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Shawn dissertation NRSF

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Dissertation by Shawn McClelland
NRSF mediated epigenetic changes
in epilepsy

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Shawn dissertation NRSF

  1. 1. Dissertation Defense<br />Shawn McClelland<br />NRSF<br />mediated epigenetic processes in neuroplasticity<br />Committee Members:<br /> Dr. Tallie Z. Baram<br /> Dr. Martin Smith<br /> Dr. Marcelo Wood<br />
  2. 2. What makes us who we are?<br />Genes<br />Changes induced <br />by the environment<br />
  3. 3. Neuroplasticity<br />the ability of the brain to change as a result of environmental input<br />
  4. 4. Why focus on Epilepsy*? <br />- Third most common chronic brain disorder<br />- The most common chronic brain disorder in children & young adults<br />- >1% of the population <br />- Enormous personal, social & fiscal impact<br />* Generation of spontaneous seizures by the brain<br />
  5. 5. Epilepsy or Epilepsies<br />Numerous Mechanisms<br /> for the Epilepsies:<br /> Genetic<br /> Acquired<br /> Gene-environment interaction<br />
  6. 6. Temporal Lobe Epilepsy (TLE)<br />Involves the hippocampus<br />Most common epilepsy in adults<br />Can be severe and intractable to medicines<br />Not genetic<br />
  7. 7. Temporal Lobe Epilepsy (TLE)<br />Risk factors<br />- Trauma<br />- Long Seizures (Status Epilepticus)<br />- Brain infection<br />
  8. 8. Insult<br />Acute damage<br />Latent period<br />Progressive damage<br />Hyperexcitability<br />Seizures<br />Epilepsy after severe Head Trauma<br />Epilepsy after severe Head Trauma<br />Circuit reorganization<br />But most epilepsy does not follow trauma….<br />
  9. 9. Temporal Lobe Epilepsy (TLE)<br />Risk factors<br />- Trauma<br />- Long Seizures (Status Epilepticus)<br />- Brain infection<br />
  10. 10. How does the brain become epileptic after insults that have little cell death?<br />- No clear structural changes<br />- Change is in the function of the brain cells<br />- Genes that neurons express govern function<br />Which gene or genes are changed to make neurons epileptic?<br />Toth et al., J Neurosci, 1998; Bender et al., 2003; Dube et al, TiNS 2007<br />
  11. 11. 0.2<br />Ci/gm<br />*<br />0.1<br />m<br />*<br />0.0<br />Control<br />HT-activity<br />KA-activity<br />HCN1 expression is repressed after epilepsy-provoking insults<br />Chen, 2001; Brewster 2002,2005; Santoro & Baram 2003; Dyhrfjeld-Johnsen, 2008 <br />
  12. 12. Dysregulation of HCN1 is common in many animal models of epilepsy<br />Chen, Baram, Soltesz, Nature Med, 2001<br />Brewster…Baram J Neurosci. 2002<br />Ludwig …Biel, EMBO J. 2003 <br />Budde….Pape. J Neurosci. 2005 <br />Kamal… de Graan, Ramakers, Eur J Neurosci, 2006<br />Kuisle…Baram, Luthi, J Physiol. 2006<br />Schridde, Strauss… van Luijtelaar 2006<br />Shin & Chetkovich, JBC, 2007<br />Jung…Poolos, J Neurosci. 2007, 2010 <br />Zhang…Sanchez, Epilepsia, 2007<br />Powell… O’Brien, Epilepsia. 2008 <br />Dugladze...Gloveli, PNAS 2008<br />Van Gaase...deGraan, 2008<br />Marcelin...Bernard, Neurobiol Dis. 2008 <br />Lewis...Baram, Chetkovich , J Neurosci, 2009<br />Huang…Shah, J Neurosci, 2009<br />Santoro…Blumenfeld, Epilepsia 2010. <br />Hablitz & Yang, Epilepsia 2010<br />
  13. 13. How is HCN1 expression repressed during epileptogenesis?<br />- The mechanisms might be informative about epileptogenesis<br />- They might provide a ‘HOOK’ into large-scale gene-network changes that constitute the epileptogenic process <br />Chen, 2001; Santoro & Baram 2003, Bender & Baram 2008; McClelland, Ann Neurol 2011 <br />
  14. 14. hcn1 gene<br />
  15. 15. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical binding to the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changes of the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing ~30 genes, (in addition to hcn1) whose repression contribute to epilepsy.<br />
  16. 16. NRSF Neuron-Restrictive Silencer Factor<br />The original dogma<br />NRSF silences gene expression<br />- Group of genes important to neurons<br />So NRSF is found in all types of cells except for neurons<br />NRSF is found in neurons!<br />Regulates, instead of silences, neuronal genes<br />Basal levels reported as low in the brain, and to increase with seizure activity<br />
  17. 17. Is NRSF involved in HCN1 suppression?<br />
  18. 18. NRSF – mechanism of gene repression<br />
  19. 19. Increased binding of NRSF to the hcn1 gene<br />is apparent following KA-SE<br />
  20. 20. Chromatin Immunoprecipitation (ChIP)<br />
  21. 21. Sick kid at the playground analogy<br />
  22. 22. Decoy NRSE-sequence<br />oligodeoxynucleotide (ODN) strategy<br />
  23. 23. Abrogation of NRSF binding to the hcn1 gene following NRSE-ODN infusion.<br />
  24. 24. Blocking NRSF binding after<br />KA-SE recovers HCN1 expression<br />
  25. 25. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical binding to the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changes of the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing ~30 genes, (in addition to hcn1) whose repression contribute to epilepsy.<br />
  26. 26. Repression of HCN1 after status epilepticus<br />is enduring<br />
  27. 27. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical binding to the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changes of the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing ~30 genes, (in addition to hcn1) whose repression contribute to epilepsy.<br />
  28. 28. An epigenetic mark of repression is selectively and persistently augmented after KA-SE at the hcn1 gene<br />Active State<br />Repressed State<br />
  29. 29. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical binding to the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changes of the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing of ~30 genes, in addition to hcn1, that together contribute to epilepsy.<br />
  30. 30. Decoy NRSE-ODNs recover HCN1<br />expression and function<br />
  31. 31. Monitor development of epilepsy<br />Continuous Video EEG<br />0 day<br />+1<br />-7 days<br />+2<br />+3<br />+4<br />+5<br />+6<br />+7<br />+8<br />+9<br />+11<br />+10<br />+12<br />+13<br />Surgery-Cannulae<br />Periodically Infuse<br />NRSE-ODNs<br />Induce Status Epilepticus<br />Kainic Acid injections<br />
  32. 32. Decoy NRSE-ODNs alter the outcome of<br />KA-induced status epilepticus.<br />
  33. 33. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical binding to the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changesof the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing ~30 genes, (in addition to hcn1) whose repression contribute to epilepsy.<br />
  34. 34. Which genes did we rescue<br />that modified the disease process?<br />MICROARRAY<br />Check the mRNA levels of 22,000 genes<br />0 day<br />+1<br />-7 days<br />+2<br />Surgery-Cannulae<br />Induce Status Epilepticus<br />Kainic Acid injections<br />Periodically Infuse<br />NRSE-ODNs<br />
  35. 35. Which genes did we rescue<br />that modified the disease process?<br />22,000 genes<br />Genes repressed<br />after insult<br />leading to epilepsy.<br />470 genes<br />30 genes<br />Genes rescued<br />Using decoy ODNs<br />
  36. 36. Decoy NRSE-ODNs alter the outcome of<br />KA-induced status epilepticus.<br />ALL<br />of these genes have putative NRSEs<br />
  37. 37. NRSF<br />mediated epigenetic processes in neuroplasticity<br />NRSF regulates hcn1 after an insult that leads to epilepsy through its physical bindingto the gene.<br />Enduring repression of hcn1 expression by NRSF involves epigenetic changesof the chromatin of the gene.<br />Blocking NRSF function after an insult that provokes epilepsy markedly attenuates the development of epilepsy.<br />The prevention of epilepsy is a result of rescuing ~30 genes, (in addition to hcn1) whose repression contribute to epilepsy.<br />
  38. 38. Temporal Lobe Epilepsy (TLE)<br />Involves the hippocampus<br />Most common epilepsy in adults<br />Can be severe and intractable to medicines<br />Not genetic<br />
  39. 39. Understanding the mechanisms involved in this type of aberrant neuroplasticity may help in other neurological disorders.<br />Neuroplasticity makes us who we are!<br />
  40. 40.
  41. 41. QUESTIONS?<br />Dissertation Defense<br />Shawn McClelland<br />NRSF<br />mediated epigenetic processes in neuroplasticity<br />
  42. 42. Patterns of NRSF expression after SE<br />
  43. 43. Patterns of NRSF expression after SE<br />
  44. 44. Patterns of NRSF expression after SE<br />
  45. 45. NRSF – Histone Modification<br />NRSF is found in neurons!<br />Direct repression, Histone modification, DNA methylation,…<br />Activity influences NRSF levels and NRSF represses a set of genes.<br />
  46. 46. ALLgenes validated by GSEA based on the Johnson paper list of genes containing an NRSE.<br />+indicates genes validated by GSEA based on MSigDB:V$NRSF_01 list of genes <br /> <br />%indicates genes (14) found with ANOVA, Benjamini-Hoch FDR < 0.25.<br />$indicates genes (6) found with one-way ANOVA, unadjusted p value < 0.05 <br />Johnson DS, Mortazavi A, Myers RM, Wold B (2007) Genome-wide mapping of in vivo protein-DNA interactions. Science 316: 1497-1502.<br />
  47. 47. Heatmap of ALL NRSE-containing genes<br />down regulated after KA-SE<br />The majority of genes containing NRSE sites are recovered by the administration of NRSE-ODNs<br />
  48. 48. Candidate Gene Analysis<br />Genes repressed in KA+SCRLD compared to Sham+SCRLD<br />Rescued Genes: increased in KA-NRSE compared to KA-SCRLD<br />4 GROUPS<br />Sham + SCRLD<br />Sham + NRSE<br />KA + SCRLD<br />KA + NRSE<br />Genes repressed in KA+NRSE compared to Sham+SCRLD<br />Also performed ANOVA on the groups.<br />
  49. 49. Gene Set Enrichment Analysis (GSEA)<br />
  50. 50. GSEAconfirms that NRSE-containing genes are most recovered by the NRSE-ODN<br />Gene sets enriched in hippocampal CA1 in KA+NRSE compared to KA+SCRLD<br />
  51. 51. We might expect<br />
  52. 52. Genes regulated by NRSF possess a distinct range of NRSF binding affinities<br />
  53. 53. Blocking NRSF binding after<br />KA-SE recovers HCN1 expression<br />

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