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Spotlight on Vanderbilt University

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HSG member Daniel Claassen of Vanderbilt University and host for HSG 2016 showcases great research going on at the Nashville-based institution.

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Spotlight on Vanderbilt University

  1. 1. Spotlight on Vanderbilt Friday, November 4 8:00-9:00am Chair: Daniel Claassen, MD Vanderbilt University
  2. 2. Welcome to Nashville! HSG 2016: DISCOVERING OUR FUTURE
  3. 3. Vanderbilt Presenters HSG 2016: DISCOVERING OUR FUTURE Aaron Bowman, PhD Associate Professor in the Department of Neurology and Pediatrics/Training Program in Environmental Toxicology, Director Jeff Conn, PhD Lee E. Limbird Professor of Pharmacology /Center for Neuroscience Drug Discovery, Director Paul Harris, PhD Professor of Biomedical Informatics/School of Engineering, Professor of Biomedical Engineering/ Office of Research Informatics, Director
  4. 4. Manganese and HD Aaron B. Bowman, PhD Departments of Pediatrics, Neurology and Biochemistry Vanderbilt University (VU) and VU Medical Center Nashville, TN
  5. 5. Huntington’s disease (HD) genetics and environment HSG 2016: DISCOVERING OUR FUTURE • Longer CAG repeats (polyQ tracts) correlate with earlier age-of-onset and faster progression • Undefined environmental factors account for majority of variability in age-of-onset after accounting for CAG repeat length Environmental Factors/Age Apparent normal neuronal function
  6. 6. Genetic x toxicant screen of 8 cytotoxic metals revealed resistance of HD striatal cells to Mn toxicity HSG 2016: DISCOVERING OUR FUTURE 0 25 50 75 100 125 0 200 400 600 Mn(II) µM PercentSurvival * * * Wild-type STHdhQ7/Q7 Mutant STHdhQ111/Q111 Mechanistic studies demonstrated a deficient Mn accumulation phenotype …see Williams et al. JNC 2010 Mutant STHdhQ111/Q111 Wild-type STHdhQ7/Q7 0 200 400 600 800 0 40 100 Manganese chloride exposure (! M) IntracellularMnlevels (fmolper100cells) * * * P<0.01 (n=5) MTT Assay Atomic Absorption Spectroscopy
  7. 7. Striatum showed decreased net Mn uptake in prodromal stage (3 months) YAC128Q mouse model of HD HSG 2016: DISCOVERING OUR FUTURE Total regional Mn by atomic absorption spectrometry Mn-exposed animals 13.9 mg/kg Mn Sub-cutaneous exposure, on days 1, 4, and 7. Collect tissue 24 hours after last exposure FVB-YAC128Q …see Williams et al. JNC 2010
  8. 8. Human-based and unbiased genomic-level data support a link between manganese biology and HD HSG 2016: DISCOVERING OUR FUTURE Andrew Tidbal et al Hum Mol Genet, 2015 Human Striatal Progenitors Statistic FVB +/+ FVB Tg- YAC128Q p < 0.0005 p < 0.001 p < 0.005 q < 0.05 Ensemble alignment 9 17 94 UCSC mm10 alignment 292 3 5 36 54 # of Mn-responsive genes by p-value or q-value 1 week subcutaneous Mn exposure Mouse HD model In collaboration with Dr. Michael Aschner and Nancy Parmalee Mouse Striatal Progenitors
  9. 9. Hypothesis HSG 2016: DISCOVERING OUR FUTURE HD genotype disrupts neuronal Mn transport and homeostatic mechanisms to impair Mn biology and block Mn toxicity ATM is a specific Mn-activated kinase that phosphorylates target proteins such as p53 Chan et al 2000. JBC Canman et al 1998. Science
  10. 10. Mn activates p53 phosphorylation, this response is impaired in HD striatal neuroprogenitors HSG 2016: DISCOVERING OUR FUTURE Human Andrew Tidball et al Hum Mol Genet, 2015
  11. 11. ATM inhibition blocks Mn-dependent phosphorylation of p53 (and other ATM targets) in human HD ISLT1 progenitors HSG 2016: DISCOVERING OUR FUTURE Andrew Tidball et al Hum Mol Genet, 2015 Manganese (Mn2+) n=6 essential metal and neurotoxicant “In Cell” Western Blots KU = KU55933 an ATM kinase inhibitor
  12. 12. ATM auto-phosphorylation responds to Mn exposure, and this response is deficient in HD cells HSG 2016: DISCOVERING OUR FUTURE Andrew Tidball et al Hum Mol Genet, 2015
  13. 13. Bypassing the Mn deficit in mouse striatal model normalizes ATM-P53 responsiveness HSG 2016: DISCOVERING OUR FUTURE Andrew Tidball et al Hum Mol Genet, 2015 KB-R7943 (KB-R) blocks activity of Sodium-Calcium Exchanger (NCX1); and is known to block Mn efflux in mouse tissues
  14. 14. Cross-talk and co-regulation of the p53/AKT/mTOR pathways and Mn in HD pathobiology HSG 2016: DISCOVERING OUR FUTURE IGF/PI3K ATM/p53 AKT/mTORmutHTT Mn For more see poster by Miles Bryan 0 13 25 50 75 Mn µM (24hr) . p-p53(Ser15) p-AKT(Ser473) p-S6(Ser235/236) Total Protein Wild-type STHdhQ7/Q7
  15. 15. Hypothesis HSG 2016: DISCOVERING OUR FUTURE HD genotype disrupts neuronal Mn transport and homeostatic mechanisms to impair Mn biology and block Mn toxicity Urea and citrulline-nitric oxide cycles are disrupted in HD models and patients (e.g. increased blood citrulline levels); arginase is a rate limiting enzyme Arg1, Arg2 and AGMAT are Mn-dependent urea hydrolases Arg1 is absent in prodromal YAC128Q HD mouse striatum
  16. 16. Arginase pathway related metabolites are altered in HD mouse model; Mn-exposure ameliorates this phenotype HSG 2016: DISCOVERING OUR FUTURE Urea Bichell, Wegrzynowicz and Bowman et al Unpublished Data
  17. 17. Basal arginase activity is reduced in prodromal HD striatum; yet both in vivo Mn-exposure & in vitro Mn-activation normalize activity between wild-type and HD HSG 2016: DISCOVERING OUR FUTURE Bichell, Wegrzynowicz and Bowman et al Unpublished Data WT HD WT HD Vehicle Mn-exposed WT HD WT HD Vehicle Mn-exposed Ex vivo (basal) activity Mn NOT ADDED to the enzyme assay Mn-activated maximal activity Mn ADDED to the enzyme assay 0 10 20 30 40 ngUrea/ugprotein * **** **** 0 20 40 60 80 100 ngUrea/ugprotein ** *** FVB genetic background; n=18 vehicle, n=12 Mn-exposed (7 day subQ exposure) p<0.05 p<0.05 p<0.05
  18. 18. HD and Mn exhibit disease by toxicant/nutrient interaction effects impacting brain urea and citrullin-NO cycles HSG 2016: DISCOVERING OUR FUTURE Bichell, Wegrzynowicz and Bowman et al Unpublished Data Citrul- line urea ADC ODC Orni- thine ARG Putre- cine AGM Manganese Metabolite Holoenzyme Nitric Oxide Antizyme NOS Argi- nine Poly- amine Agma- tine Crea- tine Glutamine GS NO
  19. 19. A continuum of possible HD-Mn pathophysiological links HSG 2016: DISCOVERING OUR FUTURE (1) Disruptions in neuronal Mn balance are a downstream response to HD pathobiology with only a minimal role in disease. (2) Disruption in neuronal Mn balance is one of several pathways impacted by HD pathobiology, mitigation would address a subset of HD symptoms (3) HD mutations directly impact neuronal Mn handling, which disrupts Mn-dependent processes to cause HD pathobiology; perhaps via interactions with proteins that function in Mn transport and homeostatic processes. HD mutation Decreased Mn transport HD pathophysiology 1 Decreased Mn transportHD pathophysiology 1 HD pathophysiology 2 HD pathophysiology 3?
  20. 20. Conclusions HSG 2016: DISCOVERING OUR FUTURE • Human and mouse models of HD exhibit deficits in Mn- dependent and Mn-responsive neuronal/cellular processes • Increasing neuronal Mn levels ameliorates the Mn-deficit related phenotypes; while HD genotype blocks at least some Mn- dependent signals/toxicity. • However, in published data we’ve seen Mn-exposure exacerbates other Mn neurotoxicities (e.g. see J. Madison et al 2012 PLoS One)
  21. 21. Future Directions and Implications HSG 2016: DISCOVERING OUR FUTURE 1. Characterize the relationship of the P53/AKT/mTOR pathway to regulation of neuronal Mn homeostasis 2. Utilize small molecules to dissect Mn transport 3. Distinguish which HD phenotypes are upstream or downstream of the defect in Mn handling 4. Determine the role of wild-type HTT gene in Mn-biology 5. Does dietary manganese influence HD pathobiology Excess systemic manganese, based on current data, may worsen some aspects of HD pathology, however, dietary deficiency of manganese may also exacerbate Mn-relevant pathology. FDA has an established Reference Daily Intake (RDI) for manganese (2.3 mg for adults and children >4)
  22. 22. Future directions: Identifying selective small molecule modifiers of HD-Mn biology HSG 2016: DISCOVERING OUR FUTURE vehicle VU 0063088 VU 0047355 VU 0025173 VU 0150155 VU 0003765 VU 0135086 VU 0004838 VU 0057971 0 250 500 750 1000 1250 1500 1750 2000 2250 2500 Q7 Q111 * * * * * * * * * Sig different from vehicle # Sig different from corresponding Q7 # # *# *# * * * nM Manganese extracted Control (Q7) HD (Q111) Vanderbilt High-Throughput Screening Core A screen of >40,000 small molecules identified ~41 lead chemicals that modify sub-cellular manganese levels. Kumar et al 2014 Nat. Sci. Reports A subset of these alter cellular Mn levels selectively in control cells, but not the HD cell model (mouse striatal cells) – suggesting they are hitting a target affected by the disease gene
  23. 23. Acknowledgements HSG 2016: DISCOVERING OUR FUTURE My Lab (contributors to this work) Andrew Tidball, Terry Jo Bichell, Kevin Kumar, Kyle Horning, Miles Bryan, Asad Al Aboud, Bingying Han, Anna Pfalzer, Piyush Joshi, Rachana Nitin, Michael Uhouse, Jack Feist, Mihir Odak Vanderbilt University & VUMC Diana Neely, Kevin Ess, David Weaver, Jens Meiler, Peter Hedera, Daniel Claassen, Chaz Hong, Edward Lowe Einstein College of Medicine Michael Aschner, Nancy Parmalee UNC Greensboro Keith Erikson Funding: NIEHS, RO1 ES016931 and RO1 ES010563 RO1 Pilot Grant from Vanderbilt Center in Molecular Toxicology Peterson Foundation for Parkinsons PK Hope is Alive Vanderbilt HTS Core (Rey Redha, Josh Bauer, Paige Vinson)
  24. 24. Selective allosteric modulators of M4 muscarinic receptors for treatment of Huntington’s Disease P. Jeffrey Conn Department of Pharmacology Vanderbilt Center for Neuroscience Drug Discovery :
  25. 25. Cortex Pre-symptomatic Huntington’s Normal Striatum Symptomatic Huntington’s Cortex Striatum Cortex Striatum Htt mutations may induce excessive activity at Cortico- Striatal synapses long before appearance of HD symptoms Hypothesis: early increases in cortico-striatal transmission contributes to cell death and behavioral deficits that appear at symptomatic stages.
  26. 26. Increased cortico-striatal EPSCs in presymptomatic 60 day old YAC128 HD mice precedes deficits at later ages Hypothesis: A drug that decreases excessive cortico-striatal activity at presymptomatic stages may reduce appearance of motor symptoms Deficits in cortico-striatal transmission appear at 5 months of age and parallels appearance of motor deficits. Normal (WT) Early HD (2 Mo YAC) Late HD Symptomatic
  27. 27. M4-KO WT Control 3mM CCh 3mM CCh in WT 3mM CCh in M4-KO %inhibitionofEPSC M4 activation inhibits glutamatergic transmission at corticostriatal synapses in HD mice Pancani et al , 2014, ACS Chem. Neurosci. WT and M4 KO YAC128 X M4 KO cross Pancani et al , 2015, PNAS
  28. 28. Positive Allosteric Modulators of M4 receptors? Will it be possible to develop selective drug-like molecules that selectively amplify activity of the M4 receptor?
  29. 29. 160,000 compounds 817 primary hits 10 µM singlicate 10 point CRC Mol Pharm Characterization 41 confirmed PAMs Optimization of novel M1 PAMs as research tools and drug candidates
  30. 30. Cholinergic inhibition of glutamatergic transmission at corticostriatal synapses is mediated by M4 mAChRs Stim Rec Will chronic administration of the drug candidate that amplifies M4 signaling reduce development of HD pathology and symptoms?
  31. 31. Chronic treatment with VU0467154 reverses loss of cortico- striatal activity in 5 mo old HD mice VU046154 dosed daily from 2 – 5 months Normal HD
  32. 32. Chronic treatment with VU0467154 reverses motor deficits in 5 mo. old YAC128 mice # Locomotor activity Rotorod performance Exploratory behavior Ongoing studies evaluating potential neuroprotective effect of M4 PAM
  33. 33. HTS Hit-to-Lead Lead Optimization Candidate IND PhI mGlu5 NAMs mGlu5 PAMs M4 PAMs GLP-1 M4 NAM M1 PAMs VCNDD Programs and Pipeline mGlu4 PAMs mGlu3 NAM mGlu2 NAM mGlu1 PAM mGlu3 PAM Schizophrenia, Alzheimer’s PD-LID, Refractory depression Schizophrenia, Alzheimer’s Parkinson’s Huntington’s, Schizophrenia Schizophrenia Schizophrenia Diabetes Refractory depression Refractory depression, cognition Parkinson’s Projected entry into clinical studies 1Q2018
  34. 34. Vanderbilt Center for Neuroscience Drug Discovery Supported by NIMH, NINDS, Huntington's Disease Foundation and CHDI In Vivo Carrie Jones Jerri Rook Nellie Byun Analisa Thompson Michael Bubser Jonathan W. Dickerson Rebekah L. Collier Mol Pharm/ Colleen Niswender Alice Rodriguez Daryl Venable Doug Sheffler Joy Marlo Ashley Brady Meredith Noetzel Ephys Tristano Pancani Dan Foster Mark Moehle Zixiu Xiang Adam Walker Ayan Ghoshal Xiaohui Lv Kari A. Johnson Med Chem Craig Lindsley Kyle Emmitte Michael Wood Shaun Stauffer Kyle Emmitte Cody Wenthur DMPK Tom Bridges Scott Daniels Annie Blobaum Matt Mulder Ryan Morrison Frank Byers
  35. 35. Why REDCap? The Software Platform
  36. 36. Security Rules
  37. 37. Security Rules 2004 Gap Assessment Researchers were using sub-standard methods to manage data for research studies/trials projects (pilot, R01, PPG)
  38. 38. Visual Status Data Validation Numerous Field Types + Text (Free) (Number) (Phone) (Zip) (Date) +TextArea +Select +Radio +File Branching LogicAuto-Variable Coding Human Readable Labels PDFs 40
  39. 39. Exports Raw Data + Stats Script Files (Labels, Coding Embedded De- Identification Tools 41
  40. 40. Why REDCap? REDCap filled a critical gap at Vanderbilt Easy way to do the right thing ... Security Rules
  41. 41. Early QI Survey (N=20) – Did REDCap improve your science? Yes (N=20) Improving Data Improves Science Improves Health 43
  42. 42. Improving Data Improves Science Improves Health 2004 Survey --- N = 20 Early Adopters REDCap Improves Science! (100%) Empowered users creating projects solidifies the data management plan before enrolling the first subject. 44
  43. 43. But here’s the cool part …
  44. 44. So … how’s it working so far? It’s fantastic, but could you add some sort of widget to … Every Time! Long ago … before we had real professionals doing this work ...
  45. 45. Used systems get better … End-users will tell you what you need to know to improve if you listen
  46. 46. Why the REDCap consortium
  47. 47. blah, blah, blah and let me know if you’re interested in collaborating …
  48. 48. Did you ever think we would get to … 2 20 200 2000 20000 2,084 active partners in 108 countries.
  49. 49. We didn’t think we wouldn’t …
  50. 50. So … how’s it working so far? It’s fantastic, but could you add some sort of widget to … Long ago … before we had real professionals doing this work ... 418,000 END USERS, 108 Countries
  51. 51. Power to the People REDCapCon 2016 – Paul Harris
  52. 52. LOTS of Brilliant Users + Flexible Tools = Power +
  53. 53. Typical Week Overall Local @ VU M T W T T S S Available to any student, faculty, staff member for any Vanderbilt use – no cost 55 The Research Community And The Institution
  54. 54. + Brilliant Users + Flexible Tools = Innovation
  55. 55. Jill Pulley

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