A large number of protein expression changes occur early in life and precede phenotype onset in a mouse model for huntington disease

Abstract

Huntington disease (HD) is fatal in humans within 15-20 years of symptomatic disease. Although late stage HD has been studied extensively, protein expression changes that occur at the early stages of disease and during disease progression have not been reported. In this study, we used a large two-dimensional gel/mass spectrometry-based proteomics approach to investigate HD-induced protein expression alterations and their kinetics at very early stages and during the course of disease. The murine HD model R6/2 was investigated at 2, 4, 6, 8, and 12 weeks of age, corresponding to absence of disease and early, intermediate, and late stage HD. Unexpectedly the most HD stage-specific protein changes (71-100%) as well as a drastic alteration (almost 6% of the proteome) in protein expression occurred already as early as 2 weeks of age. Early changes included mainly the up-regulation of proteins involved in glycolysis/gluconeogenesis and the down-regulation of the actin cytoskeleton. This suggests a period of highly variable protein expression that precedes the onset of HD phenotypes. Although an up-regulation of glycolysis/gluconeogenesis-related protein alterations remained dominant during HD progression, late stage alterations at 12 weeks showed an up-regulation of proteins involved in proteasomal function. The early changes in HD coincide with a peak in protein alteration during normal mouse development at 2 weeks of age that may be responsible for these massive changes. Protein and mRNA data sets showed a large overlap on the level of affected pathways but not single proteins/mRNAs. Our observations suggest that HD is characterized by a highly dynamic disease pathology not represented by linear protein concentration alterations over the course of disease.

Fig. 1.

Scheme of disease progression in the HD mouse model R6/2. Pathological hallmarks are marked on the time scale by arrows, and the observed phenotype is indicated on top of each arrow.

Fig. 2.

Relative protein concentration changes during HD progression. Total brain extracts of R6/2 mice were studied at 2, 4, 6, 8, and 12 weeks of age. Protein concentration alterations were calculated for all significantly altered protein isoforms (p < 0.05, <0.9- or >1.1-fold change). The total spot volume (spot intensity × spot area) on each 2-D gel was set to 100%. The y axis indicates the amount of protein changed relative to a total protein concentration of 100%. Asterisks indicate statistical significance (p < 0.05). Black bars indicate up-regulated proteins, and gray bars indicate down-regulated proteins. The numbers above each bar indicate percentage of change. The line above the bars indicates the sum of up- and down-regulated protein concentration changes that is also indicated by numbers.

Fig. 3.

Longitudinal changes of protein expression during development. Changes in protein expression per day in terms of number of proteins (A) and protein concentration (B) were determined. Total brain extracts of embryonic day 16 and 18 and neonate P0, P7, P14, and P28 wild-type C57BL/6 mice were compared. Altered protein numbers and concentration were calculated for all significantly altered protein isoforms (p < 0.05, <0.9- or >1.1-fold change). For a complete list of all alterations see Table VI.