Preclinical Studies for Designing Rational Therapies for Epilepsy in Tuberous Sclerosis Complex
Summit on Drug Discovery in TSC and Related DisordersWashington D.C.
July 7, 2011
Michael Wong, MD, PhDDepartment of Neurology, Pediatrics, and Anatomy &
NeurobiologyWashington University School of Medicine
Saint Louis, MO
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.
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
Abnormal Circuits(e.g. tubers, disrupted circuitry in perituberal cortex
+
Abnormal Cells (e.g. giant cells, gliosis)Abnormal Molecules(e.g. neurotransmitter receptors, ion channels)
Hyperexcitability/Seizures
?
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
PTENUpstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Rapamycin
Atorvastatin
Wortmannin
Metformin
ConventionalAntiepilepticDrugs (AEDs)
Neuroprotective/Antiproliferative/Other drugs
TSC – Role of Glia?
Subependymal giant cell astrocytoma
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)
“Giant cells” in a cortical tuber
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
LF
RF
RH
LF
RF
RH
5 s
0.5 mV
Uhlmann et al. 2002
Generalized Cortical Onset
Hippocampal Onset
Tsc1GFAPCKO mice: Seizure Localization and Frequency
Erbayat-Altay et al. 2007
• 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)
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
AstrocyteHamartin
Tuberin
mTOR
S6K/S6, eIF4E
Rheb
Protein synthesis
Cell growth/proliferation
Tsc1
Uhlmann et al. 2002
Tsc1 CKOControlControl Tsc1 CKO
P-S6K
Control Tsc1 CKO
Cel
l num
ber,
x104
Uhlmann et al. 2002
Tsc1GFAPCKO mice have megencephaly due to glial proliferation
• 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.
Control Tsc1GFAPCKOControl Tsc1GFAPCKO
GFAP-Astrocyte labeling
• 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)
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)
G
G
G
G
G
G
GGG
G
GG
Presynaptic Neuron Postsynaptic Neuron
G
G G
GG
Astrocyte
GLT-1/GLAST
AMPAReceptors
EPSP
Tsc1 HamartinTuberin
mTOR
S6K/S6, eIF4E
Rheb
Protein synthesis
?
G
G
G
G
G
AMPA/NMDAReceptors
EPSP
Excitotoxic Cell Death
Seizure
GLT1 and GLAST expression is decreased in astrocytes from Tsc1GFAPCKO mice
Wong et al. 2003
Cont
rol
Tsc1
CKO
Cont
rol
Tsc1
CKO
Cortex Cerebellum
GLT-1
GLAST
tubulin
Brain
GLT-1
tubulin
GLAST
tubulin
Tsc1
CKO
Cont
rol
Astrocyte Culture
Control
DHK
Control
TBOA
Control
DHK
Tsc1 CKO
TBOA
20 pA50 ms
Glutamate transporter current
Control Tsc1 CKO
Curre
nt d
ensit
y (p
A/pF
)
0.0
0.5
1.0
1.5
2.0
*
Functional glutamate transporter currents are decreased in astrocytes from Tsc1GFAPCKO mice
Astrocytes in Brain Slices
400 pA500 ms
D-Asp
ControlDHK
TBOA
Control
Tsc1 CKO
100 pA500 ms
D-AspTBOA
DHKControl
Astrocyte Culture
0100200300400500600700800900
Peak
Cur
rent
(pA)
Glutamate transporter current
Control Tsc1 CKO
Wong et al. 2003
*
Extracellular glutamate is elevated in Tsc1GFAPCKO mice, measured by in vivo microdialysis
Zeng et al. 2007
Control Tsc1 CKO
Neocortex
Hippocampus
Tsc1GFAPCKO mice have excitotoxic neuronal death in neocortex and hippocampus: TUNEL
Zeng et al. 2007
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
PTENUpstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmittersOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Rapamycin
Atorvastatin
Wortmannin
Metformin
Neuroprotective/Antiproliferative/Other drugs
ConventionalAntiepilepticDrugs (AEDs)
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
PTENUpstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmittersOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Rapamycin
Atorvastatin
Wortmannin
Metformin
Ceftriaxone Neuroprotective/Antiproliferative/Other drugs
Ceftriaxone restores normal Glt-1 astrocyte glutamate transporter levels in Tsc1GFAPCKO mice .
Zeng et al. 2010
Ceftriaxone decreases the abnormally elevated extracellular glutamate levels in Tsc1GFAPCKO mice .
Zeng et al. 2010
Ceftriaxone reduces neuronal death, but not glial proliferation in Tsc1GFAPCKO mice .
Neuronal death(Fluoro-Jade B)
Glial proliferation(GFAP)
Zeng et al. 2010
Ceftriaxone reduces seizure frequency and prolongs survival moderately, but does not prevent epilepsy or premature death .
Zeng et al. 2010
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
PTENUpstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmittersOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Rapamycin
Ceftriaxone
Early rapamycin treatment prevents glial proliferation and increased brain size in Tsc1GFAPCKO mice .
Zeng et al., 2008
Cont-Veh Cont-Rap KO-Veh KO-Rap
Early rapamycin treatment increases astrocyte Glt-1 expression of Tsc1GFAPCKO mice.
Zeng et al., 2008
Early rapamycin treatment prevents development of epilepsy and prolongs survival of Tsc1GFAPCKO mice .
Zeng et al., 2008
Late rapamycin treatment decreases seizure frequency and prolongs survival of already symptomatic Tsc1GFAPCKO mice .
Zeng et al., 2008
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.
Clinical trials: mTOR inhibition reduces astrocytoma growth and seizure frequency in TSC patients.
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
PTENUpstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Rapamycin
Atorvastatin
Wortmannin
Metformin
Ceftriaxone, Conventional antiepileptic drugs (AEDs) Neuroprotective/
Antiproliferative/Other drugs
???
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
Upstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Feedbackinhibition
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
Upstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Feedbackinhibition
Rapamycin
FOXOs, BAD, p27↓ apoptosis↑ cell proliferation
Epileptogenesis in TSC
HamartinTuberin
TSC1TSC2
PI3K
AktAMPK
LKβ1/STRADα
Energy/nutrient deprivation
Growth factors/nutrientstimulation
Upstream signalingmechanisms
mTOR
Rheb-GTP
S6K/S64E-BP1/eIF4E
Other pathways
Downstream signalingmechanisms
Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy
Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis
Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits
Epileptogenic/Ictogenicmechanisms
Feedbackinhibition
FOXOs, BAD, p27↓ apoptosis↑ cell proliferation
Dual PI3K/mTORinhibitor
Dual PI3K/mTORinhibitor
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.
Wong LabEbru Erbayat-AltayVered GazitLaura JansenYannan OuyangNicholas RensingLin XuLinghui ZengBo Zhang
David Gutmann Kevin EssErik Uhlmann
David HoltzmanJohn CirritoAdam Bero
SupportNINDS/NIH K02 NS045583NINDS/NIH R01 NS056872 Tuberous Sclerosis Alliance Citizens United for Research in Epilepsy (CURE)McDonnell Center
Peter Crino – Univ. Penn.
David Kwiatkowski – Harvard
Steven Mennerick
David Wozniak
Kel Yamada
Collaborators