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

AktAMPK

LKβ1/STRADα

Energy/nutrient deprivation

Growth factors/nutrientstimulation

Upstream signalingmechanisms

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

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

S6K/S6, eIF4E

Protein synthesis

Cell growth/proliferation

Uhlmann et al. 2002

Tsc1 CKOControlControl Tsc1 CKO

Control Tsc1 CKO

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

Presynaptic Neuron Postsynaptic Neuron

Astrocyte

GLT-1/GLAST

AMPAReceptors

Tsc1 HamartinTuberin

S6K/S6, eIF4E

Protein synthesis

AMPA/NMDAReceptors

Excitotoxic Cell Death

Seizure

GLT1 and GLAST expression is decreased in astrocytes from Tsc1GFAPCKO mice

Wong et al. 2003

Cortex Cerebellum

tubulin

Astrocyte Culture

Control

Tsc1 CKO

20 pA50 ms

Glutamate transporter current

Control Tsc1 CKO

Functional glutamate transporter currents are decreased in astrocytes from Tsc1GFAPCKO mice

Astrocytes in Brain Slices

400 pA

500 ms

ControlDHK

Control

Tsc1 CKO

100 pA

500 ms

DHKControl

Astrocyte Culture

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

AktAMPK

LKβ1/STRADα

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Molecular AbnormalitiesIon ChannelsNeurotransmittersOxidative Stress/Autophagy

Rapamycin

Atorvastatin

Wortmannin

Metformin

ConventionalAntiepilepticDrugs (AEDs)

Hamartin

Tuberin

TSC1TSC2

AktAMPK

LKβ1/STRADα

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

AktAMPK

LKβ1/STRADα

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

AktAMPK

LKβ1/STRADα

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Rapamycin

Atorvastatin

Wortmannin

Metformin

Ceftriaxone, Conventional antiepileptic drugs (AEDs)

Hamartin

Tuberin

TSC1TSC2

AktAMPK

LKβ1/STRADα

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Feedbackinhibition

Hamartin

Tuberin

TSC1TSC2

AktAMPK

LKβ1/STRADα

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Feedbackinhibition

Rapamycin

FOXOs, BAD, p27↓ apoptosis↑ cell proliferation

Hamartin

Tuberin

TSC1TSC2

AktAMPK

LKβ1/STRADα

Rheb-GTP

S6K/S64E-BP1/eIF4E

Other pathways

Feedbackinhibition

FOXOs, BAD, p27↓ apoptosis↑ cell proliferation

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

, Dear I am writing today as a [mom/dad] of a child with a very complex and obscure disease: tuberous sclerosis complex (TSC). A disease that creates tumors

Tuberous Sclerosis With Renal AML

Tuberous Sclerosis Complex (TSC): Genetics and Care Guidelines

Epilepsy surgery and tuberous sclerosis complex: special ... · Epilepsy surgery for medically refractory seizures among patients with tuberous sclerosis complex (TSC) is a well-accepted

DC/DC Converter - Traco Power · tsc 2072 4 a tsc 1272 15 a tsc 3072 12 a tsc 1372 30 a tsc 5072 20 a tsc 1572 42 a 12 vdc tsc 0672 26 a tsc 3572 56 a (11 – 13 vdc) tsc 6072 40

Mutational and functional study of Tuberous Sclerosis ......O complexo da esclerose tuberosa (TSC) é um distúrbio autossômico dominante causado por variantes patogênicas em um

Tuberous sclerosis complex for the pulmonologist

The clinical profile of tuberous sclerosis complex (TSC) in the … clinical profile.pdf · The clinical proﬁle of tuberous sclerosis complex (TSC) in the United Kingdom: A retrospective

31 tuberous facts sclerosis - TSC Canada

Tuberous Sclerosis Association (TSA) Annual Report 2018 ... · announcement was followed by intense campaigning from people living with TSC, their families and the TSA to reverse

Presentation and Diagnosis of Tuberous Sclerosis Complex ......on behalf of the Tuberous Sclerosis Complex Autism Center of Excellence Research Network OBJECTIVES: Tuberous sclerosis

Tuberous Sclerosis Lancet 2008

INFANTILE SPASMS & TUBEROUS - Epilepsy Foundation · What You Need to Know about Infantile Spasms and TSC Infantile spasms can be a devastating type of seizures for infants and young

Tuberous Sclerosis Complex (TSC): Genetics and Care …reg.abcsignup.com/files/{07D0901F-86B6-4CD0...40/355 (11.3%) Individuals with TSC Followed Long-Term. Diagnosis of TSC • Diagnosing

Sleep in Tuberous Sclerosis Complex

Tuberous Sclerosis - Orrin Devinsky, MD

A novel mouse model of tuberous sclerosis complex (TSC ... · eye-specific Tsc1-ablation disrupts visual-pathway development Iwan Jones, Anna-Carin Hägglund, Gunilla Törnqvist,

Tuberous sclerosis: a genetic - BMJ

Tuberous Sclerosis Complexdownload.e-bookshelf.de/download/0000/6036/97/L-G... · 2016. 4. 8. · 1.3 Hereditary Nature of TSC 6 1.4 Molecular Mechanisms in TSC 7 1.5 The Future of

Natural clusters of tuberous sclerosis complex (TSC ......RESEARCH Open Access Natural clusters of tuberous sclerosis complex (TSC)-associated neuropsychiatric disorders (TAND): new