Loss of CNTNAP2 Alters Human Cortical Excitatory Neuron Differentiation and Neural Network Development

BACKGROUND: Loss-of-function mutations in the contactin-associated protein-like 2 (CNTNAP2) gene are causal for neurodevelopmental disorders, including autism, schizophrenia, epilepsy and intellectual disability. CNTNAP2 encodes CASPR2, a single-pass transmembrane protein that belongs to the neurexin family of cell adhesion molecules. These proteins have a variety of functions in developing neurons, including connecting presynaptic and postsynaptic neurons, and mediating signalling across the synapse. METHODS: To study the effect of loss of CNTNAP2 function on human cerebral cortex development, and how this contributes to the pathogenesis of neurodevelopmental disorders, we generated human iPSCs from one neurotypical control donor null for full-length CNTNAP2, modelling cortical development from neurogenesis through to neural network formation in vitro. RESULTS: CNTNAP2 is particularly highly expressed in the first two populations of early-born excitatory cortical neurons, and loss of CNTNAP2 shifted the relative proportions of these two neuronal types. Live imaging of excitatory neuronal growth showed that loss of CNTNAP2 reduced neurite branching and overall neuronal complexity. At the network level, developing cortical excitatory networks null for CNTNAP2 had complex changes in activity compared to isogenic controls: an initial period of relatively reduced activity compared with isogenic controls, followed by a lengthy period of hyperexcitability, and then a further switch to reduced activity. CONCLUSIONS: Complete loss of CNTNAP2 contributes to the pathogenesis of neurodevelopmental disorders through complex changes in several aspects of human cerebral cortex excitatory neuron development that culminate in aberrant neural network formation and function

Myeloid-derived miR-223 regulates intestinal inflammation via repression of the NLRP3 inflammasome

MicroRNA (miRNA)-mediated RNA interference regulates many immune processes, but how miRNA circuits orchestrate aberrant intestinal inflammation during inflammatory bowel disease (IBD) is poorly defined. Here, we report that miR-223 limits intestinal inflammation by constraining the nlrp3 inflammasome. miR-223 was increased in intestinal biopsies from patients with active IBD and in preclinical models of intestinal inflammation. miR-223-/y mice presented with exacerbated myeloid-driven experimental colitis with heightened clinical, histopathological, and cytokine readouts. Mechanistically, enhanced NLRP3 inflammasome expression with elevated IL-1β was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1β or NLRP3 abrogated this phenotype. Generation of a novel mouse line, with deletion of the miR-223 binding site in the NLRP3 3′ untranslated region, phenocopied the characteristics of miR-223-/y mice. Finally, nanoparticle-mediated overexpression of miR-223 attenuated experimental colitis, NLRP3 levels, and IL-1β release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation

MiR-200c Regulates Noxa Expression and Sensitivity to Proteasomal Inhibitors

The pro-apoptotic p53 target Noxa is a BH3-only protein that antagonizes the function of selected anti-apoptotic Bcl-2 family members. While much is known regarding the transcriptional regulation of Noxa, its posttranscriptional regulation remains relatively unstudied. In this study, we therefore investigated whether Noxa is regulated by microRNAs. Using a screen combining luciferase reporters, bioinformatic target prediction analysis and microRNA expression profiling, we identified miR-200c as a negative regulator of Noxa expression. MiR-200c was shown to repress basal expression of Noxa, as well as Noxa expression induced by various stimuli, including proteasomal inhibition. Luciferase reporter experiments furthermore defined one miR-200c target site in the Noxa 3′UTR that is essential for this direct regulation. In spite of the miR-200c:Noxa interaction, miR-200c overexpression led to increased sensitivity to the clinically used proteasomal inhibitor bortezomib in several cell lines. This apparently contradictory finding was reconciled by the fact that in cells devoid of Noxa expression, miR-200c overexpression had an even more pronounced positive effect on apoptosis induced by proteasomal inhibition. Together, our data define miR-200c as a potentiator of bortezomib-induced cell death. At the same time, we show that miR-200c is a novel negative regulator of the pro-apoptotic Bcl-2 family member Noxa

A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases

The NOD-like receptor (NLR) family, pyrin domain-containing protein 3 (NLRP3) inflammasome is a component of the inflammatory process, and its aberrant activation is pathogenic in inherited disorders such as cryopyrin-associated periodic syndrome (CAPS) and complex diseases such as multiple sclerosis, type 2 diabetes, Alzheimer's disease and atherosclerosis. We describe the development of MCC950, a potent, selective, small-molecule inhibitor of NLRP3. MCC950 blocked canonical and noncanonical NLRP3 activation at nanomolar concentrations. MCC950 specifically inhibited activation of NLRP3 but not the AIM2, NLRC4 or NLRP1 inflammasomes. MCC950 reduced interleukin-1 beta (IL-1 beta) production in vivo and attenuated the severity of experimental autoimmune encephalomyelitis (EAE), a disease model of multiple sclerosis. Furthermore, MCC950 treatment rescued neonatal lethality in a mouse model of CAPS and was active in ex vivo samples from individuals with Muckle-Wells syndrome. MCC950 is thus a potential therapeutic for NLRP3-associated syndromes, including autoinflammatory and autoimmune diseases, and a tool for further study of the NLRP3 inflammasome in human health and disease

VEST - Venice Symbolic Tagger

The protein NLRP3 has emerged as a central regulator in the inflammatory process, being implicated directly in hereditary cryopyrinopathies, and indirectly in diseases such as gout, Type 2 diabetes and atherosclerosis. NLRP3 is an important regulator of caspase-1, the enzyme that processes the immature form of IL-1? into the active protein. The control of NLRP3 has therefore become a focus of research with evidence for redox regulation, ubiquitination and regulation by miRNA-223, kinases and calcium all emerging as controllers of NLRP3. As our knowledge expands the prospect for precise pharmacological targeting of NLRP3 will improve and could lead to substantial clinical utility

MiR-200c potentiates proteasome inhibitor-mediated cell death.

<p>(<b>A</b>) HCT116 cells were transfected with pre-miR-200c or pre-miR-control oligos for 24 hours, treated with indicated concentrations of bortezomib for an additional 24 hours and processed for immunoblotting for GAPDH, Noxa, cleaved PARP and cleaved caspase 3. Protein size in kilodaltons (kDa) is also shown. (<b>B</b>) HCT116 cells were transfected with pre-miR-200c or pre-miR-control oligos for 48 hours and apoptosis was assessed by Annexin V/PI staining and FACS analysis. (<b>C</b>) HCT116 cells were treated with 20 nM bortezomib as in (A) and apoptosis was assessed by Annexin V/PI staining and FACS analysis. Graphs show the mean of percentages of Annexin V-positive cells, including PI-positive and PI-negative, from three independent experiments.</p

MiR-200c represses Noxa protein under both normal conditions and during cellular stress.

<p>(<b>A</b>) HEK293 cells were transfected with a plasmid encoding the miR-200c microRNA cluster or an empty vector control. Cells were collected at indicated timepoints, whole cell extracts were prepared and subjected to immunblotting for Noxa. (<b>B</b>) The indicated cell lines were transfected with pre-miR-200c or pre-miR-control oligos for 48 hours, whole cell extracts were prepared and analyzed for Noxa protein expression. (<b>C</b>) HEK293 cells were transfected with the indicated expression constructs for 24 and 48 hours respectively, and Noxa mRNA expression was determined by qRT-PCR analysis. Noxa mRNA expression was normalized to that of GAPDH. (<b>D</b>) HEK293 cells were transfected with indicated expression constructs (<i>left panel</i>) or oligonucleotides (<i>right panel</i>). 24 hours post-transfection, cells were treated with the indicated concentration of MG132 for an additional 3 or 24 hours, and processed for Noxa immunoblotting as in (A). GAPDH was used as a loading control in all immunoblotting experiments. Protein size in kilodaltons (kDa) is also shown.</p

MiR-200c is a candidate Noxa-regulating microRNA.

<p>(<b>A</b>) The Noxa 3′UTR is repressed in MCF7 cells. The pMIR-REPORT (<i>upper panel</i>) and psiCHECK2 (<i>lower panel</i>) vectors with the full length Noxa 3′UTR downstream of luciferase or empty vector controls were introduced into the indicated cell lines. Luciferase activity was normalized to the activity of an external Renilla luciferase plasmid (<i>upper panel</i>) or to an internal Firefly luciferase (<i>lower panel</i>). (<b>B</b>) Expression profiling of microRNAs predicted to target the Noxa 3′UTR. (<b>C</b>) The repressive element is located in the distal part of the Noxa 3′UTR. The full length Noxa 3′UTR luciferase plasmid or the indicated deletion mutants were introduced into MCF7 cells and luciferase activity was measured as in (A). A schematic representation of the different 3′UTR constructs used in this study is also shown. The location of target sites of the three Noxa-regulating candidates is included in the scheme. (<b>D</b>) Expression of Noxa inversely correlates with that of miR-200c. MiR-200c expression was determined by qRT-PCR analysis in the indicated cell lines. Expression was normalized to that of the small nucleolar RNA RNU48 (<i>upper panel</i>). Protein extracts were prepared in parallel and subjected to immunoblotting for endogenous Noxa (<i>lower panel)</i>. GAPDH was used as a loading control. Protein size in kilodaltons (kDa) is also shown.</p