ATOMIC FORCE MICROSCOPYOF HUNTINGTIN AGGREGATES

Tomas T Ding and Peter T Lansbury JrCenter for Neurologic Diseases, Brigham and Women’s Hospital

65 Landsdowne street, Cambridge, MA 02139

Jin Wang and James F GusellaMolecular Genetics Unit, Massachusetts General Hospital

149 13Th Street, Charlestown, MA 02129

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 Huntington’s 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-peptide and -synuclein, may follow a nucleation-dependent polymerization pathway . Because both amyloid- 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 Huntington’s 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 Alzheimer’s and Parkinson’s 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 -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.

Atomic force microscopy (AFM)

Creates a relief map of the surface.Higher features are representedwith brighter colors.

Examples:

Carbon atoms

-Amyloid fibril

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1086420Grain height (nm)

IndividualParticles:

Populations:

Red cursors (horizontal):131 nmGreen cursors (height): 8.1 nm-Amyloid fibril

Mutant (A53T) -Synuclein(protofibrils)

Cross-section

Histogram

Q41 2h Q41 98h

n: 2342, coverage: 33%particle height: 5±3 nm

n:2526, coverage 8.6%particle height: 3±2 nm

Huntingtin exon 1 incubated at 37°. The particleswere counted and heights measured.

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1086420 Particle Height (nm)

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98h

“Preliminary conclusion”: Particle-size distribution changes over time

Height distribution histograms taken at 2h and 98h.Note that this is relative amounts; the total particlenumber is the same for the two distributions.

Q41

Cursors: 18 nm.Height 2.5 nm.Mean width 25 nm.Volume (10-18 cm3).Calculated weightcorresponds to56 Q41 monomers.

Annular Q41protofibrils?

For comparison: -Synuclein Protofibrils.

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1086420Grain height (nm) EM by Hilal A. Lashuel,

Harvard Medical School.Scale bar 100 nm.

“Spherical” (left) andAnnular protofibrils formedby the -synuclein A53T Mutant. This protein isAssociated with familial Parkinson’s disease. The A30P mutant and wild type-synuclein also formannular structures.

Annular A53T (from AFM):Diameter 10-12 nmHeight 1.9-2.3 nm.

Height: 3±1nmN: 2993

2.5 square100nm square

Summary

Aggregating huntingtin rapidlyforms spherical aggregates witha size distribution that changes overtime.

Preliminary data suggest that Q41 isCapable of forming annular “toxin-like” Structures similar to those formed by -synuclein (and -amyloid).