"Be sustainable": EOSC-Life recommendations for implementation of FAIR principles in life science data handling.

The main goals and challenges for the life science communities in the Open Science framework are to increase reuse and sustainability of data resources, software tools, and workflows, especially in large-scale data-driven research and computational analyses. Here, we present key findings, procedures, effective measures and recommendations for generating and establishing sustainable life science resources based on the collaborative, cross-disciplinary work done within the EOSC-Life (European Open Science Cloud for Life Sciences) consortium. Bringing together 13 European life science research infrastructures, it has laid the foundation for an open, digital space to support biological and medical research. Using lessons learned from 27 selected projects, we describe the organisational, technical, financial and legal/ethical challenges that represent the main barriers to sustainability in the life sciences. We show how EOSC-Life provides a model for sustainable data management according to FAIR (findability, accessibility, interoperability, and reusability) principles, including solutions for sensitive- and industry-related resources, by means of cross-disciplinary training and best practices sharing. Finally, we illustrate how data harmonisation and collaborative work facilitate interoperability of tools, data, solutions and lead to a better understanding of concepts, semantics and functionalities in the life sciences.

INFRAFRONTIER: mouse model resources for modelling human diseases.

Over the last decade, INFRAFRONTIER has positioned itself as a world-class Research Infrastructure for the generation, phenotyping, archiving, and distribution of mouse models in Europe. The INFRAFRONTIER network consists of 22 partners from 15 countries, and is continuously enhancing and broadening its portfolio of resources and services that are offered to the research community on a non-profit basis. By bringing together European rodent model expertise and providing valuable disease model services to the biomedical research community, INFRAFRONTIER strives to push the accessibility of cutting-edge human disease modelling technologies across the European research landscape. This article highlights the latest INFRAFRONTIER developments and informs the research community about its extensively utilised services, resources, and technical developments, specifically the intricacies of the INFRAFRONTIER database, use of Curated Disease Models, overview of the INFRAFRONTIER Cancer and Rare Disease resources, and information about its main state-of-the-art services.

Mouse Mutants of Gpr37 and Gpr37l1 Receptor Genes: Disease Modeling Applications.

The vertebrate G protein-coupled receptor 37 and G protein-coupled receptor 37-like 1 (GPR37 and GPR37L1) proteins have amino acid sequence homology to endothelin and bombesin-specific receptors. The prosaposin glycoprotein, its derived peptides, and analogues have been reported to interact with and activate both putative receptors. The GPR37 and GPR37L1 genes are highly expressed in human and rodent brains. GPR37 transcripts are most abundant in oligodendrocytes and in the neurons of the substantia nigra and hippocampus, while the GPR37L1 gene is markedly expressed in cerebellar Bergmann glia astrocytes. The human GPR37 protein is a substrate of parkin, and its insoluble form accumulates in brain samples from patients of inherited juvenile Parkinson's disease. Several Gpr37 and Gpr37l1 mouse mutant strains have been produced and applied to extensive in vivo and ex vivo analyses of respective receptor functions and involvement in brain and other organ pathologies. The genotypic and phenotypic characteristics of the different mouse strains so far published are reported and discussed, and their current and proposed applications to human disease modeling are highlighted.

Gpr37l1/prosaposin receptor regulates Ptch1 trafficking, Shh production, and cell proliferation in cerebellar primary astrocytes.

Mammalian cerebellar astrocytes critically regulate the differentiation and maturation of neuronal Purkinje cells and granule precursors. The G protein-coupled receptor 37-like 1 (Gpr37l1) is expressed by Bergmann astrocytes and interacts with patched 1 (Ptch1) at peri-ciliary membranes. Cerebellar primary astrocyte cultures from wild-type and Gpr37l1 null mutant mouse pups were established and studied. Primary cilia were produced by cultures of both genotypes, as well as Ptch1 and smoothened (Smo) components of the sonic hedgehog (Shh) mitogenic pathway. Compared to wild-type cells, Gpr37l1-/- astrocytes displayed striking increases in proliferative activity, Ptch1 protein expression and internalization, intracellular cholesterol content, ciliary localization of Smo, as well as a marked production of active Shh. Similar effects were reproduced by treating wild-type astrocytes with a putative prosaptide ligand of Gpr37l1. These findings indicate that Gpr37l1-Ptch1 interactions specifically regulate Ptch1 internalization and trafficking, with consequent stimulation of Shh production and activation of proliferative signaling.

Genetic ablation of Gpr37l1 delays tumor occurrence in Ptch1+/- mouse models of medulloblastoma.

The G-protein coupled receptor 37-like 1 (Gpr37l1) is specifically expressed in most astrocytic glial cells, including cerebellar Bergmann astrocytes and interacts with patched 1 (Ptch1), a co-receptor of the sonic hedgehog (Shh)-smoothened (Smo) signaling complex. Gpr37l1 null mutant mice exhibit precocious post-natal cerebellar development, with altered Shh-Smo mitogenic cascade and premature down-regulation of granule cell precursor (GCP) proliferation. Gpr37l1 expression is downregulated in medulloblastoma (MB) and upregulated in glioma and glioblastoma tumors. Shh-associated MBs originate postnatally, from dysregulated hyperproliferation of GCPs in developing cerebellum's external granular layer (EGL), as shown in heterozygous Ptch1+/- knock-out mouse strains that model human MB occurrence and progression. This study investigates cerebellar MB phenotypes in newly produced Gpr37l1, Ptch1 double mutant mice. Natural history analysis shows that Gpr37l1 genetic ablation, in Ptch1+/- model animals, results in marked deferment of post-natal tumor occurrence and decreased incidence of more aggressive tumor types. It is also associated with the delayed and diminished presence of more severe types of hyperplastic lesions in Ptch1+/- mice. Consistently, during early post-natal development Gpr37l1-/-;Ptch1+/- pups exhibit reduction in cerebellar GCP proliferation and EGL thickness and a precocious, sustained expression of wingless-type MMTV integration site member 3 (Wnt3), a specific inhibitor of Shh-induced neuronal mitogenesis, in comparison with Ptch1+/- heterozygous single mutants. These findings highlight the specific involvement of Gpr37l1 in modulating postnatal cerebellar Shh-Ptch1-Smo mitogenic signaling in both normal and pathological conditions. The novel Gpr37l1-/-;Ptch1+/- mouse models may thus be instrumental in the detailed characterization of the initial phases of Shh-associated MB insurgence and development.

Primary Cilia in the Murine Cerebellum and in Mutant Models of Medulloblastoma.

Cellular primary cilia crucially sense and transduce extracellular physicochemical stimuli. Cilium-mediated developmental signaling is tissue and cell type specific. Primary cilia are required for cerebellar differentiation and sonic hedgehog (Shh)-dependent proliferation of neuronal granule precursors. The mammalian G-protein-coupled receptor 37-like 1 is specifically expressed in cerebellar Bergmann glia astrocytes and participates in regulating postnatal cerebellar granule neuron proliferation/differentiation and Bergmann glia and Purkinje neuron maturation. The mouse receptor protein interacts with the patched 1 component of the cilium-associated Shh receptor complex. Mice heterozygous for patched homolog 1 mutations, like heterozygous patched 1 humans, have a higher incidence of Shh subgroup medulloblastoma (MB) and other tumors. Cerebellar cells bearing primary cilia were identified during postnatal development and in adulthood in two mouse strains with altered Shh signaling: a G-protein-coupled receptor 37-like 1 null mutant and an MB-susceptible, heterozygous patched homolog 1 mutant. In addition to granule and Purkinje neurons, primary cilia were also expressed by Bergmann glia astrocytes in both wild-type and mutant animals, from birth to adulthood. Variations in ciliary number and length were related to the different levels of neuronal and glial cell proliferation and maturation, during postnatal cerebellar development. Primary cilia were also detected in pre-neoplastic MB lesions in heterozygous patched homolog 1 mutant mice and they could represent specific markers for the development and analysis of novel cerebellar oncogenic models.

Modulation of Dhh signaling and altered Sertoli cell function in mice lacking the GPR37-prosaposin receptor.

The mammalian G-protein-coupled receptor 37 (GPR37) is expressed in brain, in adult testis, and during the early phase of gonad differentiation. Somatic Sertoli cells (SCs) are located within the seminiferous tubules where they support the germinal epithelium. An adequate number of SCs is required for the complete prepubertal differentiation of germ cells and adult fertility. This study shows that Gpr37 and its ligand prosaposin are both postnatally expressed by SCs, whose proliferation and maturation are affected in Gpr37-null mutant mice during postnatal testicular development. Mutant pups show a delayed timing in sperm cell development, with a partial arrest of spermatocytes at the meiotic pachytene (e.g., 1.5-fold increase in Gpr37(-/-) P21 pups) and their increased apoptosis (e.g., 1.8-fold and 3.5-fold increase in Gpr37(-/-) P21 and adult mice, respectively). Mutant adults have reduced testis weight (wild type, 299 ± 5 mg; knockout, 258 ± 16 mg; P < 0.05) and epididymal sperm count and motility (e.g., 1.5-fold and 1.45-fold decrease in Gpr37(-/-) mice, respectively). Lack of Gpr37 results in the reduction in androgen receptor levels during prepubertal testis development, alongside the altered expression of SC maturation markers. It also affects the prepubertal testis expression of desert hedgehog (Dhh) mitogenic cascade components (Dhh, 1.3-fold increase in Gpr37(-/-) P10 and P21 pups; Gli2, 1.4-fold and 1.6-fold increase in Gpr37(-/-) P10 and P21 pups, respectively) including patched homolog 1 (1.3-fold increase in Gpr37(-/-) P10 and P21 pups), which is found localized in prepubertal SCs and is associated with Gpr37 in cultured primary SC samples. These results indicate that Gpr37 is a specific modulator of murine testis Dhh mitogenic signaling and SC proliferation and maturation.

Precocious cerebellum development and improved motor functions in mice lacking the astrocyte cilium-, patched 1-associated Gpr37l1 receptor.

In the developing cerebellum, the proliferation and differentiation of glial and neuronal cell types depend on the modulation of the sonic hedgehog (Shh) signaling pathway. The vertebrate G-protein-coupled receptor 37-like 1 (GPR37L1) gene encodes a putative G-protein-coupled receptor that is expressed in newborn and adult cerebellar Bergmann glia astrocytes. This study shows that the ablation of the murine Gpr37l1 gene results in premature down-regulation of proliferation of granule neuron precursors and precocious maturation of Bergmann glia and Purkinje neurons. These alterations are accompanied by improved adult motor learning and coordination. Gpr37l1(-/-) mice also exhibit specific modifications of the Shh signaling cascade. Specific assays show that in Bergmann glia cells Gpr37l1 is associated with primary cilium membranes and it specifically interacts and colocalizes with the Shh primary receptor, patched 1. These findings indicate that the patched 1-associated Gpr37l1 receptor participates in the regulation of postnatal cerebellum development by modulating the Shh pathway.

Absence of the GPR37/PAEL receptor impairs striatal Akt and ERK2 phosphorylation, DeltaFosB expression, and conditioned place preference to amphetamine and cocaine.

The orphan G-protein-coupled receptor 37 (GPR37) colocalizes with the dopamine (DA) transporter (DAT) in mouse nigrostriatal presynaptic membranes, and its genetic ablation in homozygous null-mutant (GPR37-KO) mice provokes the marked increase of plasma membrane expression of DAT, alteration of psychostimulant-induced locomotor activity, and reduction of catalepsy induced by DA-receptor antagonists. We report that extracts from GPR37-KO mice displayed biochemical alterations of the nigrostriatal signaling pathways mediated by D1 and D2 dopaminergic receptors. Null-mutant mice showed an increase of the basal phosphorylation level of the D2-regulated Akt kinase. The basal phosphorylation of the D1-activated ERK2 kinase was not altered, but acute treatments with amphetamine or cocaine failed to produce its specific increase, as detected in samples from wild-type littermates. Furthermore, the chronic administration of cocaine to GPR37-KO mice did not increase the expression of the ΔFosB transcription factor isoforms. Consistently, behavioral analysis showed that null-mutant animals did not respond to the incentive properties of amphetamine or cocaine, in conditioned place preference tests. Thus, the lack of GPR37 affects both ERK2- and Akt-mediated striatal signaling pathways, impairing the biochemical and behavioral responses typically induced by acute and chronic administration of psychostimulant drugs.

Induction of macroautophagy by overexpression of the Parkinson's disease-associated GPR37 receptor.

The orphan G-protein-coupled receptor 37 (GPR37) is a substrate of parkin, and its insoluble aggregates accumulate in brain tissue samples of Parkinson's disease patients, including Lewy bodies and neurites. Parkin activates the clearance of the unfolded receptor, while the overexpression of GPR37, in the absence of parkin, can lead to unfolded protein-induced cell death. We found that overexpression of the human GPR37 receptor in HEK293 cells and consequent activation of an endoplasmic reticulum (ER) stress response had effects comparable to starvation, in inducing the cellular autophagic pathway. Treatment with specific modulators provided further evidence for the autophagic clearance of the overexpressed GPR37 protein, in detergent-soluble and -insoluble fractions, as confirmed by the conversion of the microtubule-associated protein 1, light chain 3 (LC3)-I marker to its LC3-II isoform. Furthermore, Gpr37-null mutant mice displayed consistent alterations of ER stress and autophagic pathway markers in brain tissue samples. These findings show that GPR37 overexpression per se can induce cellular autophagy, which may prevent the selective degeneration of GPR37-expressing neurons, as reported for Parkinson's and related neurodegenerative diseases.

GPR37 associates with the dopamine transporter to modulate dopamine uptake and behavioral responses to dopaminergic drugs.

The orphan G protein-coupled receptor 37 (GPR37) is a substrate of parkin; its insoluble aggregates accumulate in brain samples of Parkinson's disease patients. We report here that GPR37 interacts with the dopamine transporter (DAT) and modulates DAT activity. GPR37 and DAT were found colocalized in mouse striatal presynaptic membranes and in transfected cells and their interaction was confirmed by coimmunoprecipitation assays. Gpr37-null mutant mice showed enhanced DAT-mediated dopamine uptake in striatal membrane samples, with a significant increase in the number of plasma membrane DAT molecules. The null mutant mice also exhibited a decrease in cocaine-induced locomotor activity and in catalepsy induced by dopamine receptor antagonists. These results reveal the specific role of GPR37, a putative peptidergic G protein-coupled receptor, in modulating the functional expression of DAT and the behavioral responses to dopaminergic drugs.

Altered dopamine signaling and MPTP resistance in mice lacking the Parkinson's disease-associated GPR37/parkin-associated endothelin-like receptor.

GPR37 is an orphan G protein-coupled receptor expressed in mammalian brain, and its insoluble aggregates are found in the brain samples of juvenile Parkinson's disease patients. We have produced a Gpr37 knock-out mouse strain and identified several phenotypic features that are similar to those reported for mutants of genes encoding components of synaptic dopamine vesicles. Our results reveal an unanticipated role of GPR37 in regulating substantia nigra-striatum dopaminergic signaling. Gpr37(-/-) mice are viable, with normal brain development and anatomy, but they exhibit reduced striatal dopamine content, enhanced amphetamine sensitivity, and specific deficits in motor behavior paradigms sensitive to nigrostriatal dysfunction. These functional alterations are not associated with any substantial loss of substantia nigra neurons or degeneration of striatal dopaminergic afferences, the main histological marks of Parkinson's disease. The inactivation of GPR37, in fact, has protective effects on substantia nigra neurons, causing resistance to treatment with the Parkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.