Psoriasis is an autoimmune skin disease that is associated with aberrant activity of immune cells and keratinocytes. In mice, topical application of TLR7/8 agonist IMQ leads to a skin disorder resembling human psoriasis. Recently, it was shown that the IL-23/ IL-17 axis plays a deciding role in the pathogenesis of human psoriasis, as well as in the mouse model of IMQ-induced psoriasis-like skin disease. A consequence of IL-17A production in the skin includes increased expression and production of IL-6, resulting in the recruitment of neutrophils and other myelomonocytic cells to the site of inflammation. To further investigate and characterize the exact role of IL-6 signaling in myelomonocytic cells during experimental psoriasis, we generated mice lacking the IL-6 receptor alpha specifically in myelomonocytic cells (IL-6RαΔmyel). Surprisingly, disease susceptibility of these mice was not affected in this model. Our study shows that classical IL-6 signaling in myelomonocytic cells does not play an essential role for disease development of IMQ-induced psoriasis-like skin disease.

An important cause of obesity-induced insulin resistance is chronic systemic inflammation originating in visceral adipose tissue (VAT). VAT inflammation is associated with the accumulation of proinflammatory macrophages in adipose tissue, but the immunological signals that trigger their accumulation remain unknown. We found that a phenotypically distinct population of tissue-resident natural killer (NK) cells represented a crucial link between obesity-induced adipose stress and VAT inflammation. Obesity drove the upregulation of ligands of the NK cell-activating receptor NCR1 on adipocytes; this stimulated NK cell proliferation and interferon-γ (IFN-γ) production, which in turn triggered the differentiation of proinflammatory macrophages and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN-γ prevented the accumulation of proinflammatory macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and insulin resistance in response to obesity-induced adipocyte stress.

Human neural stem cells (hNSCs) hold great promise for the treatment of neurological diseases. Considerable progress has been made to induce neural differentiation in the cell culture in vitro and upon transplantation in vivo [2] in order to explore restoration of damaged neuronal circuits. However, in vivo conventional strategies are limited to post mortem analysis. Here, we apply our developed first fate mapping platform to monitor neuronal differentiation in vivo by magnetic resonance imaging, bioluminescence imaging, and fluorescence imaging. Ferritin, Luciferase and GFP under neuronal-specific promoters for immature and mature neurons, respectively, were used to generate transgenic hNSCs. Differentiation-linked imaging reporter expression was validated in vitro. The time profile of spontaneous neuronal maturation after transplantation into mouse brain cortex demonstrated early neuronal differentiation within 6 weeks. Fully mature neurons expressing synaptogenesis were observed only after three months or longer. Our trimodal fate mapping strategy represents a unique non-invasive tool to monitor the time course of neuronal differentiation of transplanted stem cells in vivo.

In the insect antennal lobe different types of local interneurons mediate complex excitatory and inhibitory interactions between the glomerular pathways to structure the spatiotemporal representation of odors. Mass spectrometric and immunohistochemical studies have shown that in local interneurons classical neurotransmitters are likely to colocalize with a variety of substances that can potentially act as cotransmitters or neuromodulators. In the antennal lobe of the cockroach Periplaneta americana, gamma-aminobutyric acid (GABA) has been identified as the potential inhibitory transmitter of spiking type I local interneurons, whereas acetylcholine is most likely the excitatory transmitter of nonspiking type IIa1 local interneurons. This study used whole-cell patch clamp recordings combined with single-cell labeling and immunohistochemistry to test if the GABAergic type I local interneurons and the cholinergic type IIa1 local interneurons express allatotropin and tachykinin-related neuropeptides (TKRPs). These are two of the most abundant types of peptides in the insect antennal lobe. GABA-like and choline acetyltransferase (ChAT)-like immunoreactivity were used as markers for GABAergic and cholinergic neurons, respectively. About 50% of the GABA-like immunoreactive (-lir) spiking type I local interneurons were allatotropin-lir, and ∼ 40% of these neurons were TKRP-lir. About 20% of nonspiking ChAT-lir type IIa1 local interneurons were TKRP-lir. Our results suggest that in subpopulations of GABAergic and cholinergic local interneurons, allatotropin and TKRPs might act as cotransmitters or neuromodulators. To unequivocally assign neurotransmitters, cotransmitters, and neuromodulators to identified classes of antennal lobe neurons is an important step to deepen our understanding of information processing in the insect olfactory system.

Skeletal muscle accumulates ceramides in obesity, which contribute to the development of obesity-associated insulin resistance. However, it remained unclear which distinct ceramide species in this organ contributes to instatement of systemic insulin resistance. Here, ceramide profiling of high-fat diet (HFD)-fed animals revealed increased skeletal muscle C18:0 ceramide content, concomitant with increased expression of ceramide synthase (CerS)1. Mice lacking CerS1, either globally or specifically in skeletal muscle (CerS1ΔSkM), exhibit reduced muscle C18:0 ceramide content and significant improvements in systemic glucose homeostasis. CerS1ΔSkM mice exhibit improved insulin-stimulated suppression of hepatic glucose production, and lack of CerS1 in skeletal muscle improves systemic glucose homeostasis via increased release of Fgf21 from skeletal muscle. In contrast, muscle-specific deficiency of C16:0 ceramide-producing CerS5 and CerS6 failed to protect mice from obesity-induced insulin resistance. Collectively, these results reveal the tissue-specific function of distinct ceramide species during the development of obesity-associated insulin resistance.

Although mitochondrial dysfunction is often accompanied by excessive reactive oxygen species (ROS) production, we previously showed that an increase in random somatic mtDNA mutations does not result in increased oxidative stress. Normal levels of ROS and oxidative stress could also be a result of an active compensatory mechanism such as a mild increase in proton leak. Uncoupling protein 2 (UCP2) was proposed to play such a role in many physiological situations. However, we show that upregulation of UCP2 in mtDNA mutator mice is not associated with altered proton leak kinetics or ROS production, challenging the current view on the role of UCP2 in energy metabolism. Instead, our results argue that high UCP2 levels allow better utilization of fatty acid oxidation resulting in a beneficial effect on mitochondrial function in heart, postponing systemic lactic acidosis and resulting in longer lifespan in these mice. This study proposes a novel mechanism for an adaptive response to mitochondrial cardiomyopathy that links changes in metabolism to amelioration of respiratory chain deficiency and longer lifespan.

Effector CD4+ T cell subsets, whose differentiation is facilitated by distinct cytokine cues, amplify the corresponding type of inflammatory response. Regulatory T (Treg) cells integrate environmental cues to suppress particular types of inflammation. In this regard, STAT3, a transcription factor essential for T helper 17 (Th17) cell differentiation, is necessary for Treg cell-mediated control of Th17 cell responses. Here, we showed that anti-inflammatory interleukin-10 (IL-10), and not proinflammatory IL-6 and IL-23 cytokine signaling, endowed Treg cells with the ability to suppress pathogenic Th17 cell responses. Ablation of the IL-10 receptor in Treg cells resulted in selective dysregulation of Th17 cell responses and colitis similar to that observed in mice harboring STAT3-deficient Treg cells. Thus, Treg cells limit Th17 cell inflammation by serving as principal amplifiers of negative regulatory circuits operating in immune effector cells.

Behavioral and physiological studies have shown that local interneurons are pivotal for processing odor information in the insect antennal lobe. They mediate inhibitory and excitatory interactions between the glomerular pathways and ultimately shape the tuning profile of projection neurons. To identify putative cholinergic local interneurons in the antennal lobe of Periplaneta americana, an antibody raised against the biosynthetic enzyme choline acetyltransferase (ChAT) was applied to individual morphologically and electrophysiologically characterized local interneurons. In nonspiking type IIa1 local interneurons, which were classified in this study, we found ChAT-like immunoreactivity suggesting that they are most likely excitatory. This is a well-defined population of neurons that generates Ca(2+) -driven spikelets upon depolarization and stimulation with odorants, but not Na(+) -driven action potentials, because they lack voltage-activated transient Na(+) currents. The nonspiking type IIa2 and type IIb local interneurons, in which Ca(2+) -driven spikelets were absent, had no ChAT-like immunoreactivity. The GABA-like immunoreactive, spiking type I local interneurons had no ChAT-like immunoreactivity. In addition, we showed that uniglomerular projection neurons with cell bodies located in the ventral portion of the ventrolateral somata group and projections along the inner antennocerebral tract exhibited ChAT-like immunoreactivity. Assigning potential transmitters and neuromodulators to distinct morphological and electrophysiological types of antennal lobe neurons is an important prerequisite for a detailed understanding of odor information processing in insects.

In contrast to other eukaryotes, which manufacture lipoic acid, an essential cofactor for several vital dehydrogenase complexes, within the mitochondrion, we show that the plastid (apicoplast) of the obligate intracellular protozoan parasite Toxoplasma gondii is the only site of de novo lipoate synthesis. However, antibodies specific for protein-attached lipoate reveal the presence of lipoylated proteins in both, the apicoplast and the mitochondrion of T. gondii. Cultivation of T. gondii-infected cells in lipoate-deficient medium results in substantially reduced lipoylation of mitochondrial (but not apicoplast) proteins. Addition of exogenous lipoate to the medium can rescue this effect, showing that the parasite scavenges this cofactor from the host. Exposure of T. gondii to lipoate analogues in lipoate-deficient medium leads to growth inhibition, suggesting that T. gondii might be auxotrophic for this cofactor. Phylogenetic analyses reveal the secondary loss of the mitochondrial lipoate synthase gene after the acquisition of the plastid. Our studies thus reveal an unexpected metabolic deficiency in T. gondii and raise the question whether the close interaction of host mitochondria with the parasitophorous vacuole is connected to lipoate supply by the host.

A major component of obesity-related insulin resistance is the establishment of a chronic inflammatory state with invasion of white adipose tissue by mononuclear cells. This results in the release of pro-inflammatory cytokines, which in turn leads to insulin resistance in target tissues such as skeletal muscle and liver. To determine the role of insulin action in macrophages and monocytes in obesity-associated insulin resistance, we conditionally inactivated the insulin receptor (IR) gene in myeloid lineage cells in mice (IR(Deltamyel)-mice). While these animals exhibit unaltered glucose metabolism on a normal diet, they are protected from the development of obesity-associated insulin resistance upon high fat feeding. Euglycemic, hyperinsulinemic clamp studies demonstrate that this results from decreased basal hepatic glucose production and from increased insulin-stimulated glucose disposal in skeletal muscle. Furthermore, IR(Deltamyel)-mice exhibit decreased concentrations of circulating tumor necrosis factor (TNF) alpha and thus reduced c-Jun N-terminal kinase (JNK) activity in skeletal muscle upon high fat feeding, reflecting a dramatic reduction of the chronic and systemic low-grade inflammatory state associated with obesity. This is paralleled by a reduced accumulation of macrophages in white adipose tissue due to a pronounced impairment of matrix metalloproteinase (MMP) 9 expression and activity in these cells. These data indicate that insulin action in myeloid cells plays an unexpected, critical role in the regulation of macrophage invasion into white adipose tissue and in the development of obesity-associated insulin resistance.

The red flour beetle Tribolium castaneum is an emerging insect model organism representing the largest insect order, Coleoptera, which encompasses several serious agricultural and forest pests. Despite the ecological and economic importance of beetles, most insect olfaction studies have so far focused on dipteran, lepidopteran, or hymenopteran systems.

Social learning is fundamental to human interactions, yet its computational and physiological mechanisms are not well understood. One prominent open question concerns the role of neuromodulatory transmitters. We combined fMRI, computational modelling and genetics to address this question in two separate samples (N = 35, N = 47). Participants played a game requiring inference on an adviser's intentions whose motivation to help or mislead changed over time. Our analyses suggest that hierarchically structured belief updates about current advice validity and the adviser's trustworthiness, respectively, depend on different neuromodulatory systems. Low-level prediction errors (PEs) about advice accuracy not only activated regions known to support 'theory of mind', but also the dopaminergic midbrain. Furthermore, PE responses in ventral striatum were influenced by the Met/Val polymorphism of the Catechol-O-Methyltransferase (COMT) gene. By contrast, high-level PEs ('expected uncertainty') about the adviser's fidelity activated the cholinergic septum. These findings, replicated in both samples, have important implications: They suggest that social learning rests on hierarchically related PEs encoded by midbrain and septum activity, respectively, in the same manner as other forms of learning under volatility. Furthermore, these hierarchical PEs may be broadcast by dopaminergic and cholinergic projections to induce plasticity specifically in cortical areas known to represent beliefs about others.

Double-strand breaks (DSBs) are the most toxic type of DNA lesions. Cells repair these lesions using either end protection- or end resection-coupled mechanisms. To study DSB repair choice, we present the Color Assay Tracing-Repair (CAT-R) to simultaneously quantify DSB repair via end protection and end resection pathways. CAT-R introduces DSBs using CRISPR/Cas9 in a tandem fluorescent reporter, whose repair distinguishes small insertions/deletions from large deletions. We demonstrate CAT-R applications in chemical and genetic screens. First, we evaluate 21 compounds currently in clinical trials which target the DNA damage response. Second, we examine how 417 factors involved in DNA damage response influence the choice between end protection and end resection. Finally, we show that impairing nucleotide excision repair favors error-free repair, providing an alternative way for improving CRISPR/Cas9-based knock-ins. CAT-R is a high-throughput, versatile assay to assess DSB repair choice, which facilitates comprehensive studies of DNA repair and drug efficiency testing.

The wake-active orexin system plays a central role in the dynamic regulation of glucose homeostasis. Here we show orexin receptor type 1 and 2 are predominantly expressed in dorsal raphe nucleus-dorsal and -ventral, respectively. Serotonergic neurons in ventral median raphe nucleus and raphe pallidus selectively express orexin receptor type 1. Inactivation of orexin receptor type 1 in serotonin transporter-expressing cells of mice reduced insulin sensitivity in diet-induced obesity, mainly by decreasing glucose utilization in brown adipose tissue and skeletal muscle. Selective inactivation of orexin receptor type 2 improved glucose tolerance and insulin sensitivity in obese mice, mainly through a decrease in hepatic gluconeogenesis. Optogenetic activation of orexin neurons in lateral hypothalamus or orexinergic fibers innervating raphe pallidus impaired or improved glucose tolerance, respectively. Collectively, the present study assigns orexin signaling in serotonergic neurons critical, yet differential orexin receptor type 1- and 2-dependent functions in the regulation of systemic glucose homeostasis.

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

Insulin acts on neurons and glial cells to regulate systemic glucose metabolism and feeding. However, the mechanisms of insulin access in discrete brain regions are incompletely defined. Here we show that insulin receptors in tanycytes, but not in brain endothelial cells, are required to regulate insulin access to the hypothalamic arcuate nucleus. Mice lacking insulin receptors in tanycytes (IR∆Tan mice) exhibit systemic insulin resistance, while displaying normal food intake and energy expenditure. Tanycytic insulin receptors are also necessary for the orexigenic effects of ghrelin, but not for the anorexic effects of leptin. IR∆Tan mice exhibit increased agouti-related peptide (AgRP) neuronal activity, while displaying blunted AgRP neuronal adaptations to feeding-related stimuli. Lastly, a highly palatable food decreases tanycytic and arcuate nucleus insulin signalling to levels comparable to those seen in IR∆Tan mice. These changes are rooted in modifications of cellular stress responses and of mitochondrial protein quality control in tanycytes. Conclusively, we reveal a critical role of tanycyte insulin receptors in gating feeding-state-dependent regulation of AgRP neurons and systemic insulin sensitivity, and show that insulin resistance in tanycytes contributes to the pleiotropic manifestations of obesity-associated insulin resistance.

Local interneurons (LNs) mediate complex interactions within the antennal lobe, the primary olfactory system of insects, and the functional analog of the vertebrate olfactory bulb. In the cockroach Periplaneta americana, as in other insects, several types of LNs with distinctive physiological and morphological properties can be defined. Here, we combined whole-cell patch-clamp recordings and Ca2+ imaging of individual LNs to analyze the role of spiking and nonspiking LNs in inter- and intraglomerular signaling during olfactory information processing. Spiking GABAergic LNs reacted to odorant stimulation with a uniform rise in [Ca2+]i in the ramifications of all innervated glomeruli. In contrast, in nonspiking LNs, glomerular Ca2+ signals were odorant specific and varied between glomeruli, resulting in distinct, glomerulus-specific tuning curves. The cell type-specific differences in Ca2+ dynamics support the idea that spiking LNs play a primary role in interglomerular signaling, while they assign nonspiking LNs an essential role in intraglomerular signaling.

Ca2+ functions as an important intracellular signal for a wide range of cellular processes. These processes are selectively activated by controlled spatiotemporal dynamics of the free cytosolic Ca2+. Intracellular Ca2+ dynamics are regulated by numerous cellular parameters. Here, we established a new way to determine neuronal Ca2+ handling properties by combining the 'added buffer' approach [1] with perforated patch-clamp recordings [2]. Since the added buffer approach typically employs the standard whole-cell configuration for concentration-controlled Ca2+ indicator loading, it only allows for the reliable estimation of the immobile fraction of intracellular Ca2+ buffers. Furthermore, crucial components of intracellular signaling pathways are being washed out during prolonged whole-cell recordings, leading to cellular deterioration. By combining the added buffer approach with perforated patch-clamp recordings, these issues are circumvented, allowing the precise quantification of the cellular Ca2+ handling properties, including immobile as well as mobile Ca2+ buffers.

Nociceptin/orphanin-FQ (N/OFQ) is a recently appreciated critical opioid peptide with key regulatory functions in several central behavioral processes including motivation, stress, feeding, and sleep. The functional relevance of N/OFQ action in the mammalian brain remains unclear due to a lack of high-resolution approaches to detect this neuropeptide with appropriate spatial and temporal resolution. Here we develop and characterize NOPLight, a genetically encoded sensor that sensitively reports changes in endogenous N/OFQ release. We characterized the affinity, pharmacological profile, spectral properties, kinetics, ligand selectivity, and potential interaction with intracellular signal transducers of NOPLight in vitro. Its functionality was established in acute brain slices by exogeneous N/OFQ application and chemogenetic induction of endogenous N/OFQ release from PNOC neurons. In vivo studies with fiber photometry enabled a direct recording of binding by N/OFQ receptor ligands, as well as the detection of natural or chemogenetically-evoked endogenous N/OFQ release within the paranigral ventral tegmental area (pnVTA). In summary, we show that NOPLight can be used to detect N/OFQ opioid peptide signal dynamics in tissue and freely-behaving animals.

Reactive gliosis is a common pathological hallmark of CNS pathology resulting from neurodegeneration and neuroinflammation. In this study we investigate the capability of a novel monoamine oxidase B (MAO-B) PET ligand to monitor reactive astrogliosis in a transgenic mouse model of Alzheimer`s disease (AD). Furthermore, we performed a pilot study in patients with a range of neurodegenerative and neuroinflammatory conditions.