Technical Reference #3069

3069.

Quantitative Relationships between Huntingtin Levels; Polyglutamine Length; Inclusion Body Formation; and Neuronal Death Provide Novel Insight into Huntington’s Disease Molecular Pathogenesis Jason Miller; Montserrat Arrasate; Benjamin A. Shaby; Siddhartha Mitra; Eliezer Masliah; and Steven Finkbeiner, University of California San Francisco, Journal of Neuroscience, 30(3069), (2010)
Link To Paper

Abstract:
An expanded polyglutamine (polyQ) stretch in the protein huntingtin (htt) induces self-aggregation into inclusion bodies (IBs) and causes Huntington’s disease (HD). Defining precise relationships between early observable variables and neuronal death at the molecular and cellular levels should improve our understanding ofHDpathogenesis. Here we used an automated microscope that tracks thousands of neurons individually over their entire lifetime to quantify interconnected relationships between early variables such as htt levels polyQ length and IB formation and neuronal death in a primary striatal model of HD. The resulting model revealed that mutant htt increases the risk of death by tonically interfering with homeostatic coping mechanisms rather than producing accumulated damage to the neuron htt toxicity is saturable the rate-limiting steps for inclusion body formation and death can be traced to different conformational changes in monomeric htt and IB formation reduces the impact of the starting levels of htt of a neuron on its risk of death. Finally the model that emerges from our quantitative measurements places critical limits on the potential mechanisms by which mutant htt might induce neurodegeneration which should help direct future research.

Materials & Methods:
Immunocytochemistry confocal microscopy and electron microscopy. Immunocytochemistry was performed as described (Brooks et al. 2004) using a 12 min 4% paraformaldehyde/4% sucrose fixation. Images were collected on a Zeiss LSM 510 confocal microscope with Zeiss software (Zeiss). For electron microscopy primary striatal cell cultures were plated in glass-bottom dishes (MatTek). Plates were fixed in 4% paraformaldehyde for 15 min. Cells were then further fixed in 2% paraformaldehyde and 1% glutaraldehyde and then fixed in osmium tetraoxide and embedded in Epon Araldite. Once the resin hardened blocks with the cells were detached from the coverslips and mounted into a resin block for sectioning with an ultramicrotome (Leica) at 90 nm thickness. Grids containing the attached ultrathin sections were analyzed with a ZeissOM 10 electron microscope (Zeiss). Cells were randomly acquired from three grids.

Microscopic Technique
Confocal microscopy, Electron microscopy

Cell Type(s)
primary striatal cell cultures