Intracellular distribution of differentially phosphorylated dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A).

The gene encoding dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A) is located within the Down syndrome (DS) critical region of chromosome 21. DYRK1A interacts with a plethora of substrates in the cytosol, cytoskeleton, and nucleus. Its overexpression is a contributing factor to the developmental alterations and age-associated pathology observed in DS. We hypothesized that the intracellular distribution of DYRK1A and cell-compartment-specific functions are associated with DYRK1A posttranslational modifications. Fractionation showed that, in both human and mouse brain, almost 80% of DYRK1A was associated with the cytoskeleton, and the remaining DYRK1A was present in the cytosolic and nuclear fractions. Coimmunoprecipitation revealed that DYRK1A in the brain cytoskeleton fraction forms complexes with filamentous actin, neurofilaments, and tubulin. Two-dimensional gel analysis of the fractions revealed DYRK1A with distinct isoelectric points: 5.5-6.5 in the nucleus, 7.2-8.2 in the cytoskeleton, and 8.7 in the cytosol. Phosphate-affinity gel electrophoresis demonstrated several bands of DYRK1A with different mobility shifts for nuclear, cytoskeletal, and cytosolic DYRK1A, indicating modification by phosphorylation. Mass spectrometry analysis disclosed one phosphorylated site in the cytosolic DYRK1A and multiple phosphorylated residues in the cytoskeletal DYRK1A, including two not previously described. This study supports the hypothesis that intracellular distribution and compartment-specific functions of DYRK1A may depend on its phosphorylation pattern.

Effect of DYRK1A activity inhibition on development of neuronal progenitors isolated from Ts65Dn mice.

Overexpression of dual-specificity tyrosine-(Y)-phosphorylation-regulated kinase 1A (DYRK1A), encoded by a gene located in the Down syndrome (DS) critical region, is considered a major contributor to developmental abnormalities in DS. DYRK1A regulates numerous genes involved in neuronal commitment, differentiation, maturation, and apoptosis. Because alterations of neurogenesis could lead to impaired brain development and mental retardation in individuals with DS, pharmacological normalization of DYRK1A activity has been postulated as DS therapy. We tested the effect of harmine, a specific DYRK1A inhibitor, on the development of neuronal progenitor cells (NPCs) isolated from the periventricular zone of newborn mice with segmental trisomy 16 (Ts65Dn mice), a mouse model for DS that overexpresses Dyrk1A by 1.5-fold. Trisomy did not affect the ability of NPCs to expand in culture. Twenty-four hours after stimulation of migration and neuronal differentiation, NPCs showed increased expression of Dyrk1A, particularly in the trisomic cultures. After 7 days, NPCs developed into a heterogeneous population of differentiating neurons and astrocytes that expressed Dyrk1A in the nuclei. In comparison with disomic cells, NPCs with trisomy showed premature neuronal differentiation and enhanced γ-aminobutyric acid (GABA)-ergic differentiation, but astrocyte development was unchanged. Harmine prevented premature neuronal maturation of trisomic NPCs but not acceleration of GABA-ergic development. In control NPCs, harmine treatment caused altered neuronal development of NPCs, similar to that in trisomic NPCs with Dyrk1A overexpression. This study suggests that pharmacological normalization of DYRK1A activity may have a potential role in DS therapy.

Increased levels of carbonic anhydrase II in the developing Down syndrome brain.

By using a proteomic approach, we found increased levels of carbonic anhydrase II (CA II) in the brain of Ts65Dn mice, a mouse model for Down syndrome (DS). Further immunoblot analyses showed that the levels of CA II are increased not only in the brain of adult Ts65Dn mice but also in the brain of infants and young children with DS. Cellular localization of the enzyme in human brain, predominantly in the oligodendroglia and primitive vessels in fetal brain and in the oligodendroglia and some GABAergic neurons postnatally, was similar in DS subjects and controls. Given the role of CA II in regulation of electrolyte and water balance and pH homeostasis, up-regulation of CA II may reflect a compensatory mechanism mobilized in response to structural/functional abnormalities in the developing DS brain. However, this up-regulation may also have an unfavorable effect by increasing susceptibility to seizures of children with DS.

Carbonic anhydrase II in the developing and adult human brain.

Carbonic anhydrase II (CA II) is one of 14 isozymes of carbonic anhydrases, zinc metalloenzymes that catalyze the reversible hydration of carbon dioxide to bicarbonate. Mutations in CA II in humans lead to osteopetrosis with renal tubular acidosis and cerebral calcifications, a disorder often associated with mental retardation. Recently, new avenues in CA II research have opened as a result of discoveries that the enzyme increases bicarbonate and proton fluxes and may play an important role in brain tissue. In the human brain, CA II was localized to oligodendrocytes, myelin, and choroid plexus epithelium. Because this conclusion was based on a few fragmentary reports, we analyzed in more detail the expression of the enzyme in human telencephalon. By immunoblotting, we found a gradual increase in CA II levels from 17 weeks' gestation to childhood and adolescence. By immunohistochemistry, CA II was found to be present not only in oligodendrocytes and choroid plexus epithelium (declining with aging in both these locations), but also in a subset of neurons mostly with GABAergic phenotype, in a few astrocytes, and transiently during brain development in the endothelial cells of microvessels. The enzyme also occurred in oligodendrocyte processes in contact with myelinating axons, myelin sheaths, and axolemma, but was either absent or appeared in minute amounts in compact myelin. These findings suggest the possible involvement of CA II in a wide spectrum of biologic processes in the developing and adult human brain and may contribute to better understanding of the pathogenesis of cerebral calcifications and mental retardation caused by CA II deficiency.

Widespread cerebellar transcriptome changes in Ts65Dn Down syndrome mouse model after lifelong running.

Our previous study showed an improvement in locomotor deficits after voluntary lifelong running in Ts65Dn mice, an animal model for Down syndrome (DS). In the present study, we employed mouse microarrays printed with 55,681 probes in an attempt to identify molecular changes in the cerebellar transcriptome that might contribute to the observed behavioral benefits of voluntary long-term running in Ts65Dn mice. Euploid mice were processed in parallel for comparative purposes in some analyses. We found that running significantly changed the expression of 4,315 genes in the cerebellum of Ts65Dn mice, over five times more than in euploid animals, up-regulating 1,991 and down-regulating 2,324 genes. Functional analysis of these genes revealed a significant enrichment of 92 terms in the biological process category, including regulation of biosynthesis and metabolism, protein modification, phosphate metabolism, synaptic transmission, development, regulation of cell death/apoptosis, protein transport, development, neurogenesis and neuron differentiation. The KEGG pathway database identified 18 pathways that are up-regulated and two that are down-regulated by running that were associated with learning, memory, cell signaling, proteolysis, regeneration, cell cycle, proliferation, growth, migration, and survival. Of six mRNA protein products we tested by immunoblotting, four showed significant running-associated changes in their levels, the most prominent in glutaminergic receptor metabotropic 1, and two showed changes that were close to significant. Thus, unexpectedly, our data point to the high molecular plasticity of Ts65Dn mouse cerebellum, which translated into humans with DS, suggests that the motor deficits of individuals with DS could markedly benefit from prolonged exercise.

Exposure to alcohol on E9 raises poststress corticosterone in mature but not old mice.

Prenatal exposure to alcohol alters postnatal function of the hypothalamic-pituitary-adrenal axis. Hyperresponsiveness to stress, or increased secretion of corticosterone, is a commonly studied effect in offspring of rats exposed to alcohol during a substantial period of gestation. No studies have reported on stress hormone secretion following alcohol exposure on a single day during embryonic development even though exposure at this time may damage the hypothalamus and pituitary. To explore the effect of an acute exposure, we used the offspring of C57BL/6J mice exposed to alcohol or saline on embryonic day (E) 9 (2.9 g/kg administered twice, 4h apart). At 7.5 or 22 months of age these mice were subjected to a 12-h restraint stress, or merely kept in the same environment without restraint. After the 12-h period, a blood sample was obtained from the retro-orbital plexus, and analyzed for the amount of corticosterone. The 7.5-month old group of alcohol-treated offspring were indeed hyperresponsive to restraint stress, but the 22-month old mice were not. Whether the normal-appearing corticosterone response of the old alcohol-exposed mice indicated adaptation to restraint, an aging-associated ceiling effect in corticosterone secretion, or an expression of pathology, cannot be decided on the basis of present data.

Long-term running alleviates some behavioral and molecular abnormalities in Down syndrome mouse model Ts65Dn.

Running may affect the mood, behavior and neurochemistry of running animals. In the present study, we investigated whether voluntary daily running, sustained over several months, might improve cognition and motor function and modify the brain levels of selected proteins (SOD1, DYRK1A, MAP2, APP and synaptophysin) in Ts65Dn mice, a mouse model for Down syndrome (DS). Ts65Dn and age-matched wild-type mice, all females, had free access to a running wheel either from the time of weaning (post-weaning cohort) or from around 7 months of age (adult cohort). Sedentary female mice were housed in similar cages, without running wheels. Behavioral testing and evaluation of motor performance showed that running improved cognitive function and motor skills in Ts65Dn mice. However, while a dramatic improvement in the locomotor functions and learning of motor skills was observed in Ts65Dn mice from both post-weaning and adult cohorts, improved object memory was seen only in Ts65Dn mice that had free access to the wheel from weaning. The total levels of APP and MAP2ab were reduced and the levels of SOD1 were increased in the runners from the post-weaning cohort, while only the levels of MAP2ab and α-cleaved C-terminal fragments of APP were reduced in the adult group in comparison with sedentary trisomic mice. Hence, our study demonstrates that Ts65Dn females benefit from sustained voluntary physical exercise, more prominently if running starts early in life, providing further support to the idea that a properly designed physical exercise program could be a valuable adjuvant to future pharmacotherapy for DS.

Rescue of synaptic failure and alleviation of learning and memory impairments in a trisomic mouse model of down syndrome.

Down syndrome (DS) is caused by the triplication of ∼240 protein-coding genes on chromosome 21 and is the most prevalent form of developmental disability. This condition results in abnormalities in many organ systems, as well as in intellectual retardation. Many previous efforts to understand brain dysfunction in DS have indicated that cognitive deficits are coincident with reduced synaptic plasticity and decreased neuronal proliferation. One therapeutic strategy for optimizing the microenvironment for neuronal proliferation and synaptic plasticity in the brain is the use of neurotrophins to restore the homeostasis of the brain biochemical milieu. Here, we show that peripheral administration of Peptide 6, an 11-mer corresponding to an active region of ciliary neurotrophic factor, amino acid residues 146 to 156, can inhibit learning and memory impairments in Ts65Dn mice, a trisomic mouse model of DS. Long-term treatment with Peptide 6 enhanced the pool of neural progenitor cells in the hippocampus and increased levels of synaptic proteins crucial for synaptic plasticity. These findings suggest a therapeutic potential of Peptide 6 in promoting functional neural integration into networks, thereby strengthening biologic substrates of memory processing.

Enhancement of dentate gyrus neurogenesis, dendritic and synaptic plasticity and memory by a neurotrophic peptide.

Pharmacological enhancement of hippocampal neurogenesis is a therapeutic approach for improvement of cognition in learning and memory disorders such as Alzheimer's disease. Here we report the development of an 11-mer peptide that we designed based on a biologically active region of the ciliary neurotrophic factor. This peptide, Peptide 6, induced proliferation and increased survival and maturation of neural progenitor cells into neurons in the dentate gyrus of normal adult C57BL6 mice. Furthermore, Peptide 6 increased the MAP2 and synaptophysin immunoreactivity in the dentate gyrus. Thirty-day treatment of the mice with a slow release bolus of the peptide implanted subcutaneously improved reference memory of the mice in Morris water maze. Peptide 6 has a plasma half life of over 6 h, is blood-brain barrier permeable, and acts by competitively inhibiting the leukemia inhibitory factor signaling. The fact that Peptide 6 is both neurogenic and neurotrophic and that this peptide is effective when given peripherally, demonstrates its potential for prevention and treatment of learning and memory disorders.

Upregulation of phosphorylated alphaB-crystallin in the brain of children and young adults with Down syndrome.

Our previous proteomic studies disclosed upregulation of alphaB-crystallin, a small heat shock protein, in the brain tissue of Ts65Dn mice, a mouse model for Down syndrome (DS). To validate data obtained in model animals, we studied at present the levels and distribution of total alphaB-crystallin and its forms phosphorylated at Ser-45 and Ser-59 in the brain tissues of DS subjects and age-matched controls at 4 months to 23 years of age. On immunoblots from frontal cortex and white matter, alphaB-crystallin and its form phosphorylated at Ser-59 were detectable already in infants, whereas alphaB-crystallin phosphorylated at Ser-45 appeared in small amounts in older children. Although the levels of total alphaB-crystallin were modestly increased in DS subjects, the amounts of both phosphorylated forms were much higher (up to approximately 550%) in the group of older children and young adults with DS than in age-matched controls. Immunoreactivity to alphaB-crystallin occurred not only in a subset of oligodendrocytes and some subpial and perivascular astrocytes, which was reported earlier, but also in GFAP-positive astrocytes accumulating at the sites of ependymal injury as well as some GFAP/platelet-derived growth factor receptor alpha-positive cells in both DS and control brains, which is a novel observation. Given that the chaperone and anti-apoptotic activities of alphaB-crystallin are phosphorylation-dependent, we propose that enhanced phosphorylation of alphaB-crystallin in the brains of young DS subjects might reflect a cytoprotective mechanism mobilized in response to stress conditions induced or augmented by the effect of genes encoded by the triplicated chromosome 21.