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 signaling 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 induced pluripotent stem cells from one neurotypical control donor null for full-length CNTNAP2, modeling 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 with 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.

Tumour necrosis factor induces increased production of extracellular amyloid-ß- and α-synuclein-containing aggregates by human Alzheimer's disease neurons.

In addition to increased aberrant protein aggregation, inflammation has been proposed as a key element in the pathogenesis and progression of Alzheimer's disease. How inflammation interacts with other disease pathways and how protein aggregation increases during disease are not clear. We used single-molecule imaging approaches and membrane permeabilization assays to determine the effect of chronic exposure to tumour necrosis factor, a master proinflammatory cytokine, on protein aggregation in human-induced pluripotent stem cell-derived neurons harbouring monogenic Alzheimer's disease mutations. We report that exposure of Alzheimer's disease neurons, but not control neurons, to tumour necrosis factor induces substantial production of extracellular protein aggregates. Aggregates from Alzheimer's disease neurons are composed of amyloid-ß and α-synuclein and induce significant permeabilization of lipid membranes in an assay of pathogenicity. These findings provide support for a causal relationship between two crucial processes in Alzheimer's disease pathogenesis and suggest that targeting inflammation, particularly tumour necrosis factor, may have beneficial downstream effects on ameliorating aberrant protein aggregation and accumulation.

Malonylation of GAPDH is an inflammatory signal in macrophages.

Macrophages undergo metabolic changes during activation that are coupled to functional responses. The gram negative bacterial product lipopolysaccharide (LPS) is especially potent at driving metabolic reprogramming, enhancing glycolysis and altering the Krebs cycle. Here we describe a role for the citrate-derived metabolite malonyl-CoA in the effect of LPS in macrophages. Malonylation of a wide variety of proteins occurs in response to LPS. We focused on one of these, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In resting macrophages, GAPDH binds to and suppresses translation of several inflammatory mRNAs, including that encoding TNFα. Upon LPS stimulation, GAPDH undergoes malonylation on lysine 213, leading to its dissociation from TNFα mRNA, promoting translation. We therefore identify for the first time malonylation as a signal, regulating GAPDH mRNA binding to promote inflammation.

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-1ß was a predominant feature during the initiation of colitis with miR-223 deficiency. Depletion of CCR2+ inflammatory monocytes and pharmacologic blockade of IL-1ß 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-1ß release. Collectively, our data reveal a previously unappreciated role for miR-223 in regulating the innate immune response during intestinal inflammation.

The Induction of Pro-IL-1ß by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production.

PGE2 has been shown to increase the transcription of pro-IL-1ß. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1ß by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1ß production. We have found that in murine bone marrow-derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1ß transcription, as well as boosting the ability of LPS to induce IL-1ß mRNA and pro-IL-1ß while inhibiting the production of TNF-α. This results in an increase in mature IL-1ß production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1ß production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro-IL-1ß production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1ß, PGE2 boosted LPS-induced IL-1ß production. PGE2 did not inhibit ATP-induced mature IL-1ß production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1ß, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1ß and back to PGE2, which itself is induced by IL-1ß, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.

The RNA-binding protein Tristetraprolin (TTP) is a critical negative regulator of the NLRP3 inflammasome.

The NLRP3 inflammasome is a central regulator of inflammation in many common diseases, including atherosclerosis and type 2 diabetes, driving the production of pro-inflammatory mediators such as IL-1ß and IL-18. Due to its function as an inflammatory gatekeeper, expression and activation of NLRP3 need to be tightly regulated. In this study, we highlight novel post-transcriptional mechanisms that can modulate NLRP3 expression. We have identified the RNA-binding protein Tristetraprolin (TTP) as a negative regulator of NLRP3 in human macrophages. TTP targets AU-rich elements in the NLRP3 3'-untranslated region (UTR) and represses NLRP3 expression. Knocking down TTP in primary macrophages leads to an increased induction of NLRP3 by LPS, which is also accompanied by increased Caspase-1 and IL-1ß cleavage upon NLRP3, but not AIM2 or NLRC4 inflammasome activation. Furthermore, we found that human NLRP3 can be alternatively polyadenylated, producing a short 3'-UTR isoform that excludes regulatory elements, including the TTP- and miRNA-223-binding sites. Because TTP also represses IL-1ß expression, it is a dual inhibitor of the IL-1ß system, regulating expression of the cytokine and the upstream controller NLRP3.

Analysis of Post-transcriptional Gene Regulation of Nod-Like Receptors via the 3'UTR.

Innate immune signaling is the front line of defense against pathogens, leading to an appropriate response of immune cells upon activation of their pattern recognition receptors (PRRs) by microbial products, such as Toll-like receptors (TLRs). Apart from transcriptional control, gene expression in the innate immune system is also highly regulated at the post-transcriptional level. miRNA or RNA-binding protein can bind to the 3' untranslated region (UTR) of target mRNAs and affect their mRNA stability and translation efficiency, which ultimately affects the amount of protein that is produced. In recent years, a new group of PRRs, the Nod-like receptors (NLR) have been discovered. They often cooperate with TLR signaling to induce potent inflammatory responses. Many NLRs can form inflammasomes, which facilitate the production of the potent pro-inflammatory cytokine IL-1ß and other inflammatory mediators. In contrast to TLRs, the importance of post-transcriptional regulators in the context of inflammasomes has not been well defined. This chapter describes a series of experimental approaches to determine the effect of post-transcriptional regulation for a gene of interest using the best-studied NLR, NLRP3, as an example. To start investigating post-transcriptional regulation, 3'UTR luciferase experiments can be performed to test if regulatory sequences in the 3'UTR are functional. An RNA pull-down approach followed by mass spectrometry provides an unbiased assay to identify RNA-binding proteins that target the 3'UTR. Candidate binding proteins can then be further validated by RNA immunoprecipitation (RNA-IP), where the candidate protein is isolated using a specific antibody and bound mRNAs are analyzed by qPCR.

NLRP3 at the interface of metabolism and inflammation.

The discovery of the NLRP3 (NLR family, pyrin domain containing 3) inflammasome provided an important molecular mechanism in the induction of the central pro-inflammatory cytokine interleukin-1ß (IL-1ß), via activation of caspase-1, which processes pro-IL-1ß into its mature active form. IL-1 has long been known to exert metabolic effects, most notably being implicated in insulin resistance and obesity. A key phenotype of the NLRP3-deficient mouse is insulin hypersensitivity. Over the past 5 years, a number of discoveries have been made suggesting a close interplay between NLRP3 and metabolism. Metabolic products have been shown to activate NLPR3, and disturbed mitochondria have been shown to be involved in NLRP3 function. It is possible that under normal physiology NLRP3 is homeostatic and maintains the metabolic balance. However, upon chronic activation (e.g. in obesity or hypercholesterolemia), NLRP3 becomes pathologic and promotes disease. Here, we review these findings and place them in the context of exciting new insights that are improving our understanding of the link between inflammation and metabolism. These insights are giving rise to better understanding of disease pathogenesis and might point to new therapeutic approaches.

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-1ß (IL-1ß) 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.

Modulatory mechanisms controlling the NLRP3 inflammasome in inflammation: recent developments.

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-1ß 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.

Cutting edge: miR-223 and EBV miR-BART15 regulate the NLRP3 inflammasome and IL-1ß production.

Although microRNA (miRNA) regulation of TLR signaling is well established, this has not yet been observed for NLR proteins or the inflammasomes they form. We have now validated a highly conserved miR-223 target site in the NLRP3 3'-untranslated region. miR-223 expression decreases as monocytes differentiate into macrophages, whereas NLRP3 protein increases during this time. However, overexpression of miR-223 prevents accumulation of NLRP3 protein and inhibits IL-1ß production from the inflammasome. Virus inhibition of the inflammasome is an emerging theme, and we have also identified an EBV miRNA that can target the miR-223 binding site in the NLRP3 3'-untranslated region. Furthermore, this virus miRNA can be secreted from infected B cells via exosomes to inhibit the NLRP3 inflammasome in noninfected cells. Therefore, we have identified both the first endogenous miRNA that limits NLRP3 inflammatory capacity during myeloid cell development and also a viral miRNA that takes advantage of this, limiting inflammation for its own purposes.

Proteomic screen reveals Fbw7 as a modulator of the NF-κB pathway.

Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-κB2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3ß phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-κB pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-κB2 pathway stimulation.

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.