Systemic delivery of AAV-GCDH ameliorates HLD-induced phenotype in a glutaric aciduria type I mouse model.

Glutaric aciduria type 1 (GA1) is a rare inherited metabolic disorder caused by a deficiency of glutaryl-coenzyme A dehydrogenase (GCDH), with accumulation of neurotoxic metabolites, resulting in a complex movement disorder, irreversible brain damage, and premature death in untreated individuals. While early diagnosis and a lysine restricted diet can extend survival, they do not prevent neurological damage in approximately one-third of treated patients, and more effective therapies are required. Here we report the efficacy of adeno-associated virus 9 (AAV9)-mediated systemic delivery of human GCDH at preventing a high lysine diet (HLD)-induced phenotype in Gcdh -/- mice. Neonatal treatment with AAV-GCDH restores GCDH expression and enzyme activity in liver and striatum. This treatment protects the mice from HLD-aggressive phenotype with all mice surviving this exposure; in stark contrast, a lack of treatment on an HLD triggers very high accumulation of glutaric acid, 3-hydroxyglutaric acid, and glutarylcarnitine in tissues, with about 60% death due to brain accumulation of toxic lysine metabolites. AAV-GCDH significantly ameliorates the striatal neuropathology, minimizing neuronal dysfunction, gliosis, and alterations in myelination. Magnetic resonance imaging findings show protection against striatal injury. Altogether, these results provide preclinical evidence to support AAV-GCDH gene therapy for GA1.

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.

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

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.

DYRK1A Kinase Positively Regulates Angiogenic Responses in Endothelial Cells.

Angiogenesis is a highly regulated process essential for organ development and maintenance, and its deregulation contributes to inflammation, cardiac disorders, and cancer. The Ca2+/nuclear factor of activated T cells (NFAT) signaling pathway is central to endothelial cell angiogenic responses, and it is activated by stimuli like vascular endothelial growth factor (VEGF) A. NFAT phosphorylation by dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) is thought to be an inactivating event. Contrary to expectations, we show that the DYRK family member DYRK1A positively regulates VEGF-dependent NFAT transcriptional responses in primary endothelial cells. DYRK1A silencing reduces intracellular Ca2+ influx in response to VEGF, which dampens NFAT activation. The effect is exerted at the level of VEGFR2 accumulation leading to impairment in PLCγ1 activation. Notably, Dyrk1a heterozygous mice show defects in developmental retinal vascularization. Our data establish a regulatory circuit, DYRK1A/ Ca2+/NFAT, to fine-tune endothelial cell proliferation and angiogenesis.

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

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.

Excitation/inhibition balance and learning are modified by Dyrk1a gene dosage.

Cognitive deficits in Down syndrome (DS) have been linked to increased synaptic inhibition, leading to an imbalance of excitation/inhibition (E/I). Various mouse models and studies from human brains have implicated an HSA21 gene, the serine/threonine kinase DYRK1A, as a candidate for inducing cognitive dysfunction. Here, consequences of alterations in Dyrk1a dosage were assessed in mouse models with varying copy numbers of Dyrk1a: mBACtgDyrk1a, Ts65Dn and Dp(16)1Yey (with 3 gene copies) and Dyrk1a(+/-) (one functional copy). Molecular (i.e. immunoblotting/immunohistochemistry) and behavioral analyses (e.g., rotarod, Morris water maze, Y-maze) were performed in mBACtgDyrk1a mice. Increased expression of DYRK1A in mBACtgDyrk1a induced molecular alterations in synaptic plasticity pathways, particularly expression changes in GABAergic and glutaminergic related proteins. Similar alterations were observed in models with partial trisomy of MMU16, Ts65Dn and Dp(16)1Yey, and were reversed in the Dyrk1a(+/-) model. Dyrk1a overexpression produced an increased number and signal intensity of GAD67 positive neurons, indicating enhanced inhibition pathways in three different models: mBACtgDyrk1a, hYACtgDyrk1a and Dp(16)1Yey. Functionally, Dyrk1a overexpression protected mice from PTZ-induced seizures related to GABAergic neuron plasticity. Our study shows that DYRK1A overexpression affects pathways involved in synaptogenesis and synaptic plasticity and influences E/I balance toward inhibition. Inhibition of DYRK1A activity offers a therapeutic target for DS, but its inhibition/activation may also be relevant for other psychiatric diseases with E/I balance alterations.

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.

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

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.

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.

Vascular endothelial growth factor receptor 3 directly regulates murine neurogenesis.

Neural stem cells (NSCs) are slowly dividing astrocytes that are intimately associated with capillary endothelial cells in the subventricular zone (SVZ) of the brain. Functionally, members of the vascular endothelial growth factor (VEGF) family can stimulate neurogenesis as well as angiogenesis, but it has been unclear whether they act directly via VEGF receptors (VEGFRs) expressed by neural cells, or indirectly via the release of growth factors from angiogenic capillaries. Here, we show that VEGFR-3, a receptor required for lymphangiogenesis, is expressed by NSCs and is directly required for neurogenesis. Vegfr3:YFP reporter mice show VEGFR-3 expression in multipotent NSCs, which are capable of self-renewal and are activated by the VEGFR-3 ligand VEGF-C in vitro. Overexpression of VEGF-C stimulates VEGFR-3-expressing NSCs and neurogenesis in the SVZ without affecting angiogenesis. Conversely, conditional deletion of Vegfr3 in neural cells, inducible deletion in subventricular astrocytes, and blocking of VEGFR-3 signaling with antibodies reduce SVZ neurogenesis. Therefore, VEGF-C/VEGFR-3 signaling acts directly on NSCs and regulates adult neurogenesis, opening potential approaches for treatment of neurodegenerative diseases.

The protein kinase DYRK1A regulates caspase-9-mediated apoptosis during retina development.

The precise regulation of programmed cell death is critical for the normal development of the nervous system. We show here that DYRK1A (minibrain), a protein kinase essential for normal growth, is a negative regulator of the intrinsic apoptotic pathway in the developing retina. We provide evidence that changes in Dyrk1A gene dosage in the mouse strongly alter the cellularity of inner retina layers and result in severe functional alterations. We show that DYRK1A does not affect the proliferation or specification of retina progenitor cells, but rather regulates the number of cells that die by apoptosis. We demonstrate that DYRK1A phosphorylates caspase-9 on threonine residue 125, and that this phosphorylation event is crucial to protect retina cells from apoptotic cell death. Our data suggest a model in which dysregulation of the apoptotic response in differentiating neurons participates in the neuropathology of diseases that display DYRK1A gene-dosage imbalance effects, such as Down's syndrome.

Anosmin-1 modulates the FGF-2-dependent migration of oligodendrocyte precursors in the developing optic nerve.

Oligodendrocyte precursors (OPCs) originate at specific domains within the neural tube before migrating to colonize the entire CNS. Once in their target areas, these cells differentiate into oligodendrocytes, the myelin-forming cells in the CNS. Using the embryonic mouse optic nerve as an experimental model, we have analyzed the influence of FGF-2 on OPC development. FGF-2 exerts a dose-dependent motogenic effect on the migration of plp-dm20+ and it also acts as a chemoattractant on these cells. These effects produced by FGF-2 are principally mediated by the FGFR1 receptor, which is expressed by OPCs. Anosmin-1 is the protein that is defective in the X-linked form of human Kallmann syndrome. This protein is expressed by retinal axons and it also interacts with FGFR1, thereby impairing the migration of OPCs. Because both Anosmin-1 and FGF-2 are present in the optic nerve in vivo, we propose a model whereby the relative concentration of these two proteins modulates the migration of OPCs during development through their interaction with FGFR1. This FGF-2/FGFR1/Anosmin-1 system may be relevant in the context of demyelinating diseases.

VEGF-C is a trophic factor for neural progenitors in the vertebrate embryonic brain.

Vascular endothelial growth factor C (VEGF-C) was first identified as a regulator of the vascular system, where it is required for the development of lymphatic vessels. Here we report actions of VEGF-C in the central nervous system. We detected the expression of the VEGF-C receptor VEGFR-3 in neural progenitor cells in Xenopus laevis and mouse embryos. In Xenopus tadpole VEGF-C knockdowns and in mice lacking Vegfc, the proliferation of neural progenitors expressing VEGFR-3 was severely reduced, in the absence of intracerebral blood vessel defects. In addition, Vegfc-deficient mouse embryos showed a selective loss of oligodendrocyte precursor cells (OPCs) in the embryonic optic nerve. In vitro, VEGF-C stimulated the proliferation of OPCs expressing VEGFR-3 and nestin-positive ventricular neural cells. VEGF-C thus has a new, evolutionary conserved function as a growth factor selectively required by neural progenitor cells expressing its receptor VEGFR-3.

Coordinated functions of Netrin-1 and Class 3 secreted Semaphorins in the guidance of reciprocal septohippocampal connections.

An essential characteristic of the CNS function is the formation of reciprocal connections between brain areas. Although the mechanisms controlling the establishment of neuronal connections are being determined, very little is known about the development of reciprocal connections, which often course along identical pathways. Here, we show that Netrin-1, expressed along the fimbria, chemoattracts both septohippocampal and hippocamposeptal fibers. Moreover, we show that both Semaphorins 3A and 3F expressed in regions nearby the septum prevent the growth of septal axons into these regions. Blocking experiments with recombinant ecto-Neuropilins indicate that both Semaphorins 3A and 3F act cooperatively in the repulsion of septal axons. Furthermore, netrin-1-deficient mice develop a reduced septohippocampal projection. We conclude that the coordinated actions of Netrin-1 and Semaphorins 3A and 3F cooperate in the development of septohippocampal and hippocamposeptal connections, indicating that the same molecular cues serve the construction of reciprocal connections in both directions of growth.