Absence of ATM leads to altered NK cell function in mice.

Ataxia-telangiectasia (A-T) is a rare disorder caused by genetic defects of A-T mutated (ATM) kinase, a key regulator of stress response, and characterized by neurodegeneration, immunodeficiency, and high incidence of cancer. Here we investigated NK cells in a mouse model of A-T (Atm-/-) showing that they are strongly impaired at killing tumor cells due to a block of early signaling events. On the other hand, in Atm-/- littermates with thymic lymphoma NK cell cytotoxicity is enhanced as compared with ATM-proficient mice, possibly via tumor-produced TNF-α. Results also suggest that expansion of exhausted NKG2D+ NK cells in Atm-/- mice is driven by low-level expression of stress-inducible NKG2D ligands, whereas development of thymoma expressing the high-affinity MULT1 ligand is associated with NKG2D down-regulation on NK cells. These results expand our understanding of immunodeficiency in A-T and encourage exploring NK cell biology in A-T patients in the attempt to identify cancer predictive biomarkers and novel therapeutic targets.

Identification of the GlialCAM interactome: the G protein-coupled receptors GPRC5B and GPR37L1 modulate megalencephalic leukoencephalopathy proteins.

Megalencephalic Leukoencephalopathy with subcortical Cysts (MLC) is a type of vacuolating leukodystrophy, which is mainly caused by mutations in MLC1 or GLIALCAM. The two MLC-causing genes encode for membrane proteins of yet unknown function that have been linked to the regulation of different chloride channels such as the ClC-2 and VRAC. To gain insight into the role of MLC proteins, we have determined the brain GlialCAM interacting proteome. The proteome includes different transporters and ion channels known to be involved in the regulation of brain homeostasis, proteins related to adhesion or signaling as several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptor GPR37L1. Focusing on these two GPCRs, we could validate that they interact directly with MLC proteins. The inactivation of Gpr37l1 in mice upregulated MLC proteins without altering their localization. Conversely, a reduction of GPRC5B levels in primary astrocytes downregulated MLC proteins, leading to an impaired activation of ClC-2 and VRAC. The interaction between the GPCRs and MLC1 was dynamically regulated upon changes in the osmolarity or potassium concentration. We propose that GlialCAM and MLC1 associate with different integral membrane proteins modulating their functions and acting as a recruitment site for various signaling components as the GPCRs identified here. We hypothesized that the GlialCAM/MLC1 complex is working as an adhesion molecule coupled to a tetraspanin-like molecule performing regulatory effects through direct binding or influencing signal transduction events.

GPR37 and Related Receptors: Disease Regulation.

The vertebrate G protein-coupled receptors 37 and 37-like 1 (GPR37 and GPR37L1) were discovered more than two decades ago, and they have been implicated in various neurological and neurodegenerative diseases, as well as in inflammatory pain and tumorigenesis [...].

Transcriptome programs involved in the development and structure of the cerebellum.

In the past two decades, mounting evidence has modified the classical view of the cerebellum as a brain region specifically involved in the modulation of motor functions. Indeed, clinical studies and engineered mouse models have highlighted cerebellar circuits implicated in cognitive functions and behavior. Furthermore, it is now clear that insults occurring in specific time windows of cerebellar development can affect cognitive performance later in life and are associated with neurological syndromes, such as Autism Spectrum Disorder. Despite its almost homogenous cytoarchitecture, how cerebellar circuits form and function is not completely elucidated yet. Notably, the apparently simple neuronal organization of the cerebellum, in which Purkinje cells represent the only output, hides an elevated functional diversity even within the same neuronal population. Such complexity is the result of the integration of intrinsic morphogenetic programs and extracellular cues from the surrounding environment, which impact on the regulation of the transcriptome of cerebellar neurons. In this review, we briefly summarize key features of the development and structure of the cerebellum before focusing on the pathways involved in the acquisition of the cerebellar neuron identity. We focus on gene expression and mRNA processing programs, including mRNA methylation, trafficking and splicing, that are set in motion during cerebellar development and participate to its physiology. These programs are likely to add new layers of complexity and versatility that are fundamental for the adaptability of cerebellar neurons.

GPR37 Receptors and Megalencephalic Leukoencephalopathy with Subcortical Cysts.

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a rare type of vacuolating leukodystrophy (white matter disorder), which is mainly caused by defects in MLC1 or glial cell adhesion molecule (GlialCAM) proteins. In addition, autoantibodies to GlialCAM are involved in the pathology of multiple sclerosis. MLC1 and GLIALCAM genes encode for membrane proteins of unknown function, which has been linked to the regulation of different ion channels and transporters, such as the chloride channel VRAC (volume regulated anion channel), ClC-2 (chloride channel 2), and connexin 43 or the Na+/K+-ATPase pump. However, the mechanisms by which MLC proteins regulate these ion channels and transporters, as well as the exact function of MLC proteins remain obscure. It has been suggested that MLC proteins might regulate signalling pathways, but the mechanisms involved are, at present, unknown. With the aim of answering these questions, we have recently described the brain GlialCAM interactome. Within the identified proteins, we could validate the interaction with several G protein-coupled receptors (GPCRs), including the orphan GPRC5B and the proposed prosaposin receptors GPR37L1 and GPR37. In this review, we summarize new aspects of the pathophysiology of MLC disease and key aspects of the interaction between GPR37 receptors and MLC proteins.

A Quantitative Assay for Ca2+ Uptake through Normal and Pathological Hemichannels.

Connexin (Cx) hemichannels (HCs) are large pore hexameric structures that allow the exchange of ions, metabolites and a variety of other molecules between the cell cytoplasm and extracellular milieu. HC inhibitors are attracting growing interest as drug candidates because deregulated fluxes through HCs have been implicated in a plethora of genetic conditions and other diseases. HC activity has been mainly investigated by electrophysiological methods and/or using HC-permeable dye uptake measurements. Here, we present an all-optical assay based on fluorometric measurements of ionized calcium (Ca2+) uptake with a Ca2+-selective genetically encoded indicator (GCaMP6s) that permits the optical tracking of cytosolic Ca2+ concentration ([Ca2+]cyt) changes with high sensitivity. We exemplify use of the assay in stable pools of HaCaT cells overexpressing human Cx26, Cx46, or the pathological mutant Cx26G45E, under control of a tetracycline (Tet) responsive element (TRE) promoter (Tet-on). We demonstrate the usefulness of the assay for the characterization of new monoclonal antibodies (mAbs) targeting the extracellular domain of the HCs. Although we developed the assay on a spinning disk confocal fluorescence microscope, the same methodology can be extended seamlessly to high-throughput high-content platforms to screen other kinds of inhibitors and/or to probe HCs expressed in primary cells and microtissues.

Atrophy, oxidative switching and ultrastructural defects in skeletal muscle of the ataxia telangiectasia mouse model.

Ataxia telangiectasia is a rare, multi system disease caused by ATM kinase deficiency. Atm-knockout mice recapitulate premature aging, immunodeficiency, cancer predisposition, growth retardation and motor defects, but not cerebellar neurodegeneration and ataxia. We explored whether Atm loss is responsible for skeletal muscle defects by investigating myofiber morphology, oxidative/glycolytic activity, myocyte ultrastructural architecture and neuromuscular junctions. Atm-knockout mice showed reduced muscle and fiber size. Atrophy, protein synthesis impairment and a switch from glycolytic to oxidative fibers were detected, along with an increase of in expression of slow and fast myosin types (Myh7, and Myh2 and Myh4, respectively) in tibialis anterior and solei muscles isolated from Atm-knockout mice. Transmission electron microscopy of tibialis anterior revealed misalignments of Z-lines and sarcomeres and mitochondria abnormalities that were associated with an increase in reactive oxygen species. Moreover, neuromuscular junctions appeared larger and more complex than those in Atm wild-type mice, but with preserved presynaptic terminals. In conclusion, we report for the first time that Atm-knockout mice have clear morphological skeletal muscle defects that will be relevant for the investigation of the oxidative stress response, motor alteration and the interplay with peripheral nervous system in ataxia telangiectasia.

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.

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.

Generation of Connexin-Expressing Stable Cell Pools.

Stable cell pools have the advantage of providing a definite, consistent, and reproducible transmission of a transgene of interest, compared to transient expression from a plasmid transfection. Stably expressing a transgene of interest in cells under induction is a powerful way to (switch on and) study a gene function in both in vitro and in vivo assays. Taking advantage of the ability of lentivirus (LV) to promote transgene delivery, and genomic integration and expression in both dividing and nondividing cells, a doxycycline-inducible transfer vector expressing a bicistronic transgene was developed to study the function of connexins in HeLa DH cells. Here, delving on connexin 32 (Cx32), we report how to use the backbone of this vector as a tool to generate stable pools to study the function of a gene of interest (GOI), especially with assays involving Ca2+ imaging, employing the GCaMP6s indicator. We describe a step-by-step protocol to produce the LV particle by transient transfection and the direct use of the harvested LV stock to generate stable cell pools. We further present step-by-step immunolabeling protocols to characterize the transgene protein expression by confocal microscopy using an antibody that targets an extracellular domain epitope of Cx32 in living cells, and in fixed permeabilized cells using high affinity anti-Cx32 antibodies. Using common molecular biology laboratory techniques, this protocol can be adapted to generate stable pools expressing any transgene of interest, for both in vitro and in vivo functional assays, including molecular, immune, and optical assays.

Reduced Cerebellar BDNF Availability Affects Postnatal Differentiation and Maturation of Granule Cells in a Mouse Model of Cholesterol Dyshomeostasis.

Niemann-Pick type C1 (NPC1) disease is a lysosomal lipid storage disorder due to mutations in the NPC1 gene resulting in the accumulation of cholesterol within the endosomal/lysosomal compartments. The prominent feature of the disorder is the progressive Purkinje cell degeneration leading to ataxia.In a mouse model of NPC1 disease, we have previously demonstrated that impaired Sonic hedgehog signaling causes defective proliferation of granule cells (GCs) and abnormal cerebellar morphogenesis. Studies conducted on cortical and hippocampal neurons indicate a functional interaction between Sonic hedgehog and brain-derived neurotrophic factor (BDNF) expression, leading us to hypothesize that BDNF signaling may be altered in Npc1 mutant mice, contributing to the onset of cerebellar alterations present in NPC1 disease before the appearance of signs of ataxia.We characterized the expression/localization patterns of the BDNF and its receptor, tropomyosin-related kinase B (TrkB), in the early postnatal and young adult cerebellum of the Npc1nmf164 mutant mouse strain.In Npc1nmf164 mice, our results show (i) a reduced expression of cerebellar BDNF and pTrkB in the first 2 weeks postpartum, phases in which most GCs complete the proliferative/migrative program and begin differentiation; (ii) an altered subcellular localization of the pTrkB receptor in GCs, both in vivo and in vitro; (iii) reduced chemotactic response to BDNF in GCs cultured in vitro, associated with impaired internalization of the activated TrkB receptor; (iv) an overall increase in dendritic branching in mature GCs, resulting in impaired differentiation of the cerebellar glomeruli, the major synaptic complex between GCs and mossy fibers.

Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.

BACKGROUND: Keratitis ichthyosis deafness (KID) syndrome is a rare disorder caused by hemichannel (HC) activating gain-of-function mutations in the GJB2 gene encoding connexin (Cx) 26, for which there is no cure, or current treatments based upon the mechanism of disease causation. METHODS: We applied Adeno Associated Virus (AAV) mediated mAb gene transfer (AAVmAb) to treat the epidermal features of KID syndrome with a well-characterized HC blocking antibody using male mice of a murine model that replicates the skin pathology of the human disease. FINDINGS: We demonstrate that in vivo AAVmAb treatment significantly reduced the size and thickness of KID lesions, in addition to blocking activity of mutant HCs in the epidermis in vivo. We also show that AAVmAb treatment eliminated abnormal keratinocyte proliferation and enlarged cell size, decreased apoptosis, and restored the normal distribution of keratin expression. INTERPRETATION: Our findings reinforce the critical role played by increased HC activity in the skin pathology associated with KID syndrome. They also underscore the clinical potential of anti-HC mAbs coupled with genetic based delivery systems for treating the underlying mechanistic basis of this disorder. Inhibition of HC activity is an ideal therapeutic target in KID syndrome, and the genetic delivery of mAbs targeted against mutant HCs could form the basis of new therapeutic interventions to treat this incurable disease. FUNDING: Fondazione Telethon grant GGP19148 and University of Padova grant Prot. BIRD187130 to FM; Foundation for Ichthyosis and Related Skin Types (FIRST) and National Institutes of Health grant EY 026911 to TWW.

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.

Transmembrane Protein TMEM230, a Target of Glioblastoma Therapy.

Glioblastomas (GBM) are the most aggressive tumors originating in the brain. Histopathologic features include circuitous, disorganized, and highly permeable blood vessels with intermittent blood flow. These features contribute to the inability to direct therapeutic agents to tumor cells. Known targets for anti-angiogenic therapies provide minimal or no effect in overall survival of 12-15 months following diagnosis. Identification of novel targets therefore remains an important goal for effective treatment of highly vascularized tumors such as GBM. We previously demonstrated in zebrafish that a balanced level of expression of the transmembrane protein TMEM230/C20ORF30 was required to maintain normal blood vessel structural integrity and promote proper vessel network formation. To investigate whether TMEM230 has a role in the pathogenesis of GBM, we analyzed its prognostic value in patient tumor gene expression datasets and performed cell functional analysis. TMEM230 was found necessary for growth of U87-MG cells, a model of human GBM. Downregulation of TMEM230 resulted in loss of U87 migration, substratum adhesion, and re-passaging capacity. Conditioned media from U87 expressing endogenous TMEM230 induced sprouting and tubule-like structure formation of HUVECs. Moreover, TMEM230 promoted vascular mimicry-like behavior of U87 cells. Gene expression analysis of 702 patients identified that TMEM230 expression levels distinguished high from low grade gliomas. Transcriptomic analysis of patients with gliomas revealed molecular pathways consistent with properties observed in U87 cell assays. Within low grade gliomas, elevated TMEM230 expression levels correlated with reduced overall survival independent from tumor subtype. Highest level of TMEM230 correlated with glioblastoma and ATP-dependent microtubule kinesin motor activity, providing a direction for future therapeutic intervention. Our studies support that TMEM230 has both glial tumor and endothelial cell intracellular and extracellular functions. Elevated levels of TMEM230 promote glial tumor cell migration, extracellular scaffold remodeling, and hypervascularization and abnormal formation of blood vessels. Downregulation of TMEM230 expression may inhibit both low grade glioma and glioblastoma tumor progression and promote normalization of abnormally formed blood vessels. TMEM230 therefore is both a promising anticancer and antiangiogenic therapeutic target for inhibiting GBM tumor cells and tumor-driven angiogenesis.

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.

A Dynamic Splicing Program Ensures Proper Synaptic Connections in the Developing Cerebellum.

Tight coordination of gene expression in the developing cerebellum is crucial for establishment of neuronal circuits governing motor and cognitive function. However, transcriptional changes alone do not explain all of the switches underlying neuronal differentiation. Here we unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. The motifs enriched in modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints by directly binding near the 3' splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, resulting in motor coordination defects, ataxia, and abnormal social behavior. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and establishes motor and cognitive circuitry in the developing cerebellum.

GPR37 Signaling Modulates Migration of Olfactory Ensheathing Cells and Gonadotropin Releasing Hormone Cells in Mice.

Gonadotropin releasing hormone (GnRH) neurons, part of the hypothalamic-pituitary-gonadal axis, regulate reproduction. Prenatally, GnRH neurons migrate into the brain from the nasal placode along terminal nerve fibers, intermixed with olfactory sensory axons and olfactory ensheathing cells (OECs). An expression analysis from embryonic GnRH neurons identified the G protein-coupled receptor 37 (GPR37 or PAEL-r). GPR37 has been linked to (1) juvenile Parkinson's disease in humans, (2) oligodendrocyte differentiation, and (3) Wnt/ß-catenin signaling during neurogenesis. In this study, the role of GPR37 was investigated in the developing GnRH/olfactory system. PCR and immunocytochemistry confirmed expression of GPR37 in migrating GnRH neurons as well as in OECs. Inhibition of GPR37 signaling in nasal explants attenuated GnRH neuronal migration and OEC movement. Examination of GPR37 deficient mice revealed a decrease in the olfactory bulb nerve layer and attenuated/delayed maturation and migration of GnRH neurons into the brain. These data demonstrate a developmental role for GPR37 signaling in neural migration. SIGNIFICANCE STATEMENT: Reproduction is controlled by gonadotrophin releasing hormone (GnRH) neurons located in the central nervous system. Embryonically, GnRH neurons originate in the nasal/olfactory placode and migrate into the brain on axonal tracks from cells in the vomeronasal organ, intermixed with olfactory sensory axons and olfactory ensheathing cells (OECs). An expression analysis from embryonic GnRH neurons identified the G protein-coupled receptor 37. Here we show that inhibition of GPR37 signaling in nasal explants and mutant mice attenuated GnRH neuronal migration. Signaling via GPR37 also perturbed OEC movement, resulting in a decrease in the olfactory bulb nerve layer in vivo. Together, these results identify a new role for GPR37 signaling during development - modulating cell migration.

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.