James Eubanks

Genetics and Development Division Department of Surgery

Toronto Western Research Institute Institute of Medical Sciences

University Health Network University of Toronto

Krembil Neuroscience Program

University of Toronto Epilepsy Research Program

Translational Strategies For Rett Syndrome: From Experimental Mice To Treating Girls and Women

What is Rett Syndrome

• Most Common Monogenetic Cause of Severe Developmental Delay In Girls Worldwide

• Affects Primarily Females, with a Frequency of about 1 in 15,000– X-linked Condition mostly caused by mutations of the MECP2 gene

– Severity Ranges Greatly In Affected Girls

• Is a Neurodevelopment and/or Neuromaintenance Condition• Rett Syndrome Is NOT a neurodegenerative disease

• Age of Onset Varies, But is Typically Between 12-18 Months

Relatively Normal Developmental Progression Prior to Symptomatic Onset

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Physical Features of Rett Syndrome

• No Obvious Signs of Neuronal Loss, However, Diminished “Higher Order” Dendritic Complexity Is Observed

• Brain Mass and Size Tend To Be Smaller (microcephaly)

• Intractable Seizures are Often Observed

• Breathing Dysrhythmia - Extreme Episodic Apneas

• Highly Impaired Locomotive Skills

• Poor Catacholaminergic Regulation– Heightened Stress Responsiveness

– Sympathetic Nervous System Overload

• Many Sensory Features Are Maintained– Visual Recognition

– Some if not All Auditory Recognition

– Some if not Considerable Memory

• Poor Prognosis and No Current Particularly Effective Treatments3

Several Mouse Models Of Rett Syndrome Have Been Developed

These Mice Lack Functional Mecp2, and Recapitulate Many Of The Cardinal Impairments Seen In Rett Syndrome Patients

Bird Mouse Jaenisch Mouse Zoghbi MeCP2-308 Mouse

For specific details, see “Katz et al., (2013) Dis Model Mech 5:733-45” 4

Behavioral Deficits In Mecp2-Deficient Mice That Have Good Face Validity To Rett

Syndrome

1. Diminished General Activity And Ambulation

2. Balance Impairment

3. Diminished Body Weight and Growth Rate

4. High Risk for Sudden and Unexpected Death

5. Hyper-Excitable Networks In Brain

6. Poor Peripheral Thermoregulation

7. Respiratory Apneas and Breathing Irregularities

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It Was Initially Thought That Rett Syndrome Might Be An Irremediable Condition. A Landmark Study

In 2007 Suggests This May Not Be The Case

Guy et al., (2007) Science 315:1143-1147

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This Reactivation Of MeCP2 Was Accomplished Using Specifically Engineered Mutant Mice

Neo-StopMeCP2 Exon 2 MeCP2 Exon 3 MeCP2 Exon 4

Lox

-P

Lox

-P

Cre RecombinaseEstrogen ReceptorROSA26 Promoter

However, MeCP2 Is Reactivated In These Mice When The “Stop-Flox” Cassette Is Removed By Cre Recombinase

The “Floxed” Neo-Stop Cassette Disrupts the Normal Structure of the Mecp2 Gene. These Mutants Express Almost No MeCP2 Protein

A Tamoxifen-Activated Form of Cre Recombinase Was Introduced by Transgenic Inter-Crossing. Administration of Tamoxifen Facilitates Nuclear Entry of Cre, Excision of the Floxed Locus, and Allows for the Reactivation Of Mecp2.

We Used This Strategy To Test The Rescue Potential of Other Rett Phenotypes In Highly Symptomatic Mice

***Our Study Was Concomitantly Conducted With Female Mutant Mice

We Were Able To Reproduce The Original Finding, And Identify Additional Deficits That Can Be Rescued In Mice

Phenotypic Severity Score Average: 9.5 Phenotypic Severity Score Average: 5.5

The Average Pre vs Post Improvement Is 3.3 On This Severity Scale (n=8)

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The Lifespan Of Male and Female “Rescue” Mice Was Significantly Lengthened

Male “Non-Rescue”

Male “Rescue”

Female “Non-Rescue”

Female “Rescue”

10Lang et al., (2014) Hum Mol Genet 23:303-18

Open Field Exploratory Behavior Was Improved In Both Male And Female “Rescue” Mice

Lang et al., (2014) Hum Mol Genet 23:303-18

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Cortical Epileptiform Discharge Activity Was Reduced In Both Male And Female “Rescue” Mice

Male “Non-Rescue”

Male “Rescue”

Female “Non-Rescue”

Female “Rescue”

Lang et al., (2014) Hum Mol Genet 23:303-18

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Daily Thermoregulatory Patterns Were Improved In “Rescue” Mice

Female Wild-Type Female “Non-Rescue” Female “Rescue”

Lang et al., (2014) Hum Mol Genet 23:303-18

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Female Pre-Rescue

Female Rescue

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Female Pre-Rescue

Female Rescue

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Given Phenotypic Improvement Is Possible, We Are Now Testing More Clinically-Relevant Therapeutic

Strategies In Mecp2-Deficient Mice

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• Drug-Based Phenotypic Rescue Investigations– Nonsense Mutation “Read-Through” Drugs

– Cholinesterase Inhibitors

– Strategies To Manage Oxidative Stress

– Strategies To Improve Microtubule Trafficking Efficiency

• Gene Replacement / Gene Correction Rescue Studies – Gene Therapy Mediated Reintroduction of Functional MeCP2 Into Brain

– Novel Genomic Recombination / Correction Strategies

These Are Rationale Based, And Include:

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Strategies To Improve Microtubule-Dependent Transport Rates

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Altered Intracellular Transport Could Contribute To These MeCP2-Deficient Cellular Phenotypes

Review Of Microtubule-Based Transport

Examples Of Transported Cargos:

1.Mitochondria2.Synaptic Vesicle Proteins3.Ribonucleoprotein Complexes4.Neurotransmitter Receptors5.Amyloid Precursor Protein6.BDNF and Other Trophins

In Addition To Transporting Cargos To Synaptic Domains, Microtubule-Based Transport Also Removes Spent Mitochondria, Proteins, etc., From

Distal Synaptic Domains

Cargo Transport By Motor Protein Is More Efficient Along Microtubules When Tubulin Is Acetylated

The Transport Of Cargo By The Motor Proteins Kinesin and Dynein Is More Efficient Along Microtubules When Tubulin Is Acetylated

Histone Deacetylase 6 (HDAC6) Is One Of The Primary Regulators Of Acetyl-Tubulin Levels In Neurons

Modified From Leroux, PNAS 2010;107:21238-21239

HDAC6 Is The “Unusual” Member Of The HDAC Family. It Resides Primarily Outside The Nucleus, And Regulates The Acetyl-State Of Several

Cytosolic and Cytoskeletal Proteins Including Tubulin

HDAC6 Levels Are Significantly Increased In The MeCP2-Deficient Mouse Cortex

HDAC6 Western blot of cortical homogenates from wild-type and

symptomatic male MeCP2-deficient mice. -actin serves as a

loading control.

The average induction of HDAC6 in the MeCP2-deficient Mouse Cortex was 530 +/- 70% above

wild-type.

The Elevated HDAC6 Correlated With A Decreased Level Of Acetyl-Tubulin In The MeCP2-Deficient Cortex

Western blots probed with Tubulin antibodies (Left Blot) Acetyl-tubulin (Lys-40) antibodies (Right Blot). GAPDH immunoreactivity is shown as a load control. The histogram shows the cumulative densitometric results for the ratio of tubulin to acetyl-tubulin for wild-type and MeCP2-null mice.

The Decreased Levels Of Acetyl-Tubulin In The MeCP2-Deficient Mouse Brain Are Consistent With The Observed

Impairment Of Axonal and Dendritic Transport, And Could Also Play A Role In Additional Deficits In Synaptic Function

Seen In The MeCP2-Deficient Brain.

This Raises The Possibility That An HDAC6 Inhibitor Might Improve The Rett-Like Phenotypes Of MeCP2-Deficient Mice

The First Question Was To Determine Whether Or Not HDAC6 Inhibition Increased Acetyl-Tubulin Levels

Western blots showing total tubulin (Left Blot) or acetyl-tubulin (Right Blot) in cortical homogenates from wild-type mice, male MeCP2-null mice, and from male MeCP2-null mice that received HDAC6 inhibitor for 11-22

days. Below each blot is its corresponding GAPDH loading control.

We Then Tested Whether Administering The HDAC6 Inhibitor To MeCP2-Deficient Mice Would Improve

Their Neural Circuit and/or Behavioral Deficits

MeCP2-Deficient Mice Display Spontaneous Epileptiform Discharges In Cortical Regions

The EEG Discharges Are Typically 6-10 Hz, Have Amplitudes 1.5-2X Baseline, and Are Typically Last Between 0.5-2 Seconds in Females, But

Can Last More Than 60 Seconds in Males

Illustration of Discharge Activity Seen In The

Somatosensory Cortex of and Male and Female

MeCP2-Deficient Mice

In Addition To Discharges, Female MeCP2-Deficient Mice Display Deficits in Balance Behavior

Female Mecp2-Deficient Mice Perform Poorly On the Accelerating Rotarod

***

Design of Translational Target Validation Study

Subjects Were Female MeCP2-Deficient MiceStudy Was Done Using Wireless Telemetric Recording Transmitters.

Implant Age Was 9-10 Months.

Somatosensory Cortex Was Site Of Electrode PlacementRecording Sessions Were Done For 24 Hours Consecutively

HDAC6-Inhibitor Dramatically Attenuates the Epileptiform Discharge Activity of MeCP2-Deficient Mice

The Incidence Of Spontaneous Epileptiform Discharges Dropped By More Than 70% In MeCP2-Deficient Mice Treated for 28 Days With HDAC6 Inhibitor.

N=9 MeCP2-Deficient

** P<0.01, Paired t-Test

And The Effect Was Surprisingly Long-Lasting

Reassessment of Some of the Same Mice 30 Days After Their Final Injection With HDAC6 Inhibitor Showed Epileptiform Discharge Activity Remained

Significantly Below Baseline (Pre-Injection) Levels

N=6 MeCP2-Deficient

** P<0.01, Paired t-Test

Further, The HDAC6 Inhibitor Improved The Performance of the MeCP2-Deficient Mice on the Accelerating Rotorod

Female MeCP2-Deficient Mice Treated 28 Days with HDAC6 Inhibitor Display Significantly Improved Performance on the Accelerating Rotorod. The Resulting

Performance Level was Equivalent To Wild-Type Mice.

MeCP2-Deficient Mice Receiving Vehicle Show No Improvement In Rotorod Performance Within the 28 Day Testing Interval

HDAC6 Inhibitor

Collectively, These Results Provide Evidence That Inhibiting the HDAC6 System Improves At Least Some Of The Phenotypic Impairments Of Adult MeCP2-Deficient Mice, And They Suggest A Novel Mechanism Through

Which Deficits In Synaptic Function Could Arise

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Summary

• We Have Identified Specific Deficits In Mecp2-Deficient Mice That Recapitulate Salient Co-Morbidities Seen In Rett Syndrome Patients– Ambulatory Deficits

– Impaired Thermoregulation (Autonomic Dysregulation)

– Spontaneous Epileptiform Discharge Activity

– Altered Social Behavior

– Heightened Anxiety-Like Behavior

• We Have Established That Each Of These Rett-Like Phenotypes Can Be Reversed Using Molecular Genetic Approaches

• We Have Implicated A Novel System In Rett Syndrome Pathogenesis

We Are Now Testing Drug and Genetic Strategies That Have More Direct Clinical Applicability In These Mecp2-Deficient Mice