Probes for INS

Emerging evidence indicates that long noncoding RNAs (lncRNAs) are actively involved in a number of developmental and tumorigenic processes. Here, the authors describe the first successful use of spherical nucleic acids as an effective nanoparticle platform for regulating lncRNAs in cells; specifically, for the targeted knockdown of the nuclear-retained metastasis associated lung adenocarcinoma transcript 1 (Malat1), a key oncogenic lncRNA involved in metastasis of several cancers. Utilizing the liposomal spherical nucleic acid (LSNA) constructs, the authors first explored the delivery of antisense oligonucleotides to the nucleus. A dose-dependent inhibition of Malat1 upon LSNA treatment as well as the consequent up-regulation of tumor suppressor messenger RNA associated with Malat1 knockdown are shown. These findings reveal the biologic and therapeutic potential of a LSNA-based antisense strategy in targeting disease-associated, nuclear-retained lncRNAs.

The striatum integrates motor behavior using a well-defined microcircuit whose individual components are independently affected in several neurological diseases. The glial cell line-derived neurotrophic factor (GDNF), synthesized by striatal interneurons, and Sonic hedgehog (Shh), produced by the dopaminergic neurons of the substantia nigra (DA SNpc), are both involved in the nigrostriatal maintenance but the reciprocal neurotrophic relationships among these neurons are only partially understood. To define the postnatal neurotrophic connections among fast-spiking GABAergic interneurons (FS), cholinergic interneurons (ACh), and DA SNpc, we used a genetically induced mouse model of postnatal DA SNpc neurodegeneration and separately eliminated Smoothened (Smo), the obligatory transducer of Shh signaling, in striatal interneurons. We show that FS postnatal survival relies on DA SNpc and is independent of Shh signaling. On the contrary, Shh signaling but not dopaminergic striatal innervation is required to maintain ACh in the postnatal striatum. ACh are required for DA SNpc survival in a GDNF-independent manner. These data demonstrate the existence of three parallel but interdependent neurotrophic relationships between SN and striatal interneurons, partially defined by Shh and GDNF. The definition of these new neurotrophic interactions opens the search for new molecules involved in the striatal modulatory circuit maintenance with potential therapeutic value.

Although hundreds of cytosolic or transmembrane molecules form the primary cilium, few secreted molecules are known to contribute to ciliogenesis. Here, homologous secreted metalloproteases ADAMTS9 and ADAMTS20 are identified as ciliogenesis regulators that act intracellularly. Secreted and furin-processed ADAMTS9 bound heparan sulfate and was internalized by LRP1, LRP2 and clathrin-mediated endocytosis to be gathered in Rab11 vesicles with a unique periciliary localization defined by super-resolution microscopy. CRISPR-Cas9 inactivation of ADAMTS9 impaired ciliogenesis in RPE-1 cells, which was restored by catalytically active ADAMTS9 or ADAMTS20 acting in trans, but not by their proteolytically inactive mutants. Their mutagenesis in mice impaired neural and yolk sac ciliogenesis, leading to morphogenetic anomalies resulting from impaired hedgehog signaling, which is transduced by primary cilia. In addition to their cognate extracellular proteolytic activity, ADAMTS9 and ADAMTS20 thus have an additional proteolytic role intracellularly, revealing an unexpected regulatory dimension in ciliogenesis.

Aberrant Notch signalling has been linked to many cancers including choroid plexus (CP) tumours, a group of rare and predominantly paediatric brain neoplasms. We developed animal models of CP tumours, by inducing sustained expression of Notch1, that recapitulate properties of human CP tumours with aberrant NOTCH signalling. Whole-transcriptome and functional analyses showed that tumour cell proliferation is associated with Sonic Hedgehog (Shh) in the tumour microenvironment. Unlike CP epithelial cells, which have multiple primary cilia, tumour cells possess a solitary primary cilium as a result of Notch-mediated suppression of multiciliate differentiation. A Shh-driven signalling cascade in the primary cilium occurs in tumour cells but not in epithelial cells. Lineage studies show that CP tumours arise from monociliated progenitors in the roof plate characterized by elevated Notch signalling. Abnormal SHH signalling and distinct ciliogenesis are detected in human CP tumours, suggesting the SHH pathway and cilia differentiation as potential therapeutic avenues.

Major depressive disorder (MDD) patients display a common but often variable set of symptoms making successful, sustained treatment difficult to achieve. Separate depressive symptoms may be encoded by differential changes in distinct circuits in the brain, yet how discrete circuits underlie behavioral subsets of depression and how they adapt in response to stress has not been addressed. We identify two discrete circuits of parvalbumin-positive (PV) neurons in the ventral pallidum (VP) projecting to either the lateral habenula or ventral tegmental area contributing to depression. We find that these populations undergo different electrophysiological adaptations in response to social defeat stress, which are normalized by antidepressant treatment. Furthermore, manipulation of each population mediates either social withdrawal or behavioral despair, but not both. We propose that distinct components of the VP PV circuit can subserve related, yet separate depressive-like phenotypes in mice, which could ultimately provide a platform for symptom-specific treatments of depression.

Pain is a multidimensional experience and negative affect, or how much the pain is "bothersome", significantly impacts the sufferers' quality of life. It is well established that the kappa opioid system contributes to depressive and dysphoric states, but whether this system contributes to the negative affect precipitated by the occurrence of chronic pain remains tenuous. Using a model of persistent pain, we show by quantitative RT-PCR, florescence in situ hybridization, western blotting and GTPgS autoradiography an upregulation of expression and the function of kappa opioid receptors (KORs) and its endogenous ligand dynorphin in the mesolimbic circuitry in animals with chronic pain compared to surgical controls. Using in vivo microdialysis and microinjection of drugs into the mesolimbic dopamine system, we demonstrate that inhibiting KORs reinstates evoked dopamine release and reward related behaviors in chronic pain animals. Chronic pain enhanced KOR agonist-induced place aversion in a sex-dependent manner. Using various place preference paradigms, we show that activation of KORs drives pain aversive states in male but not female mice. However, KOR antagonist treatment was effective in alleviating anxiogenic and depressive affective-like behaviors in both sexes. Finally, ablation of KORs from dopamine neurons using AAV-TH-cre in KORloxP mice prevented pain-induced aversive states as measured by place aversion assays. Our results strongly support the use of KOR antagonists as therapeutic adjuvants to alleviate the emotional, tonic-aversive component of chronic pain, which is argued to be the most significant component of the pain experience that impacts patients' quality of life.Significance StatementWe show that KORs are sufficient to drive the tonic-aversive component of chronic pain - the emotional component of pain that is argued to significantly impact a patient's quality of life. The impact of our study is broadly relevant to affective disorders associated with disruption of reward circuitry and thus likely contributes to many of the devastating sequelae of chronic pain, including the poor response to treatment of many patients, debilitating affective disorders (other disorders including anxiety and depression that demonstrate high co-morbidity with chronic pain) and substance abuse. Indeed, co-existing psychopathology increases pain intensity, pain-related disability and effectiveness of treatments (Jamison and Edwards, 2013).

The central amygdala (CeA) is important for fear responses to discrete cues. Recent findings indicate that the CeA also contributes to states of sustained apprehension that characterize anxiety, although little is known about the neural circuitry involved. The stress neuropeptide corticotropin releasing factor (CRF) is anxiogenic and is produced by subpopulations of neurons in the lateral CeA and the dorsolateral bed nucleus of the stria terminalis (dlBST). Here we investigated the function of these CRF neurons in stress-induced anxiety using chemogenetics in male rats that express Cre recombinase from a Crh promoter. Anxiety-like behavior was mediated by CRF projections from the CeA to the dlBST and depended on activation of CRF1 receptors and CRF neurons within the dlBST. Our findings identify a CRFCeA→CRFdlBST circuit for generating anxiety-like behavior and provide mechanistic support for recent human and primate data suggesting that the CeA and BST act together to generate states of anxiety.SIGNIFICANCE STATEMENT Anxiety is a negative emotional state critical to survival, but persistent, exaggerated apprehension causes substantial morbidity. Identifying brain regions and neurotransmitter systems that drive anxiety can help in developing effective treatment. Much evidence in rodents indicates that neurons in the bed nucleus of the stria terminalis (BST) generate anxiety-like behaviors, but more recent findings also implicate neurons of the CeA. The neuronal subpopulations and circuitry that generate anxiety are currently subjects of intense investigation. Here we show that CeA neurons that release the stress neuropeptide corticotropin-releasing factor (CRF) drive anxiety-like behaviors in rats via a pathway to dorsal BST that activates local BST CRF neurons. Thus, our findings identify a CeA→BST CRF neuropeptide circuit that generates anxiety-like behavior.

Genome-wide association studies have repeatedly mapped susceptibility loci for emphysema to genes that modify hedgehog signaling, but the functional relevance of hedgehog signaling to this morbid disease remains unclear. In the current study, we identified a broad population of mesenchymal cells in the adult murine lung receptive to hedgehog signaling, characterized by higher activation of hedgehog surrounding the proximal airway relative to the distal alveoli. Single cell RNA-sequencing showed that the hedgehog-receptive mesenchyme is composed of mostly fibroblasts with distinct proximal and distal subsets with discrete identities. Ectopic hedgehog activation in the distal fibroblasts promoted expression of proximal fibroblast markers, and promoted loss of distal alveoli and airspace enlargement of over twenty percent compared to controls. We found that hedgehog suppressed mesenchymal-derived mitogens enriched in distal fibroblasts that regulate alveolar stem cell regeneration and airspace size. Finally, single cell analysis of the human lung mesenchyme showed that segregated proximal-distal identity with preferential hedgehog activation in the proximal fibroblasts is conserved between mice and humans. In conclusion, we showed that differential hedgehog activation segregates mesenchymal identities of distinct fibroblast subsets, and disruption of fibroblast identity can alter the alveolar stem cell niche leading to emphysematous changes in the murine lung.