Trisomy-driven overexpression of DYRK1A kinase in the brain of subjects with Down syndrome

Abstract

Down syndrome (DS) is the most common genetic disorder associated with mental retardation (MR). It is believed that many of the phenotypic features of DS stem from enhanced expression of a set of genes located within the triplicated region on chromosome 21. Among those genes is DYRK1A encoding dual-specificity proline-directed serine/treonine kinase, which, as documented by animal studies, can potentially contribute to cognitive deficits in DS. Whether this contribution can be exerted through elevated levels of DYRK1A protein in the brain of DS subjects was the main goal of the present study. The levels of DYRK1A protein were measured by Western blotting in six brain structures that included cerebral and cerebellar cortices and white matter. The study involved large cohorts of DS subjects and age-matched controls representing infants and adults of different age, gender and ethnicity. Trisomic Ts65Dn mice, an animal model of DS, were also included in the study. Both in trisomic mice and in DS subjects, the brain levels of DYRK1A protein were increased approximately 1.5-fold, indicating that this protein is overexpressed in gene dosage-dependent manner. The exception was an infant group, in which there was no enhancement suggesting the existence of a developmentally regulated mechanism. We found DYRK1A to be present in every analyzed structure irrespective of age. This widespread occurrence and constitutive expression of DYRK1A in adult brain suggest an important, but diverse from developmental role played by this kinase in adult central nervous system. It also implies that overexpression of DYRK1A in DS may be potentially relevant to MR status of these individuals during their entire life span.

Fig. 1. Quantification of DYRK1A brain levels in control and trisomic Ts65Dn mice

(A): Western blot analysis of DYRK1A isolated through Ni-affinity chromatography from mouse and human brain. Whole mouse brain (lane 2) and frontal cortex of 32-year old control subject (lane 3) were lysed in 6 M GuHCl, purified by IMAC, separated on 8% tricine SDS-polyacrylamide gel and immunobloted with 1:5000 dilution of 7F3 antibody. The blot was developed by color reaction using AP-conjugated secondary antibody and BCIP/NBP substrate. Lane 1 was loaded with 20 ng of GST-497 recombinant fusion DYRK1A protein standard. (B): Representative Western-blot of three control (C)/trisomic(Ts) pairs of Ts65Dn mice. Equal loads of Ni-sepharose purified samples were resolved in 8% SDS-PAGE and immunoblotted with 1:5000 dilution of 7F3 antibody. Blots were developed by chemiluminescence using AP-conjugated secondary antibody. (C): DYRK1A levels quantified by densitometric analysis of three independent Western blots as shown in (A). The density of immunoreactive bands was normalized with 20 ng of purified GST-497 recombinant DYRK1A protein and expressed as ng/mg of total protein of brain lysate. For all three pairs, the difference was statistically significant (P < 0.05) as established by paired Student’s t test.

Fig. 2. Quantification of DYRK1A protein levels in selected structures of human brain: Summary of results obtained in 6 control and 12 DS subjects

(A) Samples of frontal (FC), temporal (TC), occipital (OC), and cerebellar (CX) cortices and two white matters dissected from corpus callosum (CC) and cerebellum (CW) were prepared and quantified by Western blotting according to the standard procedure as described earlier. The results expressed as ng/mg of total protein in brain lysate represent mean ± SD of all individual cases within each group, where each case was independently evaluated at least three times. In all six structures, the difference between the control and the DS group was statistically significant (P < 0.05), as determined by the two-tailed Student’s t test for independent samples. (B) Representative Western blot of pooled tissue from cases analyzed individually in (A). Mixing equal amounts of powdered brain tissue from each case made pools, which were then processed for Western blotting and densitometric quantification exactly in the same way as individual cases. Values of the DS : CTR ratio for each structural pool were calculated on the basis of scans of three independent Western blots.

Fig. 3. DYRK1A protein levels in frontal cortex of control and DS subjects grouped by age

Groups 1—3 y (3 and 3) and 10—30 y (4 and 5) consisted mainly of cases received from the NICHD Brain Bank, while cases of the group 40 + (5 and 9) were from the IBR Brain Bank. All samples were prepared as described in the legend of Fig. 1 The results expressed as ng/mg of total protein in brain lysate represent mean ± SD of all individual cases within each group, where each case was independently evaluated at least three times. The difference between control and DS for all but infants group was statistically significant (P < 0.05).