Reduced creatine kinase as a central and peripheral biomarker in Huntington's disease

Abstract

A major goal of current clinical research in Huntington's disease (HD) has been to identify preclinical and manifest disease biomarkers, as these may improve both diagnosis and the power for therapeutic trials. Although the underlying biochemical alterations and the mechanisms of neuronal degeneration remain unknown, energy metabolism defects in HD have been chronicled for many years. We report that the brain isoenzyme of creatine kinase (CK-BB), an enzyme important in buffering energy stores, was significantly reduced in presymptomatic and manifest disease in brain and blood buffy coat specimens in HD mice and HD patients. Brain CK-BB levels were significantly reduced in R6/2 mice by approximately 18% to approximately 68% from 21 to 91 days of age, while blood CK-BB levels were decreased by approximately 14% to approximately 44% during the same disease duration. Similar findings in CK-BB levels were observed in the 140 CAG mice from 4 to 12 months of age, but not at the earliest time point, 2 months of age. Consistent with the HD mice, there was a grade-dependent loss of brain CK-BB that worsened with disease severity in HD patients from approximately 28% to approximately 63%, as compared to non-diseased control patients. In addition, CK-BB blood buffy coat levels were significantly reduced in both premanifest and symptomatic HD patients by approximately 23% and approximately 39%, respectively. The correlation of CK-BB as a disease biomarker in both CNS and peripheral tissues from HD mice and HD patients may provide a powerful means to assess disease progression and to predict the potential magnitude of therapeutic benefit in this disorder.

Published by Elsevier B.V.

Figures

Figure 1

Western and dot blot analyses of brain and blood from R6/2 HD mice through the spectrum of disease at a premanifest stage (21 day), onset (30 day), and though early (42 day), moderate (63 day), and end stage disease (91 day). There were significant differences between mutant and littermate control mice at each disease stage, to include premanifest disease (Table 1). Blots are shown under each time point with alpha tubulin controls.

Figure 2

CK-BB immunohistochemistry of brain sections through anterior neostriatum at the level of the anterior commissure in R6/2 mice at 91 days. Gross CK-BB immunoreactivity is reduced in the R6/2 mutant mouse (B), in comparison to the wild type littermate control (A). Higher magnification of the neostriatum from each tissue section shows a marked reduction in CK-BB immunoreactivity in the mutant R6/2 mouse (D) within both the neuropil and the cytoplasm of striatal neurons (arrows), as compared to the wild type littermate control mouse (C). Magnification bar in A is 2 mm. Magnification bar in D is 100 µm.

Figure 3

Western and dot blot analyses of brain and blood from 140 CAG HD mice through the spectrum of disease at a premanifest stage (2 months), onset (4 months), though early (6 months), moderate (8 months), and severe disease (12 months). There were significant differences between mutant and littermate control mice at each disease stage starting a 4 months of age (Table 2). Blots are shown under each time point with alpha tubulin controls.

Figure 4

Open field analysis of 140 CAG full-length mutant HD mice from 1–6 months. There are significant differences in distance traveled (A), resting time (B), ambulatory time (C), and ambulatory counts (D) starting at 3 months of age. Significance was not obtained at 1 and 2 months. * p

Figure 5

CK-BB immunohistochemistry of brain sections through the anterior neostriatum at the level of the anterior commissure in 140 CAG HD mice at 8 months. Gross CK-BB immunoreactivity is reduced in the 140 CAG mutant mouse (B), in comparison to the wild type littermate control mouse (A). Higher magnification within the neostriatum from each tissue section shows a reduction in CK-BB immunoreactivity in the mutant 140 CAG mouse (D) within both the neuropil and the cytoplasm of striatal neurons (arrows), as compared to the wild type littermate control mouse (C). Magnification bar in A is 2 mm. Magnification bar in D is 100 µm.

Figure 6

Western analysis from the medial segment of the caudate nucleus (brain) of age-matched control patients and Grade 2, Grade 3, and Grade 4 HD patients. There was a significant grade-dependent reduction in CK-BB, with the greatest loss in Grade 4 HD (Table 3). Blood buffy coat CK-BB analysis from age-matched patient caregivers and premanifest and manifest HD patients showed a significant loss of CK-BB in both premanifest disease and manifest diseased HD patients, as compared to the controls (Table 3). Blots are shown under each time point with alpha tubulin controls.

Figure 7

CK-BB immunohistochemistry in the medial caudate nucleus from age-matched non-diseased control (A and C) and Grade 3 HD patient. There is marked loss of CK-BB immunoreactivity in HD (B and D), as observed in both low and high power magnification. CK-BB immunoreactivity is reduced within both the neuropil and the cytoplasm of striatal neurons (arrows), as compared to the control patient (A and C), with intense immunostaining in astrocytes in the HD specimen (asterisk in D). Combined GFAP and CK-BB immunofluorescence in the striatum from the same Grade 3 HD patient shows definitive colocalization of these proteins in astrocytes. Two dimensional analysis showed overlap of each of the antisera [GFAP, green (E); CK-BB. Red (F); and merged figures (G)]. Magnification bar in A is 100 µm. Magnification bar in E is 50 µm.