The Odad3 Gene Is Necessary for Spermatozoa Development and Male Fertility in Mice.

Odad3 gene loss-of-function mutation leads to Primary Ciliary Dyskinesia (PCD), a disease caused by motile cilia dysfunction. Previously, we demonstrated that knockout of the Odad3 gene in mice replicates several features of PCD, such as hydrocephalus, defects in left-right body symmetry, and male infertility, with a complete absence of sperm in the reproductive tract. The majority of Odad3 knockout animals die before sexual maturation due to severe hydrocephalus and failure to thrive, which precludes fertility studies. Here, we performed the expression analysis of the Odad3 gene during gonad development and in adult testes. We showed that Odad3 starts its expression during the first wave of spermatogenesis, specifically at the meiotic stage, and that its expression is restricted to the germ cells in the adult testes, suggesting that Odad3 plays a role in spermatozoa formation. Subsequently, we conditionally deleted the Odad3 gene in adult males and demonstrated that even partial ablation of the Odad3 gene leads to asthenoteratozoospermia with multiple morphological abnormalities of sperm flagella (MMAF) in mice. The analysis of the seminiferous tubules in Odad3-deficient mice revealed defects in spermatogenesis with accumulation of seminiferous tubules at the spermiogenesis and spermiation phases. Furthermore, analysis of fertility in heterozygous Odad3+/- knockout mice revealed a reduction in sperm count and motility as well as abnormal sperm morphology. Additionally, Odad3+/- males exhibited a shorter fertile lifespan. Overall, these results suggest the important role of Odad3 and Odad3 gene dosage in male fertility. These findings may have an impact on the genetic and fertility counseling practice of PCD patients carrying Odad3 loss-of-function mutations.

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.

First report of seedling stem blight of mango caused by Neofusicoccum parvum in Italy.

Seedling blight of mango (Mangifera indica L.) was observed in a commercial nursery located in Messina province (eastern Sicily, Italy) during winter of 2021. More than 30% of 3,000 seedlings, about three to six months old, of mango cv. Gomera 3 showed symptoms of basal stem blight. The symptoms started from seed, led to the decline and subsequent death of the plants. Necrotic lesions appeared at crown level two months after sowing. The stem tissues of ten symptomatic plants were cut, surface sterilized, dipped in 1.5% sodium hypochlorite for 1 min and transferred onto potato dextrose agar medium (PDA) and incubated at 25°C for four days. Approximately 60% of stem tissues developed very similar fungal colonies, resembling to Botryosphaeriaceae. A total of four representative isolates were collected through single hyphal-tip and stored at 4 °C. The internal transcriber spacer region (ITS) was amplified with primers, ITS5/ITS4 (White et al., 1990), and EF1-728F and EF1-986R (Carbone and Kohn, 1999) were used to amplify part of the translation elongation factor 1alpha gene (tef1-α), and primers Bt2a/Bt2b (Glass and Donaldson, 1995) were used for the partial ß-tubulin (tub2). The obtained sequences were deposited in GenBank with accession numbers: ON911292-95 for the ITS, ON933621-24 for tef1-α and ON933625-28 for tub2.To compare the results, 50 additional sequences were selected and inserted in the alignment according to the recent literature on the Botryosphaeriaceae (Bezerra et al., 2021; Zhang et al., 2021). Maximum parsimony analysis (MP) of concatenated dataset (ITS + tef1-α + tub2) was performed in PAUP v.4.0a. Clade support was assessed by 1,000 bootstrap replicates and Botryosphaeria dothidea was used as an outgroup. Our isolates clustered within the group of Neofusicoccum parvum (71% bootstrap value) (ex-type CMW9081). Based on these results, and morphological data (50 conidia length × width average: 18.1 × 6.6, respectively) our isolates (named MC) were identified and confirmed as Neofusicoccum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. Pathogenicity tests were also conducted on 18 mango cv. Gomera 3 seedlings. The crown roots of each seedling were mechanically wounded and a mycelial plug of the isolate MC14 was placed onto them and covered with soil. Controls (three seedlings) were inoculated with sterile PDA only. Seedlings were maintained in a growth chamber with a 12 hrs photoperiod at 25°C ± 1°C and watered regularly. After five days, stem lesions appeared externally (1.6 cm) and one month after the inoculation, all the inoculated seedlings died. However, controls did not show any obvious symptoms. Re-isolations were conducted as described above and fulfilled Koch's postulates confirming pathogenicity. Among the diseases affecting mango plants, Botryosphaeriaceae represent a serious threat in Sicily as reported by Aiello et al., 2022. The endophytic behaviour of Botryosphaeriaceae is well known, making them latent pathogens (Slippers and Wingfield, 2007). In Italy, N. parvum was detected in mango orchards since 2013 (Ismail et al. 2013), but symptoms of seedlings stem blight have never been reported in the nursery. In Sicily, an increase of Botryosphaeriaceae infection has been observed recently, especially in nurseries, where N. parvum has been identified as a most destructive pathogen (Aiello et al., 2020; Gusella et al., 2021). To our knowledge, this is the first report worldwide of N. parvum causing mango seedling blight. The high incidence of infected seedlings detected in this study highlights the potential risk during propagation in the nursery, representing a significant source of inoculum for the field.

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.

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.

A New Strategy to Improve Management of Citrus Mal Secco Disease Using Bioformulates Based on Bacillusamyloliquefaciens Strains.

The effectiveness of biological commercial products based on Bacillus amyloliquefaciens strains was evaluated through in vitro and in vivo experiments against Plenodomus tracheiphilus. The activity of bacterial cells, volatile organic compounds (VOCs), and culture filtrates of bacteria were tested in vitro against different isolates of P. tracheiphilus. Afterwards, the virulence of these isolates was evaluated on Citrus volkameriana plants to select the most virulent isolate to use in the in vivo experiments. To evaluate the effectiveness of products, C. volkameriana seedlings were pre-treated, twice with biological products and once with standard fungicides, before pathogen inoculation. Moreover, in order to determine the endophytic ability of the bacteria, the population density within the treated citrus stem was determined. Comprehensively, bacterial cells, filtrates, and VOCs were able to significantly reduce the average mycelial diameter of P. tracheiphilus, with some variability according to pathogen isolate. In planta experiments showed that the biological products on average were less effective than fungicides, although all formulates were able to significantly reduce disease incidence and symptom severity, except B. amyloliquefaciens strain D747 (Amylo-X) for symptom severity (SS) 20 days after inoculation. Bacteria were re-isolated from the internal woody tissue of treated plants, showing strong endophytic ability. This work is important as commercial biological products based on B. amyloliquefaciens strains could represent a promising and sustainable alternative for the integrated management of mal secco disease.

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.

Role of Lamin A/C as Candidate Biomarker of Aggressiveness and Tumorigenicity in Glioblastoma Multiforme.

Nuclear lamina components have long been regarded as scaffolding proteins, forming a dense fibrillar structure necessary for the maintenance of the nucleus shape in all the animal kingdom. More recently, mutations, aberrant localisation and deregulation of these proteins have been linked to several diseases, including cancer. Using publicly available data we found that the increased expression levels of the nuclear protein Lamin A/C correlate with a reduced overall survival in The Cancer Genome Atlas Research Network (TCGA) patients affected by glioblastoma multiforme (GBM). We show that the expression of the LMNA gene is linked to the enrichment of cancer-related pathways, particularly pathways related to cell adhesion and cell migration. Mimicking the modulation of LMNA in a GBM preclinical cancer model, we confirmed both in vitro and in vivo that the increased expression of LMNA is associated with an increased aggressiveness and tumorigenicity. In addition, delving into the possible mechanism behind LMNA-induced GBM aggressiveness and tumorigenicity, we found that the mTORC2 component, Rictor, plays a central role in mediating these effects.

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.

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.

Circulating miRNAs in Small Extracellular Vesicles Secreted by a Human Melanoma Xenograft in Mouse Brains.

The identification of liquid biomarkers remains a major challenge to improve the diagnosis of melanoma patients with brain metastases. Circulating miRNAs packaged into tumor-secreted small extracellular vesicles (sEVs) contribute to tumor progression. To investigate the release of tumor-secreted miRNAs by brain metastasis, we developed a xenograft model where human metastatic melanoma cells were injected intracranially in nude mice. The comprehensive profiles of both free miRNAs and those packaged in sEVs secreted by the melanoma cells in the plasma demonstrated that most (80%) of the sEV-associated miRNAs were also present in serum EVs from a cohort of metastatic melanomas, included in a publicly available dataset. Remarkably, among them, we found three miRNAs (miR-224-5p, miR-130a-3p and miR-21-5p) in sEVs showing a trend of upregulation during melanoma progression. Our model is proven to be valuable for identifying miRNAs in EVs that are unequivocally secreted by melanoma cells in the brain and could be associated to disease progression.

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.

Anomalies in Dopamine Transporter Expression and Primary Cilium Distribution in the Dorsal Striatum of a Mouse Model of Niemann-Pick C1 Disease.

The Niemann-Pick type C1 (NPC1) is a rare genetic disease characterized by the accumulation of endocytosed cholesterol and other lipids in the endosome/lysosome compartments. In the brain, the accumulation/mislocalization of unesterified cholesterol, gangliosides and sphingolipids is responsible for the appearance of neuropathological hallmarks, and progressive neurological decline in patients. The imbalance of unesterified cholesterol and other lipids, including GM2 and GM3 gangliosides, alters a number of signaling mechanisms impacting on the overall homeostasis of neurons. In particular, lipid depletion experiments have shown that lipid rafts regulate the cell surface expression of dopamine transporter (DAT) and modulate its activity. Dysregulated dopamine transporter's function results in imbalanced dopamine levels at synapses and severely affects dopamine-induced locomotor responses and dopamine receptor-mediated synaptic signaling. Recent studies begin to correlate dopaminergic stimulation with the length and function of the primary cilium, a non-motile organelle that coordinates numerous signaling pathways. In particular, the absence of dopaminergic D2 receptor stimulation induces the elongation of dorso-striatal neuron's primary cilia. This study has used a mouse model of the NPC1 disease to correlate cholesterol dyshomeostasis with dorso-striatal anomalies in terms of DAT expression and primary cilium (PC) length and morphology. We found that juvenile Npc1nmf164 mice display a reduction of dorso-striatal DAT expression, with associated alterations of PC number, length-frequency distribution, and tortuosity.

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.

Atm reactivation reverses ataxia telangiectasia phenotypes in vivo.

Hereditary deficiencies in DNA damage signaling are invariably associated with cancer predisposition, immunodeficiency, radiation sensitivity, gonadal abnormalities, premature aging, and tissue degeneration. ATM kinase has been established as a central player in DNA double-strand break repair and its deficiency causes ataxia telangiectasia, a rare, multi-system disease with no cure. So ATM represents a highly attractive target for the development of novel types of gene therapy or transplantation strategies. Atm tamoxifen-inducible mouse models were generated to explore whether Atm reconstitution is able to restore Atm function in an Atm-deficient background. Body weight, immunodeficiency, spermatogenesis, and radioresistance were recovered in transgenic mice within 1 month from Atm induction. Notably, life span was doubled after Atm restoration, mice were protected from thymoma and no cerebellar defects were observed. Atm signaling was functional after DNA damage in vivo and in vitro. In summary, we propose a new Atm mouse model to investigate novel therapeutic strategies for ATM activation in ataxia telangiectasia disease.

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.