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  • My talk today will focus on presenting to you how two oncogenic mutations in the same gene, Smoothened receptor has widely varying effects on CBL development, and both mutations cause cerebellar tumor. Thank: Bear Necessities Pediatric Cancer Foundation,Dr. Kathleen Casey and Drs. Bento Soares and Stewart Goldman of Children's Memorial Research Center,
  • several higher cognitive functions such as language, memory, executive functions, visuospatial skills, thought modulation and emotional regulation of behavior.
  • GNPs derived from uRL migrate tangentially through the rhombic lip migratory stream and the Purkinje cells migrate from VZ along radial glial fibers towards the IZ.Later at around 17.5, the Purkikje cell plate is formed and the RLS has been replaced by the External Granule Layer. Between 9 and 13 postconceptional weeks, the Purkinje cells of the cerebellar cortex and the neurons of the deep cerebellar nuclei migrate radially outward from this germinal matrix. In contrast, the neurons of the granular layer of the cerebellar cortex migrate tangentially from the germinal zone of the rhombic lips, over the cerebellar surface to form a transient external ( Ext. ) granular layer, which acts as a secondary germinal matrix. The external granular layer forms between the 10th and 11th postconceptional weeks and persists until approximately 15 months postnatal.During the 6-8 months postnatally, the EGL subsides progressively and the cerebellar lamell The cells in the external granular layer proliferate, and the granule cell neuroblasts begin to migrate inward between clusters of homophilic Purkinje cells with the presumed aid of radial glial (Bergman) fibers, forming the internal ( Int. ) granular layer (reprinted with permission from Lippincott Williams & Wilkins [27])
  • Postnatally, GNPs in the EGL undergo massive mitotic proliferation, become postmitotic and migrate inwards to form the iEGL. The last stage is inward migration along radial glial processes to the Inner Granule Layer, which is completed by P21. In humans, this timing would correlate to 6-8 months of postnatal life.
  • The SHH secreted by PC plays a key role in cerebellar development. It controls the postnatal mitotic expansion of the GNP pool and the extent of foliation is also spatially/temporally regulated by Shh.
  • In order to study the effect of the Smoothened mutations in CBL, our lab developed the SmoA1 transgenic mouse model which has the SmoA1 transgene driven by the promoter of NeuroD - which is granule neuron specific transcription factor. As you can see here, the CBL development is normal except for a slight thickening of the EGL at Day 14. 50% of these mice developed tumor which usually developed from a region near the fourth ventricle.In the The SmoA1 homozygous model which is Clinical Incidence of 85% by 5 months. Furthermore through histological analysis 94% by 2 months. This early & high incidence makes it a valuable tool for clinical trials. These tumors are serially transplantable and also show leptomeningeal spread which is a hallmark of human medulloblastoma. Clinical Incidence: Ataxia, protruded skill, tilted head/hunched posture indicative of hydrocephalus caused by ventricular obstruction or direct compression of nerves by tumor.
  • In parallel, we also developed the SmoA2 mouse model and have recently begun its characterization. The CBL pathology is completely different, showing hyperproliferation at very early time point, which increases with development and then there seems to be some attempt to organize yet the mature CBL remain completely scrambled. 50% of these mice go on to develop tumor by 4-5 months of age, but 100% have this massively disorganized cerebellum.
  • On a closer look, you can see how compared to the wildtype, the foliation pattern is completely disrupted, there is no defined cell layers but evidence of hyperproliferation and the Purkinje cells, which as I mentioned earlier, are the principal output of the CBL, seems to be lacking the normal monolayer arrangement.
  • Seeing this phenotype, our immediate question was how do they differ from WT mice behaviorally. Therefore, We did various conventional neurobehavioural assessments, by looking at their normal spontaneous activity, exploratory behavior, rotarod tests, and also physical examination. Whats fascinating, is that these mice are indistinguishable from normal unless they develop tumors. Even our team of mouse technicians who have run over 45 clinical trials and have 40 years of cumulative experience in mouse work, couldn’t tell apart a SmoA2 mouse from a wildtype.
  • We also conducted what is known as a foot printing assay as a measure of possible ataxia in these mice. The four paws were painted with four different colors and the mice were made to walk on a strip of white paper. As you see in this bottom panel, this is what a true ataxic footprint looks like. However, there was no significant difference in the SmoA2 and WT footprints were
  • While we were beginning to uncover more about the SmoA2 phenotype, we came across a similar disorganized cerebellar phenotpe in humans collectively known cerebellar dysplasias first described in the 1960s. In a study by Dr. Lucy Rorke, about 85% of apparently normal infant brains showed dysplasias. They were described histologically as clusters of neurons in white matter, focal or perivascular GNP aggregates that were mitotically active, disorganized mixed cell collections or heterotaxias and heterotopias that is cell arrangements that look apparently normal but are mislocalized. Whats interesting is that these dysplasias are thought to be precursors to development of MB,in the literature. Because the SmoA2 phenotype has striking similarites with human dysplasia, understanding the basis for the disorganized CBL in SmoA2 may shed light the on the link between cerebellar development and MB. http://webhome.idirect.com/~brainology/brainology/devpath_12_cerebellar.html
  • An MR imaging study identified cerebellar dysplasias in patients which included features like vertical foliation, abnormal arborization, heterotopia and hypertrophy.
  • We then went back to further analyse the SMoA2 histopathology and we find several shared features. Here’s an example of a scrambled brain with a developing tumor where as CBL has been completely engulfed by tumor with some normal looking tissue but scrambled. We also see heterotopias where the laminar arrangment is preserved but in ectopic locations. There are also mixed groups of neurons and glia around blood vesslels, And similar to what we saw in the MRI study, in this horizontal section, we see evidence of vertical foliation Given the similarity between the SmoA2 and human dysplasia, we further investigated the SmoA2 phenotype in 2 ways : Proliferative status of the dysplasias Status of the Shh pathway which is controls GNP proliferation . Neuronal & glial mis migration 3.
  • To look at the mitotic status of these dysplasias, we looked at the expression of Cyclin D1 in both WT and SmoA2 across three postnatal time points. At P28, there is no diference in cyclin D1 expression in SmoA2 and WT. This is consistent with what I showed you earlier, where in the SmoA2 CBL, there is some attempt to organize as it attains maturity.
  • This led us to further investigate the status of the Shh pathway in the SmoA2 mice and compare with SmoA1. We looked at 4 post natal time points,the bars represent the relative expression of each of the targets, normalized to the wild type controls for each time point. Compared to SmoA1, in the SmoA2 mice, majority of the targets are many fold elevated. But two interesting observations here are in the SmoA2 mice, at P5 where we see the massive hyperproliferation, the difference in the target gene expression is not that big. This suggests involvment of other non canonical targets of the Shh pathway. Also, at two months, consistent with the attempt of the SmoA2 cbl to organize & foliate, the target genes also go back to normal values.
  • In order to look at the nature and extent of neuronal mismigration if any, we looked at the localization Purkinje cells with Calbindin IHC. These neurons, as I mentioned previously, secretesShh, and play a critical role in Granule neuron proliferation, migration and foliation. As you can see here, starting as early as P5, this P cells are not organizied in the characteristic monolayer as seen in the WT control. With further development, the disarray persists and there seems to be no clear boundary between grey and white matter. Its therefore intriguing, that given this status of the P cells, which are the main output of the cerebellum that connects to the cortex, this mouse do not exhibit any discernible neurobehavioural abnormality.
  • Radial glial cells play a crucial role in neuronal migration. A study looking at various markers in human dysplastic cerebella, showed a predominance of glial cells. It is thought that entangled glial fibers might interfere with neuronal migration. We therefore looked at the organization of Radial glia by S100 staining in P14 CBL. As opposed to the distinct organization in the WT, in the SmoA2 CBL, we see this disorganized tangles of glial fibers. In this higher magnification, we can see the granule neuron precursors migrating along the processes, and in the heterotopic regions of the SmoA2 cerebella, this is preserved.
  • Since the stage for proliferation and migration are set in embryonic development & we observe a cerebellar pathology as early as P5 in SmoA2, we are now looking at various prenatal time points to track where the development starts deviating from normal. We started by looking at the prolferative status of CBL, & we observe a hyperproliferated EGL. We will now look at specific migratory pathways. .
  • It has been hypothesized that HCD play a contributory role in MB development. We hope to uncover links at a molecular level between cerebellar development, dysplasias and medulloblastoma.
  • As I had outlined the major neuronal migration pathways at various stages in early development which lead to the final architecture of the CBL. Each of these migration pathways are intricately regulated by various neuronal migration genes. We plan to look at neuronal and glial migration markers, across various prenatal and post natal time points spanning the entire developmental period, to narrow in on what pathway or pathways might be disrupted due to the SMoA2 phenotype.
  • We will be comparing SmoA1 and SmoA2 cerebella, using both proteomic tools, and microarray data to see what genes might be differentially expressed. We think that these two mutations lead to differences in proteomic profile and if so, we will then carry out pulldown assays with these oncogenic proteins to identify interacting proteins which could be potential drug targets.

*αí*ß Presentation Transcript

  • 1. Joyoti Dey, MPH Advisor: Dr. James M. Olson Fred Hutchinson Cancer Research Center & University of Washington Graduate Program Investigating two Oncogenic Mutations in Smoothened with vastly different effects on Cerebellar Development
  • 2.
    • 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
  • 3.
    • 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
  • 4. Cerebellar Neuronal Circuitry Wang et al. Nature Rev. Neurosci .(2001)
  • 5. 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)
  • 6. Postnatal Cerebellar Development in Mouse Ref: RIKEN Brain Institute Molecular Layer Purkinje Layer Inner Granule Layer Cellular Events Time Schedule GNP
  • 7. Postnatal Cerebellar Development in Mouse P5 P14 P28
  • 8. Sonic Hedgehog in Cerebellar Development Ref: Altaba et al. Nature Rev. Neuroscience (2002)
  • 9. Mammalian Sonic Hedgehog Pathway Ref: Rubin et al. Nature Reviews: Drug Discovery (2006) Without Shh With Shh
  • 10.
    • 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
  • 11. Oncogenic Smoothened Mutations
    • SmoA1 (S537N) : Human Sporadic Basal Cell Carcinoma
    • SmoA2 (W539L): Human Medulloblastoma
    Ref: Taipale et al.Nature (2000)
  • 12. Mouse Models with Oncogenic Smoothened Mutations Leptomeningeal Spread ND2 1kb SmoA1* His6/Myc3 P5 P14 P28 Symptomatic Tumor Hatton et al. Cancer Research (2008) Tumors serially transplantable
  • 13. Mouse Models with Oncogenic Smoothened Mutations ND2 1kb SmoA1* His6/Myc3 P5 P14 P28 Symptomatic Tumor ND2 1kb SmoA2* HA P14 P5 P28 Symptomatic Tumor
  • 14. Developmental Defects in SmoA2 Mice
    • Disrupted laminar organization
    • Disrupted foliation
    WT SmoA2
    • Early hyperproliferation
    • Ectopic clusters of Purkinje cells
    Wildtype SmoA2 P5 P5 adult adult
  • 15.
    • Spontaneous Activity
    • Locomotor Activity
    • Rotarod Test
    • Gait Assessment
    • Body Position
    • Tail position
    • Posture
    Neurobehavioral Assessment Neurobehavioral Physical Phenotype Assessment
  • 16. Assessment of Ataxia: “Footprinting” Assay No detectable Ataxia in SmoA2 mice SmoA2 Huntington’s Disease.
  • 17. Human Cerebellar Dysplasias: Correlation with PNETs 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 matter focal 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)
  • 18. 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
  • 19. SmoA2 & Human CBL Dysplasias: Shared Features Dysplasia & Contiguous Tumor Heterotopia & Heterotaxia Vertical Foliation SmoA2 Wildtype
  • 20. Hyperproliferation in SmoA2 Cerebella P5 P14 P28 Wildtype SmoA2 Cyclin D1
  • 21. Mouse postnatal timepoints Relative mRNA expression 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
  • 22. Purkinje Cell Alignment in SmoA2 Cerebella Calbindin P5 P14 P28 Wildtype SmoA2
  • 23. Radial Glia Alignment in SmoA2 Cerebella Wildtype 10X S100 SmoA2 20X
  • 24. Wildtype SmoA2 Nov 6, 2008 Abnormal Embryonic Development of SmoA2 Cerebella Ki67 E15.5 dpc
  • 25. 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
  • 26. Future Directions A connection between Human Cerebellar Dysplasias and the SmoA2 phenotype ? Human SmoA2
  • 27. Future Directions Determine the molecular basis of abnormal neuronal/glial migration ? ? ?
  • 28. Future Directions ND2 1kb SmoA1* His6/Myc3 Symptomatic Tumor ND2 1kb SmoA2* HA Investigate Molecular Differences between the SmoA1 and SmoA2 variants P5 P14 P28 P14 P5 P28 Symptomatic Tumor
  • 29. Acknowledgements Dr. Jim Olson & Our Team !
  • 30. 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