ObjectiveMicroRNA have recently been identified as regulators that modulate target gene expression and are involved in shaping the immune response. This study was undertaken to investigate the contribution of microRNA‐146a (miR‐146a), which was identified in the pilot expression profiling step, to the pathogenesis of systemic lupus erythematosus (SLE).MethodsTaqMan microRNA assays of peripheral blood leukocytes were used for comparison of expression levels of microRNA between SLE patients and controls. Transfection and stimulation of cultured cells were conducted to determine the biologic function of miR‐146a. Bioinformatics prediction and validation by reporter gene assay and Western blotting were performed to identify miR‐146a targets.ResultsProfiling of 156 miRNA in SLE patients revealed the differential expression of multiple microRNA, including miR‐146a, a negative regulator of innate immunity. Further analysis showed that underexpression of miR‐146a negatively correlated with clinical disease activity and with interferon (IFN) scores in patients with SLE. Of note, overexpression of miR‐146a reduced, while inhibition of endogenous miR‐146a increased, the induction of type I IFNs in peripheral blood mononuclear cells (PBMCs). Furthermore, miR‐146a directly repressed the transactivation downstream of type I IFN. At the molecular level, miR‐146a could target IFN regulatory factor 5 and STAT‐1. More importantly, introduction of miR‐146a into the patients' PBMCs alleviated the coordinate activation of the type I IFN pathway.ConclusionThe microRNA miR‐146a is a negative regulator of the IFN pathway. Underexpression of miR‐146a contributes to alterations in the type I IFN pathway in lupus patients by targeting the key signaling proteins. The findings provide potential novel strategies for therapeutic intervention.

Systemic lupus erythematosus is a complex autoimmune disease caused by genetic and epigenetic alterations. DNA methylation abnormalities play an important role in systemic lupus erythematosus disease processes. MicroRNAs (miRNAs) have been implicated as fine-tuning regulators controlling diverse biological processes at the level of posttranscriptional repression. Dysregulation of miRNAs has been described in various disease states, including human lupus. Whereas previous studies have shown miRNAs can regulate DNA methylation by targeting the DNA methylation machinery, the role of miRNAs in aberrant CD4+ T cell DNA hypomethylation of lupus is unclear. In this study, by using high-throughput microRNA profiling, we identified that two miRNAs (miR-21 and miR-148a) overexpressed in CD4+ T cells from both patients with lupus and lupus-prone MRL/lpr mice, which promote cell hypomethylation by repressing DNA methyltransferase 1 (DNMT1) expression. This in turn leads to the overexpression of autoimmune-associated methylation-sensitive genes, such as CD70 and LFA-1, via promoter demethylation. Further experiments revealed that miR-21 indirectly downregulated DNMT1 expression by targeting an important autoimmune gene, RASGRP1, which mediated the Ras–MAPK pathway upstream of DNMT1; miR-148a directly downregulated DNMT1 expression by targeting the protein coding region of its transcript. Additionally, inhibition of miR-21 and miR-148a expression in CD4+ T cells from patients with lupus could increase DNMT1 expression and attenuate DNA hypomethylation. Together, our data demonstrated a critical functional link between miRNAs and the aberrant DNA hypomethylation in lupus CD4+ T cells and could help to develop new therapeutic approaches.

Due to the uniform cyto-architecture of the cerebellar cortex, its overall physiological characteristics have traditionally been considered to be homogeneous. In this study, we show in awake mice at rest that spiking activity of Purkinje cells, the sole output cells of the cerebellar cortex, differs between cerebellar modules and correlates with their expression of the glycolytic enzyme aldolase C or zebrin. Simple spike and complex spike frequencies were significantly higher in Purkinje cells located in zebrin-negative than zebrin-positive modules. The difference in simple spike frequency persisted when the synaptic input to, but not intrinsic activity of, Purkinje cells was manipulated. Blocking TRPC3, the effector channel of a cascade of proteins that have zebrin-like distribution patterns, attenuated the simple spike frequency difference. Our results indicate that zebrin-discriminated cerebellar modules operate at different frequencies, which depend on activation of TRPC3, and that this property is relevant for all cerebellar functions.DOI: http://dx.doi.org/10.7554/eLife.02536.001

Highlights d Knockdown of Ptbp1 converts Mu ¨ller glia into retinal ganglion cells in mature retinas d Central projections of converted retinal ganglion cells restore visual responses d Induction of neurons with dopaminergic features in PD model mice

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Targeted integration of transgenes can be achieved by strategies based on homologous recombination (HR), microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ). The more generally used HR is inefficient for achieving gene integration in animal embryos and tissues, because it occurs only during cell division, although MMEJ and NHEJ can elevate the efficiency in some systems. Here we devise a homology-mediated end joining (HMEJ)-based strategy, using CRISPR/Cas9-mediated cleavage of both transgene donor vector that contains guide RNA target sites and ∼800 bp of homology arms, and the targeted genome. We found no significant improvement of the targeting efficiency by the HMEJ-based method in either mouse embryonic stem cells or the neuroblastoma cell line, N2a, compared to the HR-based method. However, the HMEJ-based method yielded a higher knock-in efficiency in HEK293T cells, primary astrocytes and neurons. More importantly, this approach achieved transgene integration in mouse and monkey embryos, as well as in hepatocytes and neurons in vivo, with an efficiency much greater than HR-, NHEJ- and MMEJ-based strategies. Thus, the HMEJ-based strategy may be useful for a variety of applications, including gene editing to generate animal models and for targeted gene therapies.

The recent discovery of microRNAs (miRNAs) has revealed a new layer of gene expression regulation, affecting the immune system. Here, we identify their roles in regulating human plasmacytoid dendritic cell (PDC) activation. miRNA profiling showed the significantly differential expression of 19 miRNAs in PDCs after Toll-like receptor 7 (TLR7) stimulation, among which miR-155* and miR-155 were the most highly induced. Although they were processed from a single precursor and were both induced by TLR7 through the c-Jun N-terminal kinase pathway, miR-155* and miR-155 had opposite effects on the regulation of type I interferon production by PDC. IntroductionPlasmacytoid dendritic cell (PDC) is a distinct dendritic cell type, specialized for the rapid secretion of type I interferon (type I IFN) in response to viruses. [1][2][3] It has been demonstrated that PDCs can coordinate events during the course of viral infection, autoimmune diseases, and cancer. PDCs, through their production of interferon-␣ (IFN-␣) and other cytokines, and through antigen presentation, link the innate and adaptive immune responses. 3 PDC deficiency, leading to low levels of IFN-␣ production, results in an inadequate immune response, entailing susceptibility to viral infections or cancer, whereas excessive secretion of IFN-␣ can induce hyperimmune activation, which may lead to autoimmune disease or, in the case of HIV infection, CD4 ϩ T-cell death. [2][3][4][5] Therefore, type I IFN production by PDCs must be under tight control to prevent improper immune responses, which could be harmful to the host. 2,3 PDCs express high levels of Toll-like receptor 7 (TLR7) and TLR9. The interaction between TLR7/9 and their ligands leads to the activation of the myeloid differentiation primary response gene 88/IL-1/4 receptor-associated kinase/tumor necrosis factor (TNF) receptor-associated factor 6/IB kinases (MyD88/IRAK1/4/TRAF6/ IKKs) pathway and the subsequent phosphorylation of interferonregulatory receptor 7 (IRF-7), which is translocated into the nucleus and initiates IFN-␣ transcription. 2,6 The phosphatidylinositol 3-kinase/Av-akt murine thymoma viral oncogene homolog 1/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway and p38 mitogen-activated protein kinase (MAPK) activity have also been shown to positively regulate type I IFN production. 7,8 In contrast to these positive regulators, an array of surface receptors on PDCs, such as blood dendritic cell antigen 2 (BDCA2), dendritic cell immunoreceptor (DCIR), immunoglobulin-like transcript 7 (ILT7), high-affinity immunoglobulin E receptor (Fc⑀RI), and natural killer partner 44 (NKp44), are reported to signal through a powerful immunoreceptor tyrosine-based activation motif (ITAM)-mediated, B-cell receptor (BCR)-like regulatory pathway to counter-regulate the prominent TLR signaling pathway. 2,9-13 Although the kinetics of type I IFN production by human PDCs have been investigated in detail, 14 the dynamic regulatory mechanism has not yet been clarified. Both TLR7 and TLR9 are considered closely ...

Loss-of-function mutations in the gene encoding the postsynaptic scaffolding protein SHANK2 are a highly penetrant cause of autism spectrum disorders (ASD) involving cerebellum-related motor problems. Recent studies have implicated cerebellar pathology in the aetiology of ASD. Here we evaluate the possibility that cerebellar Purkinje cells (PCs) represent a critical locus of ASD-like pathophysiology in mice lacking Shank2. Absence of Shank2 impairs both PC intrinsic plasticity and induction of long-term potentiation at the parallel fibre to PC synapse. Moreover, inhibitory input onto PCs is significantly enhanced, most prominently in the posterior lobe where simple spike (SS) regularity is most affected. Using PC-specific Shank2 knockouts, we replicate alterations of SS regularity in vivo and establish cerebellar dependence of ASD-like behavioural phenotypes in motor learning and social interaction. These data highlight the importance of Shank2 for PC function, and support a model by which cerebellar pathology is prominent in certain forms of ASD.