Neurological changes as a consequence of basement membrane defects- studies in the nidogen knockout mouse

Introduction: The basement membrane is a highly specialised extracellular matrix found underlying all endothelia and epithelia and surrounding many mesenchymal cells, in particular myocytes, peripheral nerves and adipocytes. Basement membranes have numerous physical and signalling functions that alter with the specific tissue type and stage of development. Nidogens are in mammals a family of two proteins nidogen -1 and 2. In vitro studies have indicated that the 150 kDa glycoprotein nidogen-1 may play a central role in the supramolecular organization of basement membranes through binding to a range of extracellular proteins. To elucidate their importance in the basement membrane, mice were generated with mutated alleles of the NID-1 gene. Ultrastructural analysis of kidney and skeletal muscle from mice lacking nidogen-1 failed to reveal differences in morphology and basement membrane structure. However, these mice show signs of neurological impairment with ataxia, especially of the hind limbs, and spontaneous seizure activity. Nidogen-2 staining in these animals is increased in certain basement membranes, particularly in cardiac and skeletal muscle, where it is normally found in scant amounts, suggesting that the loss of nidogen-1 may be compensated by nidogen-2. Mice have been generated lacking nidogen-1 and one or both alleles of NID-2. Mice lacking both nidogen-1 alleles and heterozygous of nidogen-2 show more severe neurological defects, than those lacking only nidogen -1. Aim of the project: To study the behavioural, electro-physiological, neurological and cellular aspects of the nidogen knockout mice (NID1 - -/NID2 + +) and (NID1 - -/NID2 + -), to gain a further insight into the function of this protein family. Results: The neurological defects were studied using rotarod tests, in vivo EEG recordings and in vitro hippocampal and neocortical field potential recordings analysing input/output relationships and short- and long term plasticity (paired-pulse behaviour and LTP). In vivo, the animals displayed massive functional deficits in the rotarod test and epileptiform discharges in EEG recordings. In vitro, in the hippocampus, 28% of the slices showed spontaneous, and another 33% evoked spontaneous epileptiform activity. Significant increases of the input/output ratio of synaptically evoked responses in CA1 and dentate gyrus, as well as of paired pulse accentuation, and loss of perforant path LTP was observed. By contrast, in the neocortex, the input/output ratio and paired-pulse accentuation were reduced. To augment the in vivo studies, mouse and human forms of nidogen-1 and-2 were cloned, recombinantly expressed and purified. Laminin was extracted from both control and nidogen knockout mice and the biochemical aspects of basement membrane deposition which seemed to be altered in the absence of nidogen was studied. A possible down-regulation of laminin and its receptors was investigated. Discussion: The results reveal the epileptic nature of the mice in the absence of nidogen-1. Alterations in synaptic plasticity and network function in the nidogen-1 null animals are indicative of a novel role for a nidogen-1, a protein found only in basement membranes. Also, it suggests that nidogen-1 is important for maintaining the structural integrity of basement membranes

Autonomous Vascular Networks Synchronize GABA Neuron Migration in the Embryonic Forebrain

GABA neurons, born in remote germinative zones in the ventral forebrain (telencephalon), migrate tangentially in two spatially distinct streams to adopt their specific positions in the developing cortex. The cell types and molecular cues that regulate this divided migratory route remains to be elucidated. Here we show that embryonic vascular networks are strategically positioned to fulfill the task of providing support as well as critical guidance cues that regulate the divided migratory routes of GABA neurons in the telencephalon. Interestingly, endothelial cells of the telencephalon are not homogeneous in their gene expression profiles. Endothelial cells of the periventricular vascular network have molecular identities distinct from those of the pial network. Our data suggest that periventricular endothelial cells have intrinsic programs that can significantly mold neuronal development and uncovers new insights into concepts and mechanisms of CNS angiogenesis from both developmental and disease perspectives

NAD+ protects against EAE by regulating CD4+ T-cell differentiation

CD4+ T cells are involved in the development of autoimmunity, including multiple sclerosis (MS). Here we show that nicotinamide adenine dinucleotide (NAD+) blocks experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by inducing immune homeostasis through CD4+IFNγ+IL-10+ T cells and reverses disease progression by restoring tissue integrity via remyelination and neuroregeneration. We show that NAD+ regulates CD4+ T-cell differentiation through tryptophan hydroxylase-1 (Tph1), independently of well-established transcription factors. In the presence of NAD+, the frequency of T-bet−/− CD4+IFNγ+ T cells was twofold higher than wild-type CD4+ T cells cultured in conventional T helper 1 polarizing conditions. Our findings unravel a new pathway orchestrating CD4+ T-cell differentiation and demonstrate that NAD+ may serve as a powerful therapeutic agent for the treatment of autoimmune and other diseases

Mast cells regulate CD4+ T-cell differentiation in the absence of antigen presentation

Producción CientíficaBackground: Given their unique capacity for antigen uptake, processing, and presentation, antigen-presenting cells (APCs) are critical for initiating and regulating innate and adaptive immune responses. We have previously shown the role of nicotinamide adenine dinucleotide (NAD+) in T-cell differentiation independently of the cytokine milieu, whereas the precise mechanisms remained unknown. Objective: The objective of this study is to further dissect the mechanism of actions of NAD+ and determine the effect of APCs on NAD+-mediated T-cell activation. Methods: Isolated dendritic cells and bone marrow–derived mast cells (MCs) were used to characterize the mechanisms of action of NAD+ on CD4+ T-cell fate in vitro. Furthermore, NAD+-mediated CD4+ T-cell differentiation was investigated in vivo by using wild-type C57BL/6, MC−/−, MHC class II−/−, Wiskott-Aldrich syndrome protein (WASP)−/−, 5C.C7 recombination-activating gene 2 (Rag2)−/−, and CD11b-DTR transgenic mice. Finally, we tested the physiologic effect of NAD+ on the systemic immune response in the context of Listeria monocytogenes infection. Results: Our in vivo and in vitro findings indicate that after NAD+ administration, MCs exclusively promote CD4+ T-cell differentiation, both in the absence of antigen and independently of major APCs. Moreover, we found that MCs mediated CD4+ T-cell differentiation independently of MHC II and T-cell receptor signaling machinery. More importantly, although treatment with NAD+ resulted in decreased MHC II expression on CD11c+ cells, MC-mediated CD4+ T-cell differentiation rendered mice resistant to administration of lethal doses of L monocytogenes. Conclusions: Collectively, our study unravels a novel cellular and molecular pathway that regulates innate and adaptive immunity through MCs exclusively and underscores the therapeutic potential of NAD+ in the context of primary immunodeficiencies and antimicrobial resistance.National Institutes of Health (grants R01NS073635 , R01MH110438 , R01HL096795 , U01HL126497 and R01AG039449)Instituto de Salud Carlos III (grant PI10/02 511)Fundación Ramón Areces (grant CIVP16A1843

Endothelial VEGF sculpts cortical cytoarchitecture

Current models of brain development support the view that VEGF, a signaling protein secreted by neuronal cells, regulates angiogenesis and neuronal development. Here we demonstrate an autonomous and pivotal role for endothelial cell-derived VEGF that has far-reaching consequences for mouse brain development. Selective deletion of Vegf from endothelial cells resulted in impaired angiogenesis and marked perturbation of cortical cytoarchitecture. Abnormal cell clusters or heterotopias were detected in the marginal zone, and disorganization of cortical cells induced several malformations, including aberrant cortical lamination. Critical events during brain development-neuronal proliferation, differentiation, and migration were significantly affected. In addition, axonal tracts in the telencephalon were severely defective in the absence of endothelial VEGF. The unique roles of endothelial VEGF cannot be compensated by neuronal VEGF and underscores the high functional significance of endothelial VEGF for cerebral cortex development and from disease perspectives

Aspects of Tryptophan and Nicotinamide Adenine Dinucleotide in Immunity: A New Twist in an Old Tale

Increasing evidence underscores the interesting ability of tryptophan to regulate immune responses. However, the exact mechanisms of tryptophan’s immune regulation remain to be determined. Tryptophan catabolism via the kynurenine pathway is known to play an important role in tryptophan’s involvement in immune responses. Interestingly, quinolinic acid, which is a neurotoxic catabolite of the kynurenine pathway, is the major pathway for the de novo synthesis of nicotinamide adenine dinucleotide (NAD+). Recent studies have shown that NAD+, a natural coenzyme found in all living cells, regulates immune responses and creates homeostasis via a novel signaling pathway. More importantly, the immunoregulatory properties of NAD+ are strongly related to the overexpression of tryptophan hydroxylase 1 (Tph1). This review provides recent knowledge of tryptophan and NAD+ and their specific and intriguing roles in the immune system. Furthermore, it focuses on the mechanisms by which tryptophan regulates NAD+ synthesis as well as innate and adaptive immune responses