Preclinical Studies for Designing Rational Therapies for Epilepsy in Tuberous Sclerosis Complex

1. Preclinical Studies for Designing Rational Therapies for Epilepsy in Tuberous Sclerosis Complex Summit on Drug Discovery in TSC and Related Disorders Washington D.C. July 7, 2011 Michael Wong, MD, PhD Department of Neurology, Pediatrics, and Anatomy & Neurobiology Washington University School of Medicine Saint Louis, MO

2. Epilepsy in TSC: Clinical Features • Epilepsy is a very common neurological manifestation of TSC, occurring in up to 90% of patients in some series (Sparagana et al., 2003; Devlin et al., 2006; Chu-Shore et al. 2009). • Seizures are often severe and disabling, and may be multiple types. • Infantile spasms occur in about one-third of patients with TSC. • Seizures are often intractable to antiepileptic drugs. ~60-80% are medically-refractory (Sparagana et al., 2003; Chu-Shore et al. 2009), as opposed to ~33% medical intractability rate in the general epilepsy population. • Seizures are often not amenable to epilepsy surgery, due to multifocal nature of seizures. • Thus, more effective treatments are needed for epilepsy in TSC, including disease-modifying or antiepileptogenic therapies.

3. Epileptogenesis in TSC • Circuit Abnormalities: role of tubers; disrupted circuits in “normal” cortex/perituberal cortex. • Cellular/Molecular abnormalities: altered neurotransmitter receptors/ion channels, cell proliferation, signaling pathways ? + Hyperexcitability/ Seizures Abnormal Cells (e.g. giant cells, gliosis) Abnormal Molecules (e.g. neurotransmitter receptors, ion channels) Abnormal Circuits (e.g. tubers, disrupted circuitry in perituberal cortex

4. Epileptogenesis in TSC TSC1 Hamartin TSC2 Tuberin Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

5. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα PTEN Akt Metformin AMPK TSC1 Hamartin TSC2 Tuberin Rheb-GTP mTOR Downstream signaling mechanisms Rapamycin Atorvastatin Wortmannin Conventional Antiepileptic Drugs (AEDs) Neuroprotective/ Antiproliferative/ Other drugs S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

6. TSC – Role of Glia? Pathological evidence of glial dysfunction • Tubers: astrocytic proliferation/astrogliosis • Giant cells with glial and neuronal features. • Brain tumors: neoplastic astrocytomas, most commonly subependymal giant cell astrocytomas (SEGAs) Subependymal giant cell astrocytoma “Giant cells” in a cortical tuber

7. Glia-Targeted Tsc1 Conditional Knock-out Mouse (Tsc1GFAPCKO mice) • Inactivation of Tsc1 in glia achieved with Cre-LoxP technology. • LoxP sites targeted to Tsc1 allele. • Cre recombinase linked to GFAP promoter • Crossing of GFAP-Cre with LoxP-Tsc1 mice results in inactivation of Tsc1 gene in glia Uhlmann et al. 2002

8. LF RF 0.5 mV RH 5 s LF RF RH Generalized Cortical Onset Tsc1GFAPCKO mice: Seizure Localization and Frequency Hippocampal Onset Uhlmann et al. 2002 Erbayat-Altay et al. 2007

9. Possible Glia-mediated Physiological Mechanisms of Neuronal Dysfunction and Epileptogenesis in Tsc1GFAPCKO mice • Circuit Physiology (“Epileptic Network”) • “Mass” effect from astrocyte proliferation on existing neuronal networks • Abnormal glia-guided neuronal migration/synaptogenesis/ neurogenesis with development of pathological neuronal networks • Cellular/Molecular Physiology (“Epileptic Neuron”) • Astrocytic regulation of synaptic neurotransmitter levels (e.g. glutamate transporters) • Astrocytic regulation of extracellular ion concentrations (e.g. inward-rectifying K channels) • Circuit Physiology (“Epileptic Network”) • “Mass” effect from astrocyte proliferation on existing neuronal networks • Abnormal glia-guided neuronal migration/synaptogenesis/ neurogenesis with development of pathological neuronal networks

10. Astrocyte Tuberin Tsc1 Hamartin Rheb mTOR S6K/S6, eIF4E Protein synthesis Cell growth/ proliferation Control Tsc1 CKO Tsc1 CKO Control P-S6K Control Tsc1 CKO Cell number, x104 Uhlmann et al. 2002

11. Control Tsc1GFAPCKO Tsc1GFAPCKO mice have megencephaly due to glial proliferation GFAP-Astrocyte labeling Control Tsc1GFAPCKO • Neuropathological examination shows a generalized increased brain size, which progresses with age. • GFAP immunostaining shows progressive increase in number of astrocytes throughout the brain. • No focal abnormalities (e.g. tubers) • Minor neuronal disorganization, especially dispersion of pyramidal cell layer in hippocampus. Uhlmann et al. 2002

12. Possible Glia-mediated Physiological Mechanisms of Neuronal Dysfunction and Epileptogenesis in Tsc1GFAPCKO mice • Circuit Physiology (“Epileptic Network”) • “Mass” effect from astrocyte proliferation on existing neuronal networks • Abnormal glia-guided neuronal migration/synaptogenesis/ neurogenesis with development of pathological neuronal networks • Cellular/Molecular Physiology (“Epileptic Neuron”) • Astrocytic regulation of synaptic neurotransmitter levels (e.g. glutamate transporters) • Astrocytic regulation of extracellular ion concentrations (e.g. inward-rectifying K channels) • Circuit Physiology (“Epileptic Network”) • “Mass” effect from astrocyte proliferation on existing neuronal networks • Abnormal glia-guided neuronal migration/synaptogenesis/ neurogenesis with development of pathological neuronal networks • Cellular/Molecular Physiology (“Epileptic Neuron”) • Astrocytic regulation of synaptic neurotransmitter levels (e.g. glutamate transporters) • Astrocytic regulation of extracellular ion concentrations (e.g. inward-rectifying K channels)

13. Astrocyte Tuberin Tsc1 Hamartin Rheb mTOR S6K/S6, eIF4E G G G G G G G G G G G G G G G G G G G G G G Protein synthesis ? GLT-1/ GLAST Excitotoxic Cell Death Seizure EPSP EPSP AMPA Receptors AMPA/NMDA Receptors Presynaptic Neuron Postsynaptic Neuron

14. Brain Astrocyte Culture CortexCerebellum Tsc1 CKO Tsc1 CKO Tsc1 CKO Control Control Control GLT-1 GLT-1 tubulin GLAST GLAST tubulin tubulin GLT1 and GLAST expression is decreased in astrocytes from Tsc1GFAPCKO mice Wong et al. 2003

15. Functional glutamate transporter currents are decreased in astrocytes from Tsc1GFAPCKO mice Control Control D-Asp TBOA TBOA DHK DHK Control Control Tsc1 CKO Tsc1 CKO D-Asp TBOA TBOA DHK DHK Control Control 2.0 20 pA 50 ms 1.5 Glutamate transporter current Glutamate transporter current 100 pA * 400 pA 900 1.0 500 ms 800 500 ms 700 600 0.5 500 Current density (pA/pF) Peak Current (pA) 400 0.0 300 200 100 0 Control Control Tsc1 Tsc1 CKO CKO Astrocytes in Brain Slices Astrocyte Culture * Wong et al. 2003

16. Extracellular glutamate is elevated in Tsc1GFAPCKO mice, measured by in vivo microdialysis Zeng et al. 2007

17. Tsc1GFAPCKO mice have excitotoxic neuronal death in neocortex and hippocampus: TUNEL Control Tsc1 CKO Neocortex Hippocampus Zeng et al. 2007

18. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα PTEN Akt Metformin AMPK TSC1 Hamartin TSC2 Tuberin Rheb-GTP mTOR Downstream signaling mechanisms Rapamycin Atorvastatin Wortmannin Conventional Antiepileptic Drugs (AEDs) Neuroprotective/ Antiproliferative/ Other drugs S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitters Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

19. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα PTEN Akt Metformin AMPK TSC1 Hamartin TSC2 Tuberin Rheb-GTP mTOR Downstream signaling mechanisms Rapamycin Atorvastatin Wortmannin Ceftriaxone Neuroprotective/ Antiproliferative/ Other drugs S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitters Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

20. Ceftriaxone restores normal Glt-1 astrocyte glutamate transporter levels in Tsc1GFAPCKO mice . Zeng et al. 2010

21. Ceftriaxone decreases the abnormally elevated extracellular glutamate levels in Tsc1GFAPCKO mice . Zeng et al. 2010

22. Ceftriaxone reduces neuronal death, but not glial proliferation in Tsc1GFAPCKO mice . Neuronal death (Fluoro-Jade B) Glial proliferation (GFAP) Zeng et al. 2010

23. Ceftriaxone reduces seizure frequency and prolongs survival moderately, but does not prevent epilepsy or premature death . Zeng et al. 2010

24. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα PTEN Akt AMPK TSC1 Hamartin TSC2 Tuberin Rheb-GTP mTOR Downstream signaling mechanisms Rapamycin Ceftriaxone S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitters Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

25. Early rapamycin treatment prevents glial proliferation and increased brain size in Tsc1GFAPCKO mice . Cont-Veh KO-Veh KO-Rap Cont-Rap Zeng et al., 2008

26. Early rapamycin treatment increases astrocyte Glt-1 expression of Tsc1GFAPCKO mice. Zeng et al., 2008

27. Early rapamycin treatment prevents development of epilepsy and prolongs survival of Tsc1GFAPCKO mice . Zeng et al., 2008

28. Late rapamycin treatment decreases seizure frequency and prolongs survival of already symptomatic Tsc1GFAPCKO mice . Zeng et al., 2008

29. Clinical Implications of Mouse Epilepsy Data • Preventive, “Anti-epileptogenic” Therapy: • Early treatment with rapamycin prevented the development of epilepsy in presymptomatic mice. Since many patients are diagnosed with TSC at a young age due to non-neurological findings or due to a positive family history, and yet 90% of patients may go on to develop epilepsy, it is reasonable to consider a clinical trial testing the ability of rapamycin to prevent epilepsy in TSC patients who have never had a seizure or in patients presenting with their first seizure or with infantile spasms. • Symptomatic, “Anti-Seizure” Therapy: • Late treatment with rapamycin decreased seizure frequency in symptomatic mice; so one could also consider using rapamycin to decrease progression of seizures in TSC patients that already have epilepsy.

30. Clinical trials: mTOR inhibition reduces astrocytoma growth and seizure frequency in TSC patients.

31. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα PTEN Akt Metformin AMPK TSC1 Hamartin TSC2 Tuberin Rheb-GTP mTOR Ceftriaxone, Conventional antiepileptic drugs (AEDs) Downstream signaling mechanisms ??? Rapamycin Atorvastatin Wortmannin Neuroprotective/ Antiproliferative/ Other drugs S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

32. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα Akt AMPK TSC1 Hamartin TSC2 Tuberin Feedback inhibition Rheb-GTP mTOR Downstream signaling mechanisms S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

33. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Upstream signaling mechanisms PI3K LKβ1/STRADα Akt AMPK TSC1 Hamartin FOXOs, BAD, p27 ↓ apoptosis ↑ cell proliferation TSC2 Tuberin Feedback inhibition Rheb-GTP mTOR Downstream signaling mechanisms Rapamycin S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

34. Epileptogenesis in TSC Energy/nutrient deprivation Growth factors/nutrient stimulation Dual PI3K/mTOR inhibitor Upstream signaling mechanisms PI3K LKβ1/STRADα Akt AMPK TSC1 Hamartin FOXOs, BAD, p27 ↓ apoptosis ↑ cell proliferation TSC2 Tuberin Feedback inhibition Rheb-GTP mTOR Downstream signaling mechanisms Dual PI3K/mTOR inhibitor S6K/S6 4E-BP1/eIF4E Other pathways Circuit Abnormalities Synaptic Reorganization Loss of Inhibitory Circuits Molecular Abnormalities Ion Channels Neurotransmitter Receptors Oxidative Stress/Autophagy Cellular Abnormalities Cell Growth/Proliferation Neuronal Death/Apoptosis Neurogenesis Epileptogenic/ Ictogenic mechanisms

35. Conclusions • The mTOR pathway is critical for epileptogenesis in mouse models of TSC and mTOR inhibitors may have both early antiepileptogenic and late symptomatic effects on epilepsy in TSC. • Modulation of downstream mechanisms of epileptogenesis, such as astrocyte glutamate transporters, may also have some, more limited, effectiveness for epilepsy, but could have fewer side effects . • Future therapies for epilepsy can continue to be designed with better, more selective efficacy and few side effects, based on rationally targeting different mechanistic levels of epileptogenesis.

36. Wong Lab Ebru Erbayat-Altay Vered Gazit Laura Jansen Yannan Ouyang Nicholas Rensing Lin Xu Linghui Zeng Bo Zhang Collaborators David Gutmann Kevin Ess Erik Uhlmann David Holtzman John Cirrito Adam Bero Steven Mennerick David Wozniak Kel Yamada Support NINDS/NIH K02 NS045583 NINDS/NIH R01 NS056872 Tuberous Sclerosis Alliance Citizens United for Research in Epilepsy (CURE) McDonnell Center Peter Crino – Univ. Penn. David Kwiatkowski – Harvard