ATOMIC FORCE MICROSCOPY

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ATOMIC FORCE MICROSCOPY OF HUNTINGTIN AGGREGATES
Tomas T Ding and Peter T Lansbury Jr Center for
Neurologic Diseases, Brigham and Womens
Hospital 65 Landsdowne street, Cambridge, MA 02139
Jin Wang and James F Gusella Molecular Genetics
Unit, Massachusetts General Hospital 149 13Th
Street, Charlestown, MA 02129
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ABSTRACT

• Background The extent of in vitro formation of
  aggregate with amyloid characteristics by the N-
  terminus of huntingtin with glutamine repeat
  lengths in the pathological range correlates
  inversely with the age of onset of Huntingtons
  disease. Furthermore, amyloid has been detected
  in HD-brain inclusions . Finally, huntingtin
  aggregation, like aggregation of other
  amyloidogenic and disease-related proteins such
  as amyloid-b peptide and a-synuclein, may follow
  a nucleation-dependent polymerization pathway .
  Because both amyloid-b peptide and a-synuclein
  form pre-fibrillar and possibly neurotoxic
  aggregates in vitro we speculate that mutant
  huntingtin assembles into protofibrils and that
  those could play a role in the onset of
  Huntingtons disease.
• Methods We utilize atomic force microscopy (AFM)
  to image huntingtin aggregates adsorbed to mica.
  In AFM a substrate is scanned with a sharp tip
  attached to a cantilever while the deflections of
  the cantilever are translated into a relief-map
  of the surface. Such maps provide us with
  3-dimensional morphological information about
  single proteins and protein aggregates.
• Results and conclusions Huntingtin fibrils
  feature a diameter of 7-10 nm, similar to the
  fibrils described in the context of Alzheimers
  and Parkinsons disease, regardless of glutamine
  repeat length. Preliminary data implies that a
  higher number of fibrils (eg per unit volume) is
  formed whereas the lengths of the fibrils remain
  relatively constant on increasing glutamine
  repeat number suggesting that the nucleation
  rate increases with increased glutamine repeat
  length. We also have indications that aggregating
  huntingtin forms pre-fibrillar aggregates in
  vitro. Thus, within 2h Q41 huntingtin produces
  spherical assemblies with an average height of
  2.4 nm, similar to the spheres detected in
  aggregating a-synuclein . Apparently, this
  distribution is not stable but changes over time,
  so after 98h the average height increased to 3.4
  nm as the measured particle density dropped from
  400 to 20 particles per square micron. Since
  huntingtin Q41 is capable of forming both fibrils
  and a protofibril-like intermediate in vitro we
  argue that other huntingtin mutants also form
  such intermediates, and that they could be
  considered as possible drug targets.

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Atomic force microscopy (AFM)
Examples
Carbon atoms
Creates a relief map of the surface. Higher
features are represented with brighter colors.
b-Amyloid fibril
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Red cursors (horizontal)131 nm Green cursors
(height) 8.1 nm
b-Amyloid fibril
Individual Particles
Cross-section
Histogram
Populations
Mutant (A53T) a-Synuclein (protofibrils)
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Q41 2h
Q41 98h
Huntingtin exon 1 incubated at 37. The
particles were counted and heights measured.
n 2342, coverage 33 particle height 53 nm
n2526, coverage 8.6 particle height 32 nm
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Preliminary conclusion Particle-size
distribution changes over time
2h
Q41
98h
Height distribution histograms taken at 2h and
98h. Note that this is relative amounts the
total particle number is the same for the two
distributions.
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Annular Q41 protofibrils?
Cursors 18 nm. Height 2.5 nm. Mean width 25
nm. Volume (10-18 cm3). Calculated
weight corresponds to 56 Q41 monomers.
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For comparison a-Synuclein Protofibrils.
Spherical (left) and Annular protofibrils
formed by the a-synuclein A53T Mutant. This
protein is Associated with familial Parkinsons
disease. The A30P mutant and wild
type a-synuclein also form annular
structures. Annular A53T (from AFM) Diameter
10-12 nm Height 1.9-2.3 nm.
100nm square
2.5m square
Height 31nm N 2993
EM by Hilal A. Lashuel, Harvard Medical
School. Scale bar 100 nm.
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Summary
Aggregating huntingtin rapidly forms spherical
aggregates with a size distribution that changes
over time. Preliminary data suggest that Q41
is Capable of forming annular toxin-like
Structures similar to those formed by
a-synuclein (and b-amyloid).