Dr. James Eubanks' Presentation

advertisement
Translational Strategies For Rett Syndrome: From
Experimental Mice To Treating Girls and Women
James Eubanks
Genetics and Development Division
Toronto Western Research Institute
University Health Network
Department of Surgery
Institute of Medical Sciences
University of Toronto
Krembil Neuroscience Program
University of Toronto Epilepsy Research Program
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
1
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 Treatments
2
Several Mouse Models Of Rett Syndrome
Have Been Developed
Bird Mouse
Jaenisch Mouse
Zoghbi MeCP2-308 Mouse
These Mice Lack Functional Mecp2, and Recapitulate Many
Of The Cardinal Impairments Seen In Rett Syndrome Patients
For specific details, see “Katz et al., (2013) Dis Model Mech 5:733-45”
3
Behavioral Deficits In Mecp2-Deficient Mice
That Have Good Face Validity To Rett Syndrome
1.
2.
3.
4.
5.
6.
7.
Diminished General Activity And Ambulation
Balance Impairment
Diminished Body Weight and Growth Rate
High Risk for Sudden and Unexpected Death
Hyper-Excitable Networks In Brain
Poor Peripheral Thermoregulation
Respiratory Apneas and Breathing Irregularities
4
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
5
Lox-P
Lox-P
This Reactivation Of MeCP2 Was Accomplished Using
Specifically Engineered Mutant Mice
Neo-Stop
MeCP2 Exon 2
MeCP2 Exon 3
MeCP2 Exon 4
The “Floxed” Neo-Stop Cassette Disrupts the Normal Structure of the
Mecp2 Gene. These Mutants Express Almost No MeCP2 Protein
However, MeCP2 Is Reactivated In These Mice When The “StopFlox” Cassette Is Removed By Cre Recombinase
ROSA26 Promoter
Estrogen Receptor
Cre Recombinase
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.
6
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)
8
The Lifespan Of Male and Female “Rescue” Mice Was
Significantly Lengthened
Female “Rescue”
Female “Non-Rescue”
Male “Rescue”
Male “Non-Rescue”
9
Lang et al., (2014) Hum Mol Genet 23:303-18
Open Field Exploratory Behavior Was Improved In
Both Male And Female “Rescue” Mice
10
Lang et al., (2014) Hum Mol Genet 23:303-18
Cortical Epileptiform Discharge Activity Was Reduced
In Both Male And Female “Rescue” Mice
Male “Non-Rescue”
Male “Rescue”
Female “Non-Rescue”
Female “Rescue”
11
Lang et al., (2014) Hum Mol Genet 23:303-18
Daily Thermoregulatory Patterns Were Improved In
“Rescue” Mice
*
38
*
37
36
Female “Non-Rescue”
*
35
34
Average Number of Daily
Temperature Cycles
Average Daily
Temperature (oC)
Female Wild-Type
12
*
Female “Rescue”
*
8
4
0
12
Lang et al., (2014) Hum Mol Genet 23:303-18
Given Phenotypic Improvement Is Possible, We Are
Now Testing More Clinically-Relevant Therapeutic
Strategies In Mecp2-Deficient Mice
13
These Are Rationale Based, And Include:
• 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
14
Strategies To Improve Microtubule-Dependent
Transport Rates
15
Altered Intracellular Transport Could Contribute To
These MeCP2-Deficient Cellular Phenotypes
Review Of Microtubule-Based Transport
Examples Of Transported Cargos:
1.
2.
3.
4.
5.
6.
Mitochondria
Synaptic Vesicle Proteins
Ribonucleoprotein Complexes
Neurotransmitter Receptors
Amyloid Precursor Protein
BDNF and Other Trophins
In Addition To Transporting Cargos To Synaptic Domains, MicrotubuleBased 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 MeCP2deficient mice. b-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 MeCP2Deficient 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
Illustration of Discharge
Activity Seen In The
Somatosensory Cortex of
and Male and Female
MeCP2-Deficient Mice
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
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 Mice
Study Was Done Using Wireless Telemetric Recording Transmitters.
Implant Age Was 9-10 Months.
Somatosensory Cortex Was Site Of Electrode Placement
Recording Sessions Were Done For 24 Hours Consecutively
HDAC6-Inhibitor Dramatically Attenuates the Epileptiform
Discharge Activity of MeCP2-Deficient Mice
N=9 MeCP2-Deficient
** P<0.01, Paired t-Test
The Incidence Of Spontaneous Epileptiform Discharges Dropped By More Than
70% In MeCP2-Deficient Mice Treated for 28 Days With HDAC6 Inhibitor.
And The Effect Was Surprisingly Long-Lasting
N=6 MeCP2-Deficient
** P<0.01, Paired t-Test
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
Further, The HDAC6 Inhibitor Improved The Performance
of the MeCP2-Deficient Mice on the Accelerating Rotorod
HDAC6
MeCP2-Deficient Mice Receiving Vehicle Show No Improvement
InInhibitor
Rotorod
Performance Within the 28 Day Testing Interval
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.
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
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
32
Download