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Joyoti Dey, MPH Advisor: Dr. James M. OlsonFred Hutchinson Cancer Research Center &
University of Washington Graduate Program
Investigating two Oncogenic Mutations in Smoothened with
vastly different effects on Cerebellar Development
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• Background
- Cerebellar Development & Neuronal Circuitry - Role of Shh in Cerebellar Development- Deregulated Shh signaling: SmoA1 & SmoA2 - Medulloblastoma mouse models
• Characterization of the SmoA2 mouse model
• Future Directions
Outline
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• Represents 10% of total brain, yet > 50% of total neurons
• Involved in fine tuning motor movements and balance
• One of the first brain structures to begin differentiation, one of the last to attain maturity
The Cerebellum
www.nih.nlm.org
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Cerebellar Neuronal Circuitry
Wang et al. Nature Rev. Neurosci .(2001)
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Embryonic Phase of Cerebellar Development• Neurons arise from two germinal matrices -
Ventricular zone & Rhombic Lip
Mouse E13.5 Mouse E17.5
EGL: External Granule Layer GNP: Granule Neuron
Precursor
Ref: Schuller et al. Cancer Cell (2008)
Ref: Hevner R, Reelin Glycoprotein (2008)
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Postnatal Cerebellar Development in Mouse
Ref: RIKEN Brain Institute
Molecular Layer
Purkinje
LayerInner Granule Layer
Cellu
lar
Even
tsTim
e S
ch
ed
ule
GNP
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Postnatal Cerebellar Development in Mouse
P5 P14 P28
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Sonic Hedgehog in Cerebellar Development
Ref: Altaba et al. Nature Rev. Neuroscience (2002)
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Mammalian Sonic Hedgehog Pathway
Without Shh
With Shh
Ref: Rubin et al. Nature Reviews: Drug Discovery (2006)
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• Basal cell carcinoma, medulloblastoma, rhabdomyosarcoma
- Mutations activating proto-oncogene Smoothened
- Mutations inactivating tumor suppressor Patched
• Medulloblastoma: most common pediatric brain tumor
- Increased activity of Shh pathway due to mutations in 20% cases
- Tumors thought to arise from granule neuron precursors
Ref: Children’s Hospital, Seattle
Shh Pathway Aberrations & Cancer
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Oncogenic Smoothened Mutations
• SmoA1 (S537N) : Human Sporadic Basal Cell Carcinoma
• SmoA2 (W539L): Human Medulloblastoma
Ref: Taipale et al.Nature (2000)
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Mouse Models with Oncogenic Smoothened MutationsND2 1kb SmoA1* His6/Myc3
P5 P14 P28 Symptomatic Tumor
Hatton et al. Cancer Research (2008)
Tumors serially transplantable
Leptomeningeal Spread
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Mouse Models with Oncogenic Smoothened Mutations
ND2 1kb SmoA1* His6/Myc3
P5 P14 P28 Symptomatic Tumor
ND2 1kb SmoA2* HA
P14P5 P28 Symptomatic Tumor
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Developmental Defects in SmoA2 Mice
• Disrupted laminar organization
• Disrupted foliation
WT
SmoA2
P5
P5
adult
adult
• Early hyperproliferation
• Ectopic clusters of Purkinje cells
Wildtype
SmoA2
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Neurobehavioral Assessment
• Spontaneous Activity• Locomotor Activity• Rotarod Test• Gait Assessment
• Body Position• Tail position• Posture
Neurobehavioral Physical Phenotype Assessment
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Assessment of Ataxia: “Footprinting” Assay
No detectable Ataxia in SmoA2 mice
SmoA2
Huntington’s Disease.
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Human Cerebellar Dysplasias: Correlation with PNETs
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Figure: Jeffrey A. Golden & Brian N. Harding. (2004). Developmental Neuropathology
Common in apparently normal infant brains
Rorke et al (1968) showed histologically, clusters of mature neurons in white matterfocal or perivascular GNPs, heterotaxias heterotopias
Possible Cause : Inappropriate progenitor cell death and/or neuronal & glial migration
Contributory Role in Medulloblastoma: Dysplasias might be targets for neoplastic transformation (Yachnis et al.(1994), Jay V.(1996)
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Bilateral, vertical, orientated folia Enlarged fourth ventricle.
Human Cerebellar Dysplasias
Right cerebellar hypertrophy &
vertical folia
Soto-Ares et al. Am J Neuroradiol (2000)
Abnormal Arborization of White Matter Heterotopia
Normal
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SmoA2 & Human CBL Dysplasias: Shared Features
Dysplasia & Contiguous Tumor
Heterotopia &
Heterotaxia
Vertical Foliation
SmoA2
Wildtype
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Hyperproliferation in SmoA2 Cerebella
P5 P14 P28
Wildtype
SmoA2
Cyclin
D1
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Mouse postnatal timepoints
Rel
ativ
e m
RN
A e
xpre
ssio
n
SmoA2
SmoA2
Expression of Shh Canonical Targets in SmoA2 & SmoA1 cerebella, compared to WT controls
SmoA1
SmoA2 >10 >17
N = 3 WT & 3 Mutant/ timepoint
P5 P14 P21 2m
P5 P14 P21 2m
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Purkinje Cell Alignment in SmoA2 Cerebella
P5 P14 P28
Wildtype
SmoA2
Calb
ind
in
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Radial Glia Alignment in SmoA2 Cerebella
Wildtype
10X
S100
SmoA2
20X
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Wildtype SmoA2
Nov 6, 2008
Abnormal Embryonic Development of SmoA2 Cerebella
Ki67
E15.5 dpc
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Conclusions
• Significant difference in cerebellar development due to SmoA1 and SmoA2 oncogenic mutations
• Severe defects in neuronal and glial migration yet no neurobehaviourial abnormalities in the SmoA2 mice
• More robust and earlier activation of Shh pathway due to SmoA2 mutation
• Several shared key features with Human Cerebellar Dyslasias
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Future Directions
A connection between Human Cerebellar Dysplasias and the SmoA2 phenotype ?
Human SmoA2
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Future Directions
Determine the molecular basis of abnormal neuronal/glial migration
? ?
?
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Future DirectionsND2 1kb SmoA1* His6/Myc3
P5 P14 P28 Symptomatic Tumor
ND2 1kb SmoA2* HA
P14P5 P28 Symptomatic Tumor
Investigate Molecular Differences between the SmoA1 and SmoA2 variants
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Acknowledgements
Dr. Jim Olson & Our Team !
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Acknowledgements
FHCRC Experimental Histopathology Core:
Dr. Julie Randolph Habecker
Dr. Sue Knoblaugh
FHCRC Scientific Imaging Core:
Dr. Julio Vasquez
FHCRC Animal Health Resources
Dr. Jon Cooper
Tapscott Lab: Laurie Snider
Vasioukhin Lab: Dr. Olga Klezovitch