Cdc42interacting protein 4 binds to huntingtin

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Cdc42-interacting protein 4 binds to huntingtin

• By Sebastien Holbert, Alpaslan Dedeoglu,
  Sandrine Humbert, Frederic Saudou, Robert J.
  Ferrante and Christian Neri

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A little bit about Huntington's Disease (HD)

• Occurs in 1 in 10,000 Americans
• Typical age of onset is between 30 and 45 years.
• Have been occurrences in children as young as 2
  developing the disease
• Average life span after developing HD is 10-13
  years
• The earlier the onset of the disease, the faster
  it progresses.

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Huntingtons Disease

• Causes degradation of neurons in specific areas
  of the brain and results in loss of cognitive and
  muscular control.
• Results from a dominant mutation in the htt gene
  that adds extra copies of the amino acid
  Glutamine (Glu or Q).
• Length of polyQ sequence is inversely correlated
  with age of disease onset

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Alleles of HD

• Those with 28 or less Glns per allele are
  considered normal.
• 29-34 Glns person will not develop HD, but there
  is a risk for future generations.
• 35-39 some individuals will develop HD, there is
  also risk for next generation.
• 40 or more development of HD.

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Gradation of HD

• Like AIDS, people do not die from the disease
  itself, but from other infections (ie pneumonia).
• Levels of disease are from 0-4, with 0
  representing a normal human and 4 representing a
  person with severe HD symptoms.
• Symptoms split into separate categories. Overall
  score is calculated by adding up the degree of
  severity (0-4) in each category.

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What they already knew

• HD caused by variable polyglutamine (polyQ or
  gln) sequence in htt protein
• Accumulation of affected protein in the brain
  leads to Neuronal Intranuclear Inclusions(NII)
• NIIs were thought to be toxic to neurons

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Main Questions

• Are there any interacting proteins with htt
  characteristic to HD?
• What system will be used to identify interacting
  proteins?
• How do the proteins interact in the development
  of the disease?
• How does this protein interact with mutant and
  non-mutant htt?

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Going on a Fishing Trip in C. elegans

• Used Y2H
• Bait used were normal N-terminal of htt, mutated
  N-terminal of htt (81-128 Glns), ataxin-3 and
  lamin C.
• Lamin C is a random bait to detect background
  noise.
• Ataxin-3 has 23 Glns (close to normal htt) which
  differentiates specific from non-specific
  binding.
• Prey was random cDNA from C. elegans.

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Size of the Catch

• Screened 3.98107 cDNAs
• Found only 1 interaction
• Gene is KO8E3.3b
• Has an N-terminal Fer-CIP4 homology domain and a
  SH3 domain at the C-terminal
• SH3 domains are crucial to multi-protein
  complexes. Mutations in this domain can lead to
  diseases.

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Homologs in Humans?

• Searched BLAST and found family of strongly
  related homologs.
• CIP4 (Cdc-42 Interacting Protein 4) has a well
  conserved C-terminal SH3 domain and an N-terminal
  Fer-CIP4 homology domain.
• This suggests that it is involved with htt (like
  the C. elegans protein).

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CIP4 Pathway

• Called Rac1/Cdc42 signaling pathway
• Small GTPase
• Initiates two different pathways with Pak1
• 1 Cell polarity, membrane protrusion, migration
• 2 Stress response, apoptosis

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Had to Make Sure

• Tested to see if CIP4 binds with htt in vitro.
• Used GST fusions and western blot.
• Results both normal and mutated htt interact
  with CIP4. All had similar binding signal.

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CIP4 in Human Brain Tissue

• Took small slices from brain tissue.
• Immunostaining was done with second antibody
  method. First antibody was rabbit anti-CIP4 and
  then used goat anti-rabbit (this one had the tag
  on it).
• CIP4 expression increases with grade of HD

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Is There Co-localization of CIP4 and inclusion
sites?

• CIP4 B. ubiquitin C. combined

• Performed double immunofluorescence
• Used ubiquitin positive inclusions and screened
  for ubiquitin expression
• CIP4 and ubiquitin are partially co-localized in
  tissue samples
• Ubiquitin and CIP4 were not co-existent in the
  tissue samples
• CIP4 immunoactivity was higher than ubiquitin
  immunoactivity

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CIP4 levels are higher in HD patients than in
Control (CT)

• Used western Blot analysis with CIP4 antibody on
  the striatum region tissue
• Data showed significant increases of CIP4 in HD
  patients vs. Control Groups
• Interpretation CIP4 is up-regulated in HD
  patients

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Are CIP4 levels increased by mutant htt?

• Over expressed htt 1-480 and htt exon 1 in mutant
  and non mutant samples
• Measured corresponding levels of CIP4 mRNA
• Neither htt 1-480 or htt exon 1 lead to
  up-regulation

Ratio CIP4 intensity- background signal
ß-actin intensity- background signal
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Effects of CIP4 on Striatal Neurons

• Induced protein overexpression by transfecting
  striatal neurons with CIP4 constructs and empty
  vectors
• Empty vectors showed similar levels of cell death
  compared to WT htt.
• Overexpression led to cell death levels similar
  to those seen with mutant htt.
• Could determine CIP4 is toxic
• to striatal neurons.
• Co-transfected CIP4 and mutant
• N-terminal htt into one neuron
• and there was no change in the
• effects.

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Conclusions

• Identified that CIP4 levels are increased in HD
  patients.
• Determined CIP4 is partially co-localized with
  NII.
• CIP4 accumulation results in striatal neuron
  death.
• Levels of CIP4 are not controlled by htt
  expression.