modeling Function • Neuronal-glial interactions • Developmental Cytokine/Chemokine Pathways • Cytokine networks • Developmental TLRs and MMPs function • Cytokine/neuroglial & • Developmental complement function neurotransmitter interaction Adaptive Developmental Synaptic Plasticity Synaptic Plasticity
NEUROBIOLOGICAL TRAJECTORIES NEUROBEHAVIORAL Neuronal migra on, cor cal & neuronal TRAJECTORIES network organiza on Language & Communica on Sociability Behavior
The Neuroimmune System
Central Nervous System Innate immunity Neuron Astroglia Microglia Microglia & astroglia
BBB Cytokines BBB Chemokines Perivascular macrophage Blood Brain Barrier Adap ve immunity Specific Modulation of immune responses T cell and an body responses Lymphocyte/monocyte trafficking
16 Microglia Astroglia
11 cases of au sm (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 ac va on in au sm
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 Psychiatry 15;68(4):368-76, 2010 Astroglial reac on as an indicator ac va on of innate immunity in au sm
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 - Oligodendrocyte func on Leukocyte trafficking Interneuron migra on
Neuromodula on
Ques on 1: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Adap ve Immunity Innate Immunity Lymphocyte and Microglia & astroglia An body produc on ac va on
? ? ?
22 Au sm: 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
Ques on 2: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD during childhood?
Adap ve Immunity Innate Immunity Lymphocyte and Microglia & astroglia An body produc on ac va on
?
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 Au sm Intramural Program S. Swedo, S. Spence, A. Thurm & others
• Prospec ve study of pa ents with ASD – Non-regressive vs. “regressive” – Clinical, behavioral and cogni ve assessment – Neuroimaging – Blood/CSF studies for evalua on of immune ac va on
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 Ques on: Are immune or neuroimmune mechanisms involved in pathogenesis of ASD? Are we able to treat microglia “ac va on”?
Adaptive Immunity Innate Immunity Lymphocyte and Microglia & astroglia Antibody production activation
? ? ?
38 How to modify microglial ac va on? • 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 central nervous system (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 pa ents with developmental regression
Post-Tx Pre-Tx 6 months
Neurobehavioral testing
Blood/CSF: cytokines, chemokines, neurotrophins, 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 Ins tute Andrew W. Zimmerman Becky Landa
Rebecca NIH-NIMH Landa Peter Emch Fund Sue Swedo for Au sm Research Sarah Spence Bart McLean Fund for Audrey Thurm Neuroimmunology Research 47