preclinical studies for designing rational therapies for epilepsy in tuberous sclerosis complex...

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

?


Hamartin

Tuberin

TSC1TSC2

Molecular AbnormalitiesIon ChannelsNeurotransmitter ReceptorsOxidative Stress/Autophagy

Cellular AbnormalitiesCell Growth/ProliferationNeuronal Death/ApoptosisNeurogenesis

Circuit AbnormalitiesSynaptic ReorganizationLoss of Inhibitory Circuits

Epileptogenic/Ictogenicmechanisms


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα

Energy/nutrient deprivation

Growth factors/nutrientstimulation

PTEN

Upstream signalingmechanisms

mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Downstream signalingmechanisms





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




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







Possible Glia-mediated Physiological Mechanisms of

Neuronal Dysfunction and Epileptogenesis in Tsc1GFAPCKO mice







G

G

G

G

G

G

G

GG

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

Contr

ol

Tsc1

CKO

Contr

ol

Tsc1

CKO

Cortex Cerebellum

GLT-1

GLAST

tubulin

Brain

GLT-1

tubulin

GLAST

tubulin

Tsc1

CKO

Contr

ol

Astrocyte Culture

Control

DHK

Control

TBOA

Control

DHK

Tsc1 CKO

TBOA

20 pA50 ms

Glutamate transporter current

Control Tsc1 CKO

Curr

ent

den

sity

(p

A/p

F)

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 pA

500 ms

D-Asp

ControlDHK

TBOA

Control

Tsc1 CKO

100 pA

500 ms

D-Asp

TBOA

DHKControl

Astrocyte Culture

0

100

200

300

400

500

600

700

800

900

Peak

Cu

rren

t (p

A)

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


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα



PTEN


mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways


Molecular AbnormalitiesIon ChannelsNeurotransmittersOxidative Stress/Autophagy




Rapamycin

Atorvastatin

Wortmannin

Metformin


ConventionalAntiepilepticDrugs (AEDs)


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα



PTEN


mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






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


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα



PTEN


mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






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.


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα



PTEN


mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






Rapamycin

Atorvastatin

Wortmannin

Metformin

Ceftriaxone, Conventional antiepileptic drugs (AEDs)


???


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα




mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






Feedbackinhibition


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα




mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






Feedbackinhibition

Rapamycin

FOXOs, BAD, p27↓ apoptosis↑ cell proliferation


Hamartin

Tuberin

TSC1TSC2

PI3K

AktAMPK

LKβ1/STRADα




mTOR

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways






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

preclinical studies for designing rational therapies for epilepsy in tuberous sclerosis complex...

Documents

tsc1 allele

loxptsc1 mice results

epilepsy surgery

inactivation of tsc1

creloxp technology

general epilepsy population

cell proliferation

role of tubers