Background

The late myelinating superficial white matter at the juncture of the cortical gray and white matter comprising the intracortical myelin and short-range association fibers has not received attention in Huntington's disease. It is an area of the brain that is late myelinating and is sensitive to both normal aging and neurodegenerative disease effects. Therefore, it may be sensitive to Huntington's disease processes.

Methods

Structural MRI data from 25 Pre-symptomatic subjects, 24 Huntington's disease patients and 49 healthy controls was run through a cortical pattern-matching program. The surface corresponding to the white matter directly below the cortical gray matter was then extracted. Individual subject's Diffusion Tensor Imaging (DTI) data was aligned to their structural MRI data. Diffusivity values along the white matter surface were then sampled at each vertex point. DTI measures with high spatial resolution across the superficial white matter surface were then analyzed with the General Linear Model to test for the effects of disease.

Results

There was an overall increase in the axial and radial diffusivity across much of the superficial white matter (p < 0.001) in Pre-symptomatic subjects compared to controls. In Huntington's disease patients increased diffusivity covered essentially the whole brain (p < 0.001). Changes are correlated with genotype (CAG repeat number) and disease burden (p < 0.001).

Conclusions

This study showed broad abnormalities in superficial white matter even before symptoms are present in Huntington's disease. Since, the superficial white matter has a unique microstructure and function these abnormalities suggest it plays an important role in the disease.

Posttranslational modifications can have profound effects on the biological and biophysical properties of proteins associated with misfolding and aggregation. However, their detection and quantification in clinical samples and an understanding of the mechanisms underlying the pathological properties of misfolding- and aggregation-prone proteins remain a challenge for diagnostics and therapeutics development. We have applied an ultrasensitive immunoassay platform to develop and validate a quantitative assay for detecting a posttranslational modification (phosphorylation at residue T3) of a protein associated with polyglutamine repeat expansion, namely Huntingtin, and characterized its presence in a variety of preclinical and clinical samples. We find that T3 phosphorylation is greatly reduced in samples from Huntington's disease models and in Huntington's disease patients, and we provide evidence that bona-fide T3 phosphorylation alters Huntingtin exon 1 protein conformation and aggregation properties. These findings have significant implications for both mechanisms of disease pathogenesis and the development of therapeutics and diagnostics for Huntington's disease.

Conformational changes in disease-associated or mutant proteins represent a key pathological aspect of Huntington's disease (HD) and other protein misfolding diseases. Using immunoassays and biophysical approaches, we and others have recently reported that polyglutamine expansion in purified or recombinantly expressed huntingtin (HTT) proteins affects their conformational properties in a manner dependent on both polyglutamine repeat length and temperature but independent of HTT protein fragment length. These findings are consistent with the HD mutation affecting structural aspects of the amino-terminal region of the protein, and support the concept that modulating mutant HTT conformation might provide novel therapeutic and diagnostic opportunities. We now report that the same conformational TR-FRET based immunoassay detects polyglutamine- and temperature-dependent changes on the endogenously expressed HTT protein in peripheral tissues and post-mortem HD brain tissue, as well as in tissues from HD animal models. We also find that these temperature- and polyglutamine-dependent conformational changes are sensitive to bona-fide phosphorylation on S13 and S16 within the N17 domain of HTT. These findings provide key clinical and preclinical relevance to the conformational immunoassay, and provide supportive evidence for its application in the development of therapeutics aimed at correcting the conformation of polyglutamine-expanded proteins as well as the pharmacodynamics readouts to monitor their efficacy in preclinical models and in HD patients.