Control of neuron numbers by DYRK1A: lessons from mouse models

Trabajo presentado en el DYRK1A, related kinases & human disease, celebrado en Saint Malo, Bretagne (Francia), del 28 de marzo al 1 de abril de 2017Neurons in the mammalian brain are generated prenatally from a heterogeneous population of progenitors that divide producing more progenitors (expansion divisions) or producing neurons (differentiative divisions). Neurons are usually generated in excess and a fraction of them die during development by physiological apoptosis. Therefore, alterations in the neurogenic potential of embryonic neural stem cells or in the activity of apoptotic cell death pathways may have a significant impact in the number of the different neuron types that integrate into functional circuits. Studies in mouse models carrying 1 or 3 functional copies of Dyrk1a have shown that DYRK1A controls brain size and neuron numbers in a dosage-dependent and region-specific manner (1). In both haploinsufficient Dyrk1a+/- embryos and transgenic embryos carrying 3 copies of mouse Dyrk1a (TgBACDyrk1a), neurogenesis is preserved in regions of the ventral mesencephalon where dopaminergic neurons involved in the control of voluntary movement and regulation of emotion are generated. However, at postnatal stages the number of these neurons were decreased in Dyrk1a+/- mice and increased in TgBACDyrk1a mice due to a dysregulation of Caspase 9-mediated cell death pathway (2). In contrast, neuron counts in the postnatal neocortex of Dyrk1a mutant mice, the region involved in higher-order brain functions, inversely correlate with DYRK1A protein levels. Examination of this structure indicated that neurogenesis is increased in the Dyrk1a+/- model and reduced in the TgBACDyrk1a model, and that variations in the division mode (proliferative vs. differentiative divisions) of the stem cells (radial glial progenitors) that give rise to cortical excitatory neurons contribute to the neurogenic defects observed in these two models. Reduced neurogenesis in TgBACDyrk1a embryos correlates with a longer cell cycle G1 phase and decreased nuclear levels of the cell cycle activator Cyclin D1 in radial glial progenitors. These defects are consistent with the ability of DYRK1A to phosphorylate T286 in Cyclin D1, which promotes its nuclear export and subsequent degradation via the ubiquitin-proteasome pathway (3). During the talk, I will present new data showing the effect of DYRK1A overexpression in the production of cortical inhibitory neurons and discuss the pathogenic effects of DYRK1A gene-dosage variations in neocortical development.This work was supported by the Spanish Ministry of Economy, Innovation and Competitiveness (MINECO), the Spanish network on Rare Diseases (CIBERER) and the Jérôme Lejeune Foundation.Peer reviewe

Upregulation of RCAN1 causes Down syndrome-like immune dysfunction

[Background]: People with Down syndrome (DS) are more susceptible to infections and autoimmune disease, but the molecular genetic basis for these immune defects remains undetermined. In this study, we tested whether increased expression of the chromosome 21 gene RCAN1 contributes to immune dysregulation. [Methods]: We investigated the immune phenotype of a mouse model that overexpresses RCAN1. RCAN1 transgenic (TG) mice exhibit T cell abnormalities that bear a striking similarity to the abnormalities described in individuals with DS. [Results]: RCAN1-TG mice display T cell developmental defects in the thymus and peripheral immune tissues. Thymic cellularity is reduced by substantial losses of mature CD4 and CD8 thymocytes and medullary epithelium. In peripheral immune organs T lymphocytes are reduced in number and exhibit reduced proliferative capacity and aberrant cytokine production. These T cell defects are stem cell intrinsic in that transfer of wild type bone marrow into RCAN1-TG recipients restored medullary thymic epithelium and T cell numbers in the thymus, spleen and lymph nodes. However, bone marrow transplantation failed to improve T cell function, suggesting an additional role for RCAN1 in the non-haemopoietic compartment. [Conclusions]: RCAN1 therefore facilitates T cell development and function, and when overexpressed, may contribute to immune dysfunction in DS.Peer Reviewe

DYRK1A controls replication-associated damage in the developing brain

Trabajo presentado en el 17th Spanish Society for Developmental Biology Meeting, celebrado en modalidad virtual del 18 al 20 de noviembre de 2020.DYRK1A (Dual specificity Tyrosine Phosphorylation Regulated Kinase 1A) is encoded by a dosage-dependent gene and it is involved in neurogenesis and neuron differentiation. The overexpression of DYRK1A causes some of the neurological alterations associated to Down syndrome while its haploinsufficiency leads to developmental delay and microcephaly in both humans and mice. Mouse Dyrk1a-/- embryos die @ at E11 limiting the use of this model to study brain development. In this work we have used a conditional knockout mouse model (Dyrk1aNes) in which Dyrk1a was deleted in neural progenitors before the onset of neurogenesis. Dyrk1aNes embryos presented a reduction of brain parenchyma that was notorious before birth (E19) and very prominent in the striatum and other ventral regions. Staining with antibodies against the active form of caspase3 (CASP3) revealed an increase in apoptotic progenitors and differentiating neurons in Dyrk1aNes embryos. Apoptosis in Dyrk1aNes mutants was already significant in the ventral telencephalon at E11 and progressively increased as neurogenesis advances, affecting dorsal telencephalic regions by E13. Alterations in the levels of the cell cycle regulators Cyclin D1 and D2 and the number of progenitors and neurons that were indicative of defects in neurogenesis were also detected in the dorsal telencephalon of E11-E12 Dyrk1aNes embryos. E11-E13 Dyrk1aNes brains showed a significant increase in cells expressing the replication stress marker gH2AX and active p53 (p53+ nuclei) in the same telencephalic regions labelled for CASP3, suggesting that apoptosis is these brains is p53-dependent and likely triggered by activation of DNA repair pathways. Collectively, these results indicate that DYRK1A is necessary for DNA repair and cell cycle progression of neural brain progenitors

Triplication of DYRK1A causes retinal structural and functional alterations in Down syndrome

Ariadna Laguna et al.Down syndrome (DS) results from the triplication of approximately 300 human chromosome 21 (Hsa21) genes and affects almost all body organs. Children with DS have defects in visual processing that may have a negative impact on their daily life and cognitive development. However, there is little known about the genes and pathogenesis underlying these defects. Here, we show morphometric in vivo data indicating that the neural retina is thicker in DS individuals than in the normal population. A similar thickening specifically affecting the inner part of the retina was also observed in a trisomic model of DS, the Ts65Dn mouse. Increased retinal size and cellularity in this model correlated with abnormal retinal function and resulted from an impaired caspase- 9-mediated apoptosis during development. Moreover, we show that mice bearing only one additional copy of Dyrk1a have the same retinal phenotype as Ts65Dn mice and normalization of Dyrk1a gene copy number in Ts65Dn mice completely rescues both, morphological and functional phenotypes. Thus, triplication of Dyrk1a is necessary and sufficient to cause the retinal phenotype described in the trisomic model. Our data demonstrate for the first time the implication of DYRK1A overexpression in a developmental alteration of the central nervous system associated with DS, thereby providing insights into the aetiology of neurosensorial dysfunction in a complex disease. © The Author 2013. Published by Oxford University Press. All rights reserved.This work was supported by the Spanish Ministry of Economy and Competitiveness (grant numbers SAF-2007-60940 and SAF-2010-17004 to M.L.A., and SAF-2010-21879 to P.dlV.), by the Generalitat de Catalunya (grant number 2009SGR1464) and by the Portuguese Foundation for Science and Technology (grant numbers PIC/IC/82986/2007 and Compete/PTDC/SAU/ORG_118380). A.L. was supported by an FI grant (AGAUR, Generalitat de Cataluya) and M.-J.B. by the CIBERER.Peer Reviewe

DYRK1A: A master regulatory protein controlling brain growth

Copy number variation in a small region of chromosome 21 containing DYRK1A produces morphological and cognitive alterations in human. In mouse models, haploinsufficiency results in microcephaly, and a human DYRK1A gain-of-function model (three alleles) exhibits increased brain volume. To investigate these developmental aspects, we used a murine BAC clone containing the entire gene to construct an overexpression model driven by endogenous regulatory sequences. We compared this new model to two other mouse models with three copies of Dyrk1a, YACtgDyrk1a and Ts65Dn, as well as the loss-of-function model with one copy (Dyrk1a +/-). Growth, viability, brain weight, and brain volume depended strongly upon gene copy number. Brain region-specific variations observed in gain-of-function models mirror their counterparts in the loss-of-function model. Some variations, such as increased volume of the superior colliculus and ventricles, were observed in both the BAC transgenic and Ts65Dn mice. Using unbiased stereology we found that, in the cortex, neuron density is inversely related to Dyrk1a copy number but, in thalamic nuclei, neuron density is directly related to copy number. In addition, six genes involved either in cell division (Ccnd1 and pAkt) or in neuronal machinery (Gap43, Map2, Syp, Snap25) were regulated by Dyrk1a throughout development, from birth to adult. These results imply that Dyrk1a expression alters different cellular processes during brain development. Dyrk1a, then, has two roles in the development process: shaping the brain and controlling the structure of neuronal components. © 2012 Elsevier Inc.Trabajo financiado por Ministerio de España de Ciencia e Innovación (SAF2010-17004).Peer Reviewe

MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy [version 2; peer review: 2 approved]

© 2020 Tosi S et al.We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, sample fiducials, or conversion of raw TIFF images, and the stitched images can be explored instantly. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-side lightsheet illumination and dual-side camera detection.The preparation of some of the datasets that were used to test MosaicExplorerJ was partially funded by project TEC2016-78052-R from the Spanish Ministry of Economy and Competitiveness and RTC2017-6600-1 from Ministry of Science, Innovation and Universities, as well as project PI17/01766 from Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III (co-financed by European Regional Development Fund (ERDF), “A way of making Europe")

MosaicExplorerJ: Interactive stitching of terabyte-size tiled datasets from lightsheet microscopy

© 2021 Tosi S et al.We introduce MosaicExplorerJ, an ImageJ macro to stitch 3D tiles from terabyte-size microscopy datasets organized on a regular 2D grid. As opposed to existing software, stitching does not require any prior information on the actual positions of the tiles, or conversion of raw TIFF images to a multi-resolution format for interactive exploration and fast processing. MosaicExplorerJ was specifically designed to process lightsheet microscopy datasets from optically cleared samples. It can handle multiple fluorescence channels, dual-sided lightsheet illumination and dual-sided camera detection.This publication was supported by COST Action NEUBIAS (CA15124), funded by COST (European Cooperation in Science and Technology). MJB acknowledges the support of Jérôme Lejeune Foundation. : The preparation of some of the datasets that were used to test MosaicExplorerJ was partially funded by project TEC2016-78052-R from the Spanish Ministry of Economy and Competitiveness and RTC2017-6600-1 from Ministry of Science, Innovation and Universities, as well as project PI17/01766 from Ministerio de Ciencia, Innovación y Universidades, Instituto de Salud Carlos III (cofinanced by European Regional Development Fund (ERDF), “A way of making Europe")

DYRK1A-mediated Cyclin D1 Degradation in Neural Stem Cells Contributes to the Neurogenic Cortical Defects in Down Syndrome

© 2015. Alterations in cerebral cortex connectivity lead to intellectual disability and in Down syndrome, this is associated with a deficit in cortical neurons that arises during prenatal development. However, the pathogenic mechanisms that cause this deficit have not yet been defined. Here we show that the human DYRK1A kinase on chromosome 21 tightly regulates the nuclear levels of Cyclin D1 in embryonic cortical stem (radial glia) cells, and that a modest increase in DYRK1A protein in transgenic embryos lengthens the G1 phase in these progenitors. These alterations promote asymmetric proliferative divisions at the expense of neurogenic divisions, producing a deficit in cortical projection neurons that persists in postnatal stages. Moreover, radial glial progenitors in the Ts65Dn mouse model of Down syndrome have less Cyclin D1, and Dyrk1a is the triplicated gene that causes both early cortical neurogenic defects and decreased nuclear Cyclin D1 levels in this model. These data provide insights into the mechanisms that couple cell cycle regulation and neuron production in cortical neural stem cells, emphasizing that the deleterious effect of DYRK1A triplication in the formation of the cerebral cortex begins at the onset of neurogenesis, which is relevant to the search for early therapeutic interventions in Down syndrome.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) [Grant numbers SAF-2010-17004, SAF2013-46676-P and CSIC-201020I003], the Jérôme Lejeune Foundation, and the Generalitat de Catalunya [Grant number 2009SGR1464]. S.N. was supported by a FI fellowship from the Generalitat de Catalunya [Fellowship number 2011B1-OG242] and by a FPU fellowship from the MINECO [Fellowship number AP2012-3064]. M.J.B. is supported by the program JAE DOC-CSIC/European Social Fund and JA by a FPI fellowship from the MINECO [Fellowship number BES2011-047472]. S.J.C and D.O. were supported by a strategic grant from the Biotechnology and Biological Sciences Research Council (BBSRC). A.L.A. was supported by a BBSRC CASE PhD studentship with AstraZeneca and P.A.L by a grant form the Association for International Cancer ResearchPeer Reviewe

DYRK1A promotes dopaminergic neuron survival in the developing brain and in a mouse model of Parkinson's disease

In the brain, programmed cell death (PCD) serves to adjust the numbers of the different types of neurons during development, and its pathological reactivation in the adult leads to neurodegeneration. Dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in neural proliferation and cell death, and its role during brain growth is evolutionarily conserved. Human DYRK1A lies in the Down syndrome critical region on chromosome 21, and heterozygous mutations in the gene cause microcephaly and neurological dysfunction. The mouse model for DYRK1A haploinsufficiency (the Dyrk1a+/− mouse) presents neuronal deficits in specific regions of the adult brain, including the substantia nigra (SN), although the mechanisms underlying these pathogenic effects remain unclear. Here we study the effect of DYRK1A copy number variation on dopaminergic cell homeostasis. We show that mesencephalic DA (mDA) neurons are generated in the embryo at normal rates in the Dyrk1a haploinsufficient model and in a model (the mBACtgDyrk1a mouse) that carries three copies of Dyrk1a. We also show that the number of mDA cells diminishes in postnatal Dyrk1a+/− mice and increases in mBACtgDyrk1a mice due to an abnormal activity of the mitochondrial caspase9 (Casp9)-dependent apoptotic pathway during the main wave of PCD that affects these neurons. In addition, we show that the cell death induced by 1-methyl-4-phenyl-1,2,3,6 tetrahydropyridine (MPTP), a toxin that activates Casp9-dependent apoptosis in mDA neurons, is attenuated in adult mBACtgDyrk1a mice, leading to an increased survival of SN DA neurons 21 days after MPTP intoxication. Finally, we present data indicating that Dyrk1a phosphorylation of Casp9 at the Thr125 residue is the mechanism by which this kinase hinders both physiological and pathological PCD in mDA neurons. These data provide new insight into the mechanisms that control cell death in brain DA neurons and they show that deregulation of developmental apoptosis may contribute to the phenotype of patients with imbalanced DYRK1A gene dosage.This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) (grant numbers SAF-2010-17004 and CSIC-201020I003 to M.L.A.), the Generalitat de Catalunya (grant number 2009SGR1464), the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III, Spain (to MV, JB and CP), and the I3 Program from the MINECO (to CP). MJB was supported by the CIBERER and by the program JAE DOC-CSIC/European Social Fund, and AL was supported by an FI grant (AGAUR, Generalitat de Catalunya).Peer Reviewe

Impaired macroglial development and axonal conductivity contributes to the neuropathology of DYRK1A-related intellectual disability syndrome

The correct development and activity of neurons and glial cells is necessary to establish proper brain connectivity. DYRK1A encodes a protein kinase involved in the neuropathology associated with Down syndrome that influences neurogenesis and the morphological differentiation of neurons. DYRK1A loss-of-function mutations in heterozygosity cause a well-recognizable syndrome of intellectual disability and autism spectrum disorder. In this study, we analysed the developmental trajectories of macroglial cells and the properties of the corpus callosum, the major white matter tract of the brain, in Dyrk1a+/− mice, a mouse model that recapitulates the main neurological features of DYRK1A syndrome. We found that Dyrk1a+/− haploinsufficient mutants present an increase in astrogliogenesis in the neocortex and a delay in the production of cortical oligodendrocyte progenitor cells and their progression along the oligodendroglial lineage. There were fewer myelinated axons in the corpus callosum of Dyrk1a+/− mice, axons that are thinner and with abnormal nodes of Ranvier. Moreover, action potential propagation along myelinated and unmyelinated callosal axons was slower in Dyrk1a+/− mutants. All these alterations are likely to affect neuronal circuit development and alter network synchronicity, influencing higher brain functions. These alterations highlight the relevance of glial cell abnormalities in neurodevelopmental disorders.This work was supported by Grants from the Spanish Government (MCIN/AEI/10.13039/501100011033, Grants SAF2016-77971-R, PID2019-105902RB-I00 and RED2018-102553-T to M.L.A. and RTI2018-098969-B-100 and PROMETEO/2019/119 to E.F.) and grants from the European Union’s Horizon 2020 programme under Grant agreement No. 899287 to E.F. and from The Jérôme Lejeune Foundation to M.J.B. I.P. received a PhD fellowship from the Spanish Ministry of Economy and Competitivity (MINECO, BES2014-069217) and E.B. received a PhD fellowship from the Spanish Ministry of Education and Science (AP2006-04190). M.J.B. was supported by the CIBERER, an initiative of the ISCIII