Virus-infected cells often exhibit dramatic cellular changes accompanied by altered mitochondrial function. The contribution of factors shaping the inner mitochondrial membrane (IMM) and cristae architecture to viral replication is insufficiently understood. Single-cell transcriptomics applying vesicular stomatitis virus infection suggests involvement of factors determining IMM architecture following infection. Consistently, inhibition of the F1FO adenosine triphosphate (ATP) synthase reduces viral replication, which is associated with oligomerization of this complex and altered IMM structure. Moreover, deletion of mitochondrial contact site and cristae organizing system (MICOS) complex by targeting MIC60 results in reduced viral replication. Generation of Mic60inv/invCD11c-Cre+ mice uncovers reduced crista junctions and viral replication in bone marrow-derived dendritic cells. Consequently, reduced viral replication in CD11c-expressing cells limits prolonged immune activation. Altogether, by linking the F1FO ATP synthase and the MICOS complex to viral replication and immune activation, we describe links between the mitochondrial structure-metabolism and the immune response against viral infection.

AIM:The Repressor Element-1 Silencing Transcription Factor (REST) is an epigenetic master regulator playing a crucial role in the nervous system. In early developmental stages, REST downregulation promotes neuronal differentiation and the acquisition of the neuronal phenotype. In addition, postnatal fluctuations in REST expression contribute to shaping neuronal networks and maintaining network homeostasis. Here we investigate the role of the early postnatal deletion of neuronal REST in the assembly and strength of excitatory and inhibitory synaptic connections. METHODS:We investigated excitatory and inhibitory synaptic transmission by patch-clamp recordings in acute neocortical slices in a conditional knockout mouse model (RestGTi) in which Rest was deleted by delivering PHP.eB adeno-associated viruses encoding CRE recombinase under the control of the human synapsin I promoter in the lateral ventricles of P0-P1 pups. RESULTS:We show that, under physiological conditions, Rest deletion increased the intrinsic excitability of principal cortical neurons in the primary visual cortex and the density and strength of excitatory synaptic connections impinging on them, without affecting inhibitory transmission. Conversely, in the presence of a pathological excitation/inhibition imbalance induced by pentylenetetrazol, Rest deletion prevented the increase in synaptic excitation and decreased seizure severity. CONCLUSION:The data indicate that REST exerts distinct effects on the excitability of cortical circuits depending on whether it acts under physiological conditions or in the presence of pathologic network hyperexcitability. In the former case, REST preserves a correct excitatory/inhibitory balance in cortical circuits, while in the latter REST loses its homeostatic activity and may become pro-epileptogenic.

Visual cortical plasticity is high during early life, but gradually decreases with development. This is due to the Otx2-driven maturation of intracortical inhibition that parallels the condensation of extracellular matrix components into perineuronal nets mainly around parvalbumin-positive GABAergic neurons. Repressor Element 1 Silencing Transcription (REST) epigenetically controls the expression of a plethora of neuron-specific genes. We demonstrate that the conditional knockout of REST in the primary visual cortex of adult mice induces a shift of ocular dominance after short-term monocular deprivation and promotes the recovery of vision in long-term deprived animals after reverse suture. These phenomena paralleled a reduction of perineuronal net density and increased expression of REST target genes, but not of the homeoprotein Otx2 in the visual cortex contralateral to the deprived eye. This shows that REST regulates adult visual cortical plasticity and is a potential therapeutic target to restore vision in adult amblyopia by enhancing V1 plasticity.

The repressor element-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) is an epigenetic master regulator that plays a crucial role during nervous system development and maturation. REST function was originally described during development, where it determines neuronal phenotype. However, recent studies showed that REST participates in several processes in the adult brain, including neuronal plasticity and epileptogenesis. In this regard, the relationships between REST and epilepsy are still controversial and need further investigation. As forebrain excitatory neurons are the common final pathway of seizure susceptibility, we investigated the role of REST in epilepsy by inducing REST conditional knockout (REST-cKO) specifically in excitatory neurons of the hippocampus. To target the excitatory neuronal population, we cloned the calcium/calmodulin-dependent protein kinase IIα minimal promoter upstream of Cre recombinase. After assessing the specificity of the promoter's expression, the transgenes were packaged in an engineered adeno-associated virus able to cross the blood–brain and blood–cerebrospinal fluid barriers and delivered in the lateral ventricles of 2-month-old RESTflox/flox mice to characterize, after 1 month, the cognitive phenotype and the seizure propensity. We show that REST-cKO mice display lower levels of anxiety in the light–dark test with respect to control mice but have unaltered motor, social, and cognitive profiles. The evaluation of the susceptibility to epileptic seizures showed that REST-cKO mice are more resistant to pentylenetetrazole-induced kindling but not to seizures induced by a single administration of the convulsant and show higher survival rates. Overall, these data suggest that the absence of REST in forebrain excitatory neurons decreases seizure susceptibility, pointing to a pro-epileptogenic role of the transcriptional repressor under conditions of pathological excitation/inhibition imbalance.

Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non-neuronal cells, including astrocytes, while it is down-regulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocyte displayed a markedly reduced activity of inward rectifying K+ channels subtype 4.1 (Kir4.1) underlying spatial K+ buffering that was associated with a decreased expression and activity of the glutamate transporter-1 (GLT-1) responsible for glutamate uptake by astrocytes. The effects of the impaired astrocyte homeostatic functions on neuronal activity were investigated by co-culturing wild-type hippocampal neurons with NRSF/REST KO astrocytes. Interestingly, neurons experienced increased neuronal excitability at high firing rates associated with decrease after hyperpolarization and increased amplitude of excitatory postsynaptic currents. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that dysfunctions of NRSF/REST expression in astrocytes may contribute to the pathogenesis of neurological disorders.

During aging, brain performances decline. Cellular senescence is one of the aging drivers and a key feature of a variety of human age-related disorders. The transcriptional repressor RE1-silencing transcription factor (REST) has been associated with aging and higher risk of neurodegenerative disorders. However, how REST contributes to the senescence program and functional impairment remains largely unknown. Here, we report that REST is essential to prevent the senescence phenotype in primary mouse neurons. REST deficiency causes failure of autophagy and loss of proteostasis, increased oxidative stress, and higher rate of cell death. Re-establishment of autophagy reverses the main hallmarks of senescence. Our data indicate that REST has a protective role in physiological aging by regulating the autophagic flux and the senescence program in neurons, with implications for neurological disorders associated with aging.

Neuroinflammation is associated with synapse dysfunction and cognitive decline in patients and animal models. One candidate for translating the inflammatory stress into structural and functional changes in neural networks is the transcriptional repressor RE1-silencing transcription factor (REST) that regulates the expression of a wide cluster of neuron-specific genes during neurogenesis and in mature neurons. To study the cellular and molecular pathways activated under inflammatory conditions mimicking the experimental autoimmune encephalomyelitis (EAE) environment, we analyzed REST activity in neuroblastoma cells and mouse cortical neurons treated with activated T cell or microglia supernatant and distinct pro-inflammatory cytokines. We found that REST is activated by a variety of neuroinflammatory stimuli in both neuroblastoma cells and primary neurons, indicating that a vast transcriptional change is triggered during neuroinflammation. While a dual activation of REST and its dominant-negative splicing isoform REST4 was observed in N2a neuroblastoma cells, primary neurons responded with a pure full-length REST upregulation in the absence of changes in REST4 expression. In both cases, REST upregulation was associated with activation of Wnt signaling and increased nuclear translocation of β-catenin, a well-known intracellular transduction pathway in neuroinflammation. Among single cytokines, IL-1β caused a potent and prompt increase in REST transcription and translation in neurons, which promoted a delayed and strong synaptic downscaling specific for excitatory synapses, with decreased frequency and amplitude of spontaneous synaptic currents, decreased density of excitatory synaptic connections, and decreased frequency of action potential-evoked Ca 2+ transients. Most important, the IL-1β effects on excitatory transmission were strictly REST dependent, as conditional deletion of REST completely occluded the effects of IL-1β activation on synaptic transmission and network excitability. Our results demonstrate that REST upregulation represents a new pathogenic mechanism for the synaptic dysfunctions observed under neuroinflammatory conditions and identify the REST pathway as therapeutic target for EAE and, potentially, for multiple sclerosis.

( TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Accumulation of TDP-43 is associated with neuronal death in the brain. How increased and disease-causing mutant forms of TDP-43 induce cell death remains unclear. Here we addressed the role of TDP-43 during neural development and show that reduced TDP-43 causes defects in neural stem/progenitor cell proliferation but not cell death. However, overexpression of wild type and TDP-43A315T proteins induce p53-dependent apoptosis of neural stem/progenitors and human induced pluripotent cell (iPS)-derived immature cortical neurons. We show that TDP-43 induces expression of the proapoptotic BH3-only genes Bbc3 and Bax, and that p53 inhibition rescues TDP-43 induced cell death of embryonic mouse, and human cortical neurons, including those derived from TDP-43(G298S) ALS patient iPS cells. Hence, an increase in wild type and mutant TDP-43 induces p53-dependent cell death in neural progenitors developing neurons and this can be rescued. These findings may have important implications for accumulated or mutant TDP-43 induced neurodegenerative diseases.

The Hsp90 family of molecular chaperones includes the cytosolic isoforms Hsp90a and Hsp90β and the mitochondrial isoform Trap1. Hsp90a/βsupport a large number of client proteins in the cytoplasm and the nucleus whereas Trap1 regulates oxidative phosphorylation in mitochondria. Many of the associated proteins and cellular processes are relevant to cancer, and there is ample pharmacological and genetic evidence to support the idea that Hsp90a/βand Trap1 are required for tumorigenesis. However, a direct and comparative genetic test in a mouse cancer model has not been done. Here we report the effects of deleting the Hsp90a or Trap1 genes in a mouse model of breast cancer. Neither Hsp90a nor Trap1 are absolutely required for mammary tumor initiation, growth and metastasis induced by the polyoma middle T-antigen as oncogene. However, they do modulate growth and lung metastasis in vivo and cell proliferation, migration and invasion of isolated primary carcinoma cells in vitro. Without Hsp90a, tumor burden and metastasis are reduced, correlating with impaired proliferation, migration and invasion of cells in culture. Without Trap1, the appearance of tumors is initially delayed, and isolated cells are affected similarly to those without Hsp90a. Analysis of expression data of human breast cancers supports the conclusion that this is a valid mouse model highlighting the importance of these molecular chaperones.

Novel hypotheses about familial and sporadic mutations are currently established by Genome-wide association screens (GWAS) for a large number of human genetic diseases. Nevertheless, our knowledge about their function is lagging behind. Due to traditional gene targeting in embryonic stem cells (ESC), along with the new repertoire of Zinc Finger Nucleases (ZFN), TALENs, Recombinase-mediated cassette exchange (RMCE) and CRISPR-Cas9 techniques, the increasing number of null, point, conditional, large deletions, inducible, reporter and hypomorphic alleles, combined with additional breeding schemes, different mutant backgrounds and several reporter lines, represent a vast repertoire of molecular tools that provides everything needed to target essentially any gene of interest in virtually any cell type, making imagination the only limiting factor. These techniques will allow us to investigate novel hypotheses in model organisms which are not necessarily limited to the mouse any longer. Correction of inherited mutations in adult human stem cells and even zygotes may be done with great efficiency in the future.

Pf1, also known as Phf12 (plant homeodomain [PHD] zinc finger protein 12), is a member of the PHD zinc finger family of proteins. Pf1 associates with a chromatin-interacting protein complex comprised of MRG15, Sin3B, and histone deacetylase 1 (HDAC1) that functions as a transcriptional modulator. The biological function of Pf1 remains largely elusive. We undertook the generation of Pf1 knockout mice to elucidate its physiological role. We demonstrate that Pf1 is required for mid- to late gestation viability. Pf1 inactivation impairs the proliferative potential of mouse embryonic fibroblasts (MEFs) and is associated with a significant decrease in bromodeoxyuridine incorporation; an increase in senescence-associated β-galactosidase (SA-β-Gal) activity, a marker of cellular senescence; and elevated levels of phosphorylated H2AX (γ-H2A.X), a marker associated with DNA double-strand breaks. Analysis of transcripts differentially expressed in wild-type and Pf1-deficient cells revealed the impact of Pf1 in multiple regulatory arms of the ribosome biogenesis pathways. Strikingly, assessment of the morphology of the nucleoli exposed an abnormal nucleolar structure in Pf1-deficient cells. Finally, proteomic analysis of the Pf1-interacting complexes highlighted proteins involved in ribosome biogenesis. Taken together, our data reveal an unsuspected function for the Pf1-associated chromatin complex in the ribosomal biogenesis and senescence pathways.

The matchless identification of mouse genes or genetic locus is critical in research and in literature to avoid misleading. With this notion in mind, the Mouse Genome and Nomenclature Committee is carrying out the implementation of the mouse nomenclature rules and guidelines. The major aim is to assign unique identifiers to each gene (structural, mitochondrial, nuclear tRNA and rRNA genes); genetic locus (ESTs, mapped genomic DNA fragments, microRNAs, and DNA variants); and chromosomal anomalies and mutations (mutant phenotypes, allelic variations, gene-trap loci, SNPs, and spontaneous and induced mutations). A background in the field and potential controversy are discussed; a summary of the rules and guidelines are given with examples as well.

The dynamic interaction of DNA methylation and transcription factor binding in regulating spatiotemporal gene expression is essential for embryogenesis, but the underlying mechanisms remain understudied. In this study, using mouse models and integration of in vitro and in vivo genetic and epigenetic analyses, we show that the binding of REST (repressor element 1 (RE1) silencing transcription factor; also known as NRSF) to its cognate RE1 sequences is temporally regulated by non-CpG methylation. This process is dependent on DNA methyltransferase 3B (DNMT3B) and leads to suppression of adult cardiac genes in developing hearts. We demonstrate that DNMT3B preferentially mediates non-CpG methylation of REST-targeted genes in the developing heart. Downregulation of DNMT3B results in decreased non-CpG methylation of RE1 sequences, reduced REST occupancy, and consequently release of the transcription suppression during later cardiac development. Together, these findings reveal a critical gene silencing mechanism in developing mammalian hearts that is regulated by the dynamic interaction of DNMT3B-mediated non-CpG methylation and REST binding.

Sphingolipids and the derived gangliosides have critical functions in spermatogenesis, thus mutations in genes involved in sphingolipid biogenesis are often associated with male infertility. We have generated a transgenic mouse line carrying an insertion in the sphingomyelin synthase gene Sms1, the enzyme which generates sphingomyelin species in the Golgi apparatus. We describe the spermatogenesis defect of Sms1(-/-) mice, which is characterized by sloughing of spermatocytes and spermatids, causing progressive infertility of male homozygotes. Lipid profiling revealed a reduction in several long chain unsaturated phosphatidylcholins, lysophosphatidylcholins and sphingolipids in the testes of mutants. Multi-Spectral Optoacoustic Tomography indicated blood-testis barrier dysfunction. A supplementary diet of the essential omega-3 docosahexaenoic acid and eicosapentaenoic acid diminished germ cell sloughing from the seminiferous epithelium and restored spermatogenesis and fertility in 50% of previously infertile mutants. Our findings indicate that SMS1 has a wider than anticipated role in testis polyunsaturated fatty acid homeostasis and for male fertility.

The timely transition from neural progenitor to post-mitotic neuron requires down-regulation and loss of the neuronal transcriptional repressor, REST. Here, we have used mice containing a gene trap in the Rest gene, eliminating transcription from all coding exons, to remove REST prematurely from neural progenitors. We find that catastrophic DNA damage occurs during S-phase of the cell cycle, with long-term consequences including abnormal chromosome separation, apoptosis, and smaller brains. Persistent effects are evident by latent appearance of proneural glioblastoma in adult mice deleted additionally for the tumor suppressor p53 protein (p53). A previous line of mice deleted for REST in progenitors by conventional gene targeting does not exhibit these phenotypes, likely due to a remaining C-terminal peptide that still binds chromatin and recruits co-repressors. Our results suggest that REST-mediated chromatin remodeling is required in neural progenitors for proper S-phase dynamics, as part of its well-established role in repressing neuronal genes until terminal differentiation.

The maintenance and control of pluripotency is of great interest in stem cell biology. The dual specificity T-box/basic-helix-loop-helix-zipper transcription factor Mga is expressed in the pluripotent cells of the inner cell mass (ICM) and epiblast of the peri-implantation mouse embryo, but its function has not been investigated previously. Here, we use a loss-of-function allele and RNA knockdown to demonstrate that Mga depletion leads to the death of proliferating pluripotent ICM cells in vivo and in vitro, and the death of embryonic stem cells (ESCs) in vitro. Additionally, quiescent pluripotent cells lacking Mga are lost during embryonic diapause. Expression of Odc1, the rate-limiting enzyme in the conversion of ornithine into putrescine in the synthesis of polyamines, is reduced in Mga mutant cells, and the survival of mutant ICM cells as well as ESCs is rescued in culture by the addition of exogenous putrescine. These results suggest a mechanism whereby Mga influences pluripotent cell survival through regulation of the polyamine pool in pluripotent cells of the embryo, whether they are in a proliferative or quiescent state.

Receptor families of the innate immune response engage in 'cross-talk' to tailor optimal immune responses against invading pathogens. However, these responses are subject to multiple levels of regulation to keep in check aberrant inflammatory signals. Here, we describe a role for the orphan receptor interleukin-17 receptor D (IL-17RD) in negatively regulating Toll-like receptor (TLR)-induced responses. Deficiency of IL-17RD expression in cells leads to enhanced pro-inflammatory signalling and gene expression in response to TLR stimulation, and Il17rd(-/-) mice are more susceptible to TLR-induced septic shock. We demonstrate that the intracellular Sef/IL-17R (SEFIR) domain of IL-17RD targets TIR adaptor proteins to inhibit TLR downstream signalling thus revealing a paradigm involving cross-regulation of members of the IL-17R and TLR families.

Recently, mutations in the mitochondrial translation optimization factor 1 gene (MTO1) were identified as causative in children with hypertrophic cardiomyopathy, lactic acidosis and respiratory chain defect. Here, we describe an MTO1-deficient mouse model generated by gene trap mutagenesis that mirrors the human phenotype remarkably well. As in patients, the most prominent signs and symptoms were cardiovascular and included bradycardia and cardiomyopathy. In addition, the mutant mice showed a marked worsening of arrhythmias during induction and reversal of anaesthesia. The detailed morphological and biochemical workup of murine hearts indicated that the myocardial damage was due to complex I deficiency and mitochondrial dysfunction. In contrast, neurological examination was largely normal in Mto1-deficient mice. A translational consequence of this mouse model may be to caution against anaesthesia-related cardiac arrhythmias which may be fatal in patients.