Fragile X mental retardation protein Fmrp expression in zebra finch brain

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Fragile X mental retardation protein (Fmrp)
expression in zebra finch brain

• Stephanie Ceman, PhD
• Dept. of Cell and Developmental Biology
• Neuroscience Program and NeuroTech Group

www.joelertola.com/ tutorials/brain/gifs/Brain.gif
commons.wikimedia.org
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CLINICAL FEATURES of Fragile X syndrome

• Most common form of inherited mental retardation
• 1/4000 males and 1/8000 females
• 1/260 women are carriers
• Mental impairment (I.Q. 20-60)
• Some autistic-like behavior poor eye contact,
  hand-flapping, anxiety
• Severe delays in speech and language skills
• Subtle facial features
• Head circumference gt 50tile
• Long face - prominent forehead jaw, large ears
  (gt2 s.d. for age)
• Features of a mild connective tissue disorder

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Fragile X Mental Retardation gene 1 FMR1
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Model for FMRP function in neurons
AXON
nucleus
Beckman Researchers working on fragile X
syndrome William Greenough Roberto Galvez Lee
Cox Work done in collaboration with David Clayton
DENDRITE
Spine maturation synaptic plasticity
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Rationale for studying Fmrp in the song bird
zebra finch
Fragile X individuals have pronounced speech and
language deficits Roberts, et. al J. Speech Lang
Hear Res, 2005 Kau, et al. Microscopy Research
and Technique, 2002 Roberts, et al. . Am J Ment
Retard., 2007 Bailey, et al. Am J Ment Retard.,
1998 Dykens, et al. J. Autism Dev. Disord.,
1993. Zebra finch is one of a handful of
vertebrates, including humans, that learns to
vocally communicate. Birdsong learning is
considered the closest animal equivalent to human
speech acquisition Bolhuis, Gahr, Nat Rev
Neurosci, 2006 Brainard, Doupe,. Nature,
2002 Doupe, Kuhl, Ann. Rev Neuroscience,
1999 Song birds, like humans, learn to vocalize
during a critical period during development
Wilbrecht Nottebohm,. Ment. Retard. Dev.
Disabil. Res. Rev., 2003 The neural circuits
that control vocal learning in zebra finches have
clear relationships to the pathways in the human
brain . Brainard and Doupe.Nature. 2002
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Ho Fmrp plays an essential role in vocal
learning

• Clone and analyze zebra finch Fmr1
• Taeniopygia guttata TguFmr1 sequence
• Develop an Fmrp-specific antibody
• Examine where Fmrp is expressed in brain by
  immuno-histochemistry
• 4. Determine whether Fmrp expression changes
  during development and/or during learning and
  memory

www.finchworld.com
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Claudia Winograd, MD/PhD student
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Alignment of Human, Zebra Finch, and Chicken
Fm rps

• H MEELVVEVRGSNGAFYKAFVKDVHEDSITVAFENNWQPDRQIPFHDV
  R
• F MEELVVEVRGSNGAFYKAFVKDVHEDSITVSFENNWQPERQIPFHDV
  R
• C MEELVVEVRGSNGAFYKAFVKDVHEDSITVSFENNWQPERQIPFHDV
  R
• H FPPPVGYNKDINESDEVEVYSRANEKEPCCWWLAKVRMIKGEFYVIE
  Y
• F FPPPAGYNKDINESDEVEVYSRANEKEPCCWWLAKVRMIKGEFYVIE
  Y
• C FPPPAGYNKDINESDEVEVYSRANEKEPCCWWLAKVRMIKGEFYVIE
  Y
• H AACDATYNEIVTIERLRSVNPNKPATKDTFHKIKLDVPEDLRQMCAK
  E
• F AACDATYNEIVTIERLRSVNPNKPATKDTFHKIKLAVPEDLRQMCAK
  E
• C AACDATYNEIVTIERLRSVNPNKPATKDTPHKIKLAVPEDLRQMCAK
  G
• H AAHKDFKKAVGAFSVTYDPENYQLVILSINEVTSKRAHMLIDMHFRS
  L
• F SAHKDFKKAVGAFSVTYDSENHQLIILSINDVTTKRANMLIDMHFRS
  L
• C SAHKDFKKAVGAFSVTYDSENHQLIILSINDVTTKRANMLIDMHFRS
  L
• H RTKLSLIMRNEEASKQLESSRQLASRFHEQFIVREDLMGLAIGTHGA
  N

• H GESQKPELKAWQGMVPFVFVGTKDSIANATVLLDYHLNYLKEVDQLR
  LER
• F ------------GMVPFVFVGTKDSITNATVLLDYHLNYLKEVDQLR
  LER
• C ------------GMVPFVFVGTKDSITNATVLLDYHLNYLKEVDQLR
  LER
• H LQIDEQLRQIGASSRPPPNRTDKEKSYVTDDGQGMGRGSRPYRNRGH
  GRR
• F LQIDEQLRQIGATSRPPPNRTDKDKGYMTDDGPGLGRGSRPYRNRGH
  SRR
• C LQIDEQLTQIGATSRPPPNRTDKDKGYMTDDGPGKGRGSRPYRNRGH
  SRR
• H GPGYTSGTNSEASNASETESDHRDELSDWSLAPTEEERESFLRRGDG
  RRR
• F GPGYASGTNSEASNASETESDHRDELSDWSAAPAEEERDNYLRRGDG
  RRR
• C GPGYASGTNSEASNASETESDHRDELSDWSAAPAEEERDNYLRRGDG
  RRR
• H GGGGRGQGGRGRGGGFKGNDDHSRTDNRPRNPREAKGRTTDGSLQIR
  VDC
• F GGGGRGQGGRGRGG-FKGNDEQSRTDNRQRNSREAKGRTADGSLENT
  ---
• C GGGGRGQGGRGRGG-FKGNDEQSRTDNRERNSREAKGRTADGSLENT
  ---

RGG
KH1
epitope
KH2
90 homology to Chicken 85 homology to Human
photo from www.ncbi.nlm.nih.gov
figure adapted from Winograd et al. 2008
Neuroscience
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Zebra finch brain regions involved with song
learning and production
Posterior Pathway Pathway for singing/Vocal Motor
pathway mammalian motor corticobulbar
pathway Lesions affect singing at any age

• Pallium
• performs functions similar to mammalian neocortex
• Organized by nuclei

HVC
RA
LMAN
nXIIts
Area X
Anterior Forebrain Pathway Pathway for learning
song mammalian cortical-basal ganglia-thalamic
cortical loop Lesions affect ability to learn song
DLM Thalamus
By immunostaining, Fmrp is expressed in neurons
in the song nuclei
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Timeline for song learning
Brainard Doupe 2002 Nature
www.eko.uj.edu.pl/ rutkowska
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Fmrp is increased in premotor nucleus RA
P30
P60
Adult
Fmrp NeuN Bar 100um
Winograd, Clayton, Ceman, 2008 Neuroscience
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TguFmrp expression in male RA changes with
development
Bar 200um
Winograd, Clayton, Ceman, 2008 Neuroscience
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What does elevated TguFmrp in the neuropil of
RA mean?

• RA is a premotor nucleus for song production
• There is rapid growth of HVC axons into RA day
  30
• Konishi and Akutagava, 1985
• Ho Fmrp plays a role in synaptogenesis
• During song learning, HVC projects to RA, which
  also receives input from LMAN that introduces
  variability into the song of juvenile birds
• Ho Fmrp is involved in the processing that occurs
  in RA, thus, is increased after singing,
  reflecting experience dependent plasticity

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Future Goals
1. Create a knockdown bird by delivering Fmrp
shRNAs into the RA of developing zebra
finches 2. Examine the effect of Fmrp loss on
song learning
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Acknowledgments
UIUC David Clayton Sarah London Julie
George Michael Miles
Tory Blackwell
Claudia Winograd
Stephanie Ceman

• Funding
• NIH HD41591, FRAXA Research Foundation, Spastic
  Paralysis and Allied Diseases of the Central
  Nervous System Research Foundation, Neuroscience
  program, CMBTG, DPNTG