Abnormalities of DYRK1A-Cytoskeleton Complexes in the Blood Cells as Potential Biomarkers of Alzheimer's Disease.

BACKGROUND: DYRK1A is implicated in mental retardation and Alzheimer's disease (AD) dementia of Down syndrome (DS) individuals. The protein is associated with cytoskeleton and altered expression has been shown to impair the cytoskeletal network via dosage effect. OBJECTIVE: Our original observations of marked reduction of cytoskeletal proteins associated with DYRK1A in brains and lymphoblastoid cell lines from DS and AD prompted an investigation whether cytoskeleton abnormalities could potentially be used as biomarkers of AD. METHODS: Our assay relied on quantification of co-immunoprecipitated cytoskeletal proteins with DYRK1A (co-IP assay) and analysis of the profile of G- and F-actin fractions obtained by high-speed centrifugations (spin-down assay). RESULTS: In co-IP assay, both DS and AD samples displayed reduced abundance of associated cytoskeletal proteins. In spin-down assay, G-actin fractions of controls displayed two closely spaced bands of actin in SDS-PAGE; while in AD and DS, only the upper band of the doublet was present. In both assays, alterations of actin cytoskeleton were present in DS, sporadic and familial AD cases, and in asymptomatic persons who later progressed to confirmed AD, but not in non-AD donors. In blind testing involving six AD and six controls, the above tests positively identified ten cases. Analysis of blood samples revealed the diversity of mild cognitive impairment (MCI) cases regarding the presence of the AD biomarker allowing distinction between likely prodromal AD and non-AD MCI cases. CONCLUSIONS: Both brain tissue and lymphocytes from DS and AD displayed similar semi-quantitative and qualitative alterations of actin cytoskeleton. Their specificity for AD-type dementia and the presence before clinical onset of the disease make them suitable biomarker candidates for early and definite diagnosis of AD. The presence of alterations in peripheral tissue points to systemic underlying mechanisms and suggests that early dysfunction of cytoskeleton may be a predisposing factor in the development of AD.

The role of overexpressed DYRK1A protein in the early onset of neurofibrillary degeneration in Down syndrome.

The gene encoding the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A (DYRK1A) is located in the Down syndrome (DS) critical region of chromosome 21. The third copy of DYRK1A is believed to contribute to abnormal brain development in patients with DS. In vitro studies showing that DYRK1A phosphorylates tau protein suggest that this kinase is also involved in tau protein phosphorylation in the human brain and contributes to neurofibrillary degeneration, and that this contribution might be enhanced in patients with DS. To explore this hypothesis, the brain tissue from 57 subjects including 16 control subjects, 21 patients with DS, and 20 patients with sporadic Alzheimer's disease (AD) was examined with two antibodies to the amino-terminus of DYRK1A (7F3 and G-19), as well as two polyclonal antibodies to its carboxy-terminus (X1079 and 324446). Western blots demonstrated higher levels of full-length DYRK1A in the brains of patients with DS when compared to control brains. Immunocytochemistry revealed that DYRK1A accumulates in neurofibrillary tangles (NFTs) in subjects with sporadic AD and in subjects with DS/AD. Overexpression of DYRK1A in patients with DS was associated with an increase in DYRK1A-positive NFTs in a gene dosage-dependent manner. Results support the hypothesis that overexpressed DYRK1A contributes to neurofibrillary degeneration in DS more significantly than in subjects with two copies of the DYRK1A gene and sporadic AD. Immunoreactivity with antibodies against DYRK1A not only in NFTs but also in granules in granulovacuolar degeneration and in corpora amylacea suggests that DYRK1A is involved in all three forms of degeneration and that overexpression of this kinase may contribute to the early onset of these pathologies in DS.

Cell type- and brain structure-specific patterns of distribution of minibrain kinase in human brain.

The minibrain kinase (Mnb/Dyrk1A) gene is localized in the Down syndrome (DS) critical region of chromosome 21. This gene encodes a proline-directed serine/threonine protein kinase (minibrain kinase-Mnb/Dyrk1A), which is required for the proliferation of distinct neuronal cell types during postembryonic neurogenesis. To study the distribution of Mnb/Dyrk1A during human brain development and aging, we raised Mnb/Dyrk1A-specific antibody (mAb 7F3) and examined 22 brains of normal subjects from 8 months to 90 years of age. We found that neurons were the only cells showing the presence of 7F3-positive product in both cell nucleus and cytoplasm. Nuclear localization supports the concept that Mnb/Dyrk1A may be involved in control of gene expression. Synaptic localization of Mnb/Dyrk1A also supports our previous studies suggesting that Mnb/Dyrk1A is a regulator of assembly of endocytic apparatus and appears to be involved in synaptic vesicle recycling and synaptic signal transmission. Accumulation of numerous 7F3-positive corpora amylacea in the memory and motor system subdivisions in subjects older than 33 years of age indicates that Mnb/Dyrk1A is colocalized with markers of astrocyte and neuron degeneration. Differences in the topography and the amount of Mnb/Dyrk1A in neurons, astrocytes, and ependymal and endothelial cells appear to reflect cell type- and brain structure-specific patterns in trafficking and utilization of Mnb/Dyrk1A.

Gene dosage-dependent association of DYRK1A with the cytoskeleton in the brain and lymphocytes of down syndrome patients.

The triplication of the DYRK1A gene encoding proline-directed serine/threonine kinase and located in the critical region of Down syndrome (DS) has been implicated in cognitive deficits and intellectual disability of individuals with DS. We investigated the effect of abnormal levels of this kinase on the cytoskeleton in brain and peripheral tissues of DS subjects. In DS tissues, the predictable approximately equal to 1.5-fold enhancement of the levels of DYRK1A protein was demonstrated. An association of DYRK1A with all 3 major cytoskeleton networks was identified using immunoprecipitation. We concentrated on the actin cytoskeleton because its association with DYRK1A was the most affected by the enzyme levels. As measured by coimmunoprecipitation in DS tissues, but not in fragile X lymphocytes, actin association with DYRK1A was reduced. This reduced association was dependent on the state of phosphorylation of cytoskeletal proteins and was present only in cells overproducing DYRK1A kinase; therefore, the effect was attributable to the DYRK1A gene dosage. Alterations of DYRK1A-actin assemblies were detected in newborn and infant groups, thereby linking DYRK1A overexpression with abnormal brain development of DS children. The identification of the actin cytoskeleton as one of cellular targets of DYRK1A action provides new insights into a gene dosage-sensitive mechanism by which DYRK1A could contribute to the pathogenesis of DS. In addition, the presence of this DS-specific cytoskeleton anomaly in lymphocytes attests to the systemic nature of some features of DS. To our knowledge, this is the first study conducted in human tissue that shows DYRK1A association with the cytoskeleton.