Down’s syndrome (DS) is a major cause of mental retardation, hypotonia and delayed development. Murine models of DS carrying large murine or human genomic fragments show motor alterations and memory deficits. The specific genes responsible for these phenotypic alterations have not yet been defined. DYRK1A, the human homolog of the Drosophilaminibrain gene, maps to the DS critical region of human chromosome 21 and is overexpressed in DS fetal brain. DYRK1A encodes a serine-threonine kinase, probably involved in neuroblast proliferation. Mutant Drosophilaminibrain flies have a reduction in both optic lobes and central brain, showing learning deficits and hypoactivity. We have generated transgenic mice (TgDyrk1A) overexpressing the full-length cDNA of Dyrk1A. TgDyrk1A mice exhibit delayed cranio-caudal maturation with functional consequences in neuromotor development. TgDyrk1A mice also show altered motor skill acquisition and hyperactivity, which is maintained to adulthood. In the Morris water maze, TgDyrk1A mice show a significant impairment in spatial learning and cognitive flexibility, indicative of hippocampal and prefrontal cortex dysfunction. In the more complex repeated reversal learning paradigm, this defect turned out to be specifically related to reference memory, whereas working memory was almost unimpaired. These alterations are comparable with those found in the partial trisomy chromosome 16 murine models of DS and suggest a causative role of DYRK1A in mental retardation and in motor anomalies of DS.

Previous studies have shown that the skeletal dihydropyridine receptor (DHPR) pore subunit Ca _V 1.1 (α1S) physically interacts with ryanodine receptor type 1 (RyR1), and a molecular signal is transmitted from α1S to RyR1 to trigger excitation-contraction (EC) coupling. We show that the β-subunit of the skeletal DHPR also binds RyR1 and participates in this signaling process. A novel binding site for the DHPR β1a-subunit was mapped to the M ³²⁰¹ to W ³⁶⁶¹ region of RyR1. In vitro binding experiments showed that the strength of the interaction is controlled by K ³⁴⁹⁵ KKRR _ _ R ³⁵⁰² , a cluster of positively charged residues. Phenotypic expression of skeletal-type EC coupling by RyR1 with mutations in the K ³⁴⁹⁵ KKRR _ _ R ³⁵⁰² cluster was evaluated in dyspedic myotubes. The results indicated that charge neutralization or deletion severely depressed the magnitude of RyR1-mediated Ca ²⁺ transients coupled to voltage-dependent activation of the DHPR. Meantime the Ca ²⁺ content of the sarcoplasmic reticulum was not affected, and the amplitude and activation kinetics of the DHPR Ca ²⁺ currents were slightly affected. The data show that the DHPR β-subunit, like α1S, interacts directly with RyR1 and is critical for the generation of high-speed Ca ²⁺ signals coupled to membrane depolarization. These findings indicate that EC coupling in skeletal muscle involves the interplay of at least two subunits of the DHPR, namely α1S and β1a, interacting with possibly different domains of RyR1.

Autosomal dominant mutations in GRIN2B are associated with severe encephalopathy, but little is known about the pathophysiological outcomes and any potential therapeutic interventions. Genetic studies have described the association between de novo mutations of genes encoding the subunits of the N-methyl-d-aspartate receptor (NMDAR) and severe neurological conditions. Here, we evaluated a missense mutation in GRIN2B, causing a proline-to-threonine switch (P553T) in the GluN2B subunit of NMDAR, which was found in a 5-year-old patient with Rett-like syndrome with severe encephalopathy. Structural molecular modeling predicted a reduced pore size of the mutant GluN2B-containing NMDARs. Electrophysiological recordings in a HEK-293T cell line expressing the mutated subunit confirmed this prediction and showed an associated reduced glutamate affinity. Moreover, GluN2B(P553T)-expressing primary murine hippocampal neurons showed decreased spine density, concomitant with reduced NMDA-evoked currents and impaired NMDAR-dependent insertion of the AMPA receptor subunit GluA1 at stimulated synapses. Furthermore, the naturally occurring coagonist d-serine restored function to GluN2B(P553T)-containing NMDARs. l-Serine dietary supplementation of the patient was hence initiated, resulting in the increased abundance of d-serine in the plasma and brain. The patient has shown notable improvements in motor and cognitive performance and communication after 11 and 17 months of l-serine dietary supplementation. Our data suggest that l-serine supplementation might ameliorate GRIN2B-related severe encephalopathy and other neurological conditions caused by glutamatergic signaling deficiency.

Dual-specificity tyrosine-phosphorylated and regulated kinase (DYRK) proteins are an evolutionarily conserved family of protein kinases, with members identified from yeast to humans, that participate in a variety of cellular processes. DYRKs are serine/threonine protein kinases that are activated by autophosphorylation on a tyrosine residue in the activation loop. The family member DYRK1A has been shown to phosphorylate several cytosolic proteins and a number of splicing and transcription factors, including members of the nuclear factor of activated T cells family. In the present study, we show that DYRK1A autophosphorylates, via an intramolecular mechanism, on Ser-520, in the PEST domain of the protein. We also show that phosphorylation of this residue, which we show is subjected to dynamic changes in vivo, mediates the interaction of DYRK1A with 14-3-3β. A second 14-3-3 binding site is present within the N-terminal of the protein. In the context of the DYRK1A molecule, neither site can act independently of the other. Bacterially produced DYRK1A and the mutant DYRK1A/S520A have similar kinase activities, suggesting that Ser-520 phosphorylation does not affect the intrinsic kinase activity on its own. Instead, we demonstrate that this phosphorylation allows the binding of 14-3-3β, which in turn stimulates the catalytic activity of DYRK1A. These findings provide evidence for a novel mechanism for the regulation of DYRK1A kinase activity.