Cell Type-specific Modulation Research Articles

Evolutionary conservation of VSX2 super-enhancer modules in retinal development.

Super-enhancers (SEs) are expansive regions of genomic DNA that regulate the expression of genes involved in cell identity and cell fate. We recently identified developmental stage- and cell type-specific modules within the murine Vsx2 SE. Here, we show that the human VSX2 SE modules have similar developmental stage- and cell type-specific activity in reporter gene assays. By inserting the human sequence of one VSX2 SE module into a mouse with microphthalmia, eye size was rescued. To understand the function of these SE modules during human retinal development, we deleted individual modules in human embryonic stem cells and generated retinal organoids. Deleting one module results in small organoids, recapitulating the small-eyed phenotype of mice with microphthalmia, while deletion of the other module led to disruptions in bipolar neuron development. This prototypical SE serves as a model for understanding developmental stage- and cell type-specific effects of neurogenic transcription factors with complex expression patterns. Moreover, by elucidating the gene regulatory mechanisms, we can begin to examine how dysregulation of these mechanisms contributes to phenotypic diversity and disease.

Cell type-specific modulation of metabolic, immune-regulatory, and anti-microbial pathways by CD101

T lymphocytes and myeloid cells express the Ig-like glycoprotein CD101, notably in the gut. Here, we investigated the cell-specific functions of CD101 during DSS-induced colitis and Salmonella enterica Typhimurium infection. Similar to conventional CD101-/- mice, animals with a Treg-specific Cd101 deletion developed more severe intestinal pathology than littermate controls in both models. While the accumulation of Th1 cytokines in a CD101-deficient environment entertained DSS-induced colitis, it impeded the replication of Salmonella as revealed by studying CD101-/- x IFN- γ-/- mice. Moreover, CD101-expressing neutrophils were capable to restrain Salmonella infection in vitro and in vivo. Both cell-intrinsic and -extrinsic mechanisms of CD101 contributed to the control of bacterial growth and spreading. The CD101-dependent containment of Salmonella infection required the expression of Irg-1 and Nox2 and the production of itaconate and reactive oxygen species. The level of intestinal microbial antigens in the sera of IBD patients correlated inversely with the expression of CD101 on myeloid cells, which is in line with the suppression of CD101 seen in mice following DSS application or Salmonella infection. Thus, depending on the experimental or clinical setting, CD101 helps to limit inflammatory insults or bacterial infections due to cell type-specific modulation of metabolic, immune-regulatory and anti-microbial pathways.

Evidence for the involvement of TRPV2 channels in the modulation of vascular tone in the mouse aorta

AimsTransient receptor potential vanilloid 2 (TRPV2) channels are expressed in both smooth muscle and endothelial cells and participate in vascular mechanotransduction and sensing of high temperatures and lipids. Nevertheless, the impact of TRPV2 channel activation by agonists on the coordinated and cell-type specific modulation of vasoreactivity is unknown. Main methodsAorta from 2- to 4-months-old male Oncins France 1 mice was dissected and mounted in tissue baths for isometric tension measurements. TRPV2 channel expression was assessed by immunofluorescence and western blot in mice aortas and in cultured A7r5 rat aortic smooth muscle cells. Key findingsTRPV2 channels were expressed in all three mouse aorta layers. Activation of TRPV2 channels with probenecid evoked endothelium-dependent relaxations through a mechanism that involved activation of smooth muscle Kir and Kv channels. In addition, TRPV2 channel inhibition with tranilast increased endothelium-independent relaxations to probenecid and this effect was abrogated by the KATP channel blocker glibenclamide, revealing that smooth muscle TRPV2 channels induce negative feedback on probenecid relaxations mediated via KATP channel inhibition. Exposure to the NO donor sodium nitroprusside increased TRPV2 channel translocation to the plasma membrane in cultured smooth muscle cells and enhanced negative feedback on probenecid relaxations. SignificanceIn conclusion, we present the first evidence that TRPV2 channels may modulate vascular tone through a balance of opposed inputs from the endothelium and the smooth muscle leading to net vasodilation. The fact that TRPV2 channel-induced activity can be amplified by NO emphasizes the pathophysiological relevance of these findings.

Sex-specific modulation of the medial prefrontal cortex by glutamatergic median raphe neurons.

A substantial proportion of raphe neurons are glutamatergic. However, little is known about how these glutamatergic neurons modulate the forebrain. We investigated how glutamatergic median raphe nucleus (MRN) input modulates the medial prefrontal cortex (mPFC), a critical component of fear circuitry. We show that vesicular glutamate transporter 3 (VGLUT3)-expressing MRN neurons activate VGLUT3- and somatostatin-expressing neurons in the mPFC. Consistent with this modulation of mPFC GABAergic neurons, activation of MRN (VGLUT3) neurons enhances GABAergic transmission in mPFC pyramidal neurons and attenuates fear memory in female but not male mice. Serotonin plays a key role in MRN (VGLUT3) neuron-mediated GABAergic plasticity in the mPFC. In agreement with these female-specific effects, we observed sex differences in glutamatergic transmission onto MRN (VGLUT3) neurons and in mPFC (VGLUT3) neuron-mediated dual release of glutamate and GABA. Our results demonstrate a cell type-specific modulation of the mPFC by MRN (VGLUT3) neurons and reveal a sex-specific role of this neuromodulation in mPFC synaptic plasticity.

(Invited) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Drug Delivery

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

Investigations of brain-wide functional and structural networks of dopaminergic and CamKIIα-positive neurons in VTA with DREADD-fMRI and neurotropic virus tracing technologies

BackgroundThe ventral tegmental area (VTA) contains heterogeneous cell populations. The dopaminergic neurons in VTA play a central role in reward and cognition, while CamKIIα-positive neurons, composed mainly of glutamatergic and some dopaminergic neurons, participate in the reward learning and locomotor activity behaviors. The differences in brain-wide functional and structural networks between these two neuronal subtypes were comparatively elucidated.MethodsIn this study, we applied a method combining Designer Receptors Exclusively Activated by Designer Drugs (DREADD) and fMRI to assess the cell type-specific modulation of whole-brain neural networks. rAAV encoding the cre-dependent hM3D was injected into the right VTA of DAT-cre or CamKIIα-cre transgenic rats. The global brain activities elicited by DREADD stimulation were then detected using BOLD-fMRI. Furthermore, the cre-dependent antegrade transsynaptic viral tracer H129ΔTK-TT was applied to label the outputs of VTA neurons.ResultsWe found that DREADD stimulation of dopaminergic neurons induced significant BOLD signal changes in the VTA and several VTA-related regions including mPFC, Cg and Septum. More regions responded to selective activation of VTA CamKIIα-positive neurons, resulting in increased BOLD signals in VTA, Insula, mPFC, MC_R (Right), Cg, Septum, Hipp, TH_R, PtA_R, and ViC_R. Along with DREADD-BOLD analysis, further neuronal tracing identified multiple cortical (MC, mPFC) and subcortical (Hipp, TH) brain regions that are structurally and functionally connected by VTA dopaminergic and CamKIIα-positive neurons.ConclusionsOur study dissects brain-wide structural and functional networks of two neuronal subtypes in VTA and advances our understanding of VTA functions.

P390 Ustekinumab modulates IL-12 and IL-23-related cell type-specific inflammatory transcriptional states in Ulcerative Colitis induction therapy

Abstract Background Ustekinumab (UST) is a monoclonal antibody specific for the p40 subunit of interleukin (IL)-12 and IL-23. In a randomised phase 3 trial (UNIFI; NCT02407236) in patients with moderate-to-severe ulcerative colitis (UC), UST was shown to be more effective for inducing and maintaining clinical remission than placebo. There has yet to be a mechanistic understanding of p40 blockade in inflammatory bowel disease. Here, we investigated the underlying molecular mechanism of action of UST in UC. Methods Paired colon biopsies for transcriptomic analysis were obtained at screening and week 8 in patients who received a single intravenous induction treatment of placebo (n=145 subjects), UST 130 mg (n=155 subjects), or a weight-range-based dose (approximated to UST 6 mg/kg of body weight; n=171 subjects). Tissue transcriptional profiles were determined using Affymetrix microarrays. Transcriptional changes were analysed in the context of cell type-specific co-expression modules derived from single cell UC data. Gene set variation analysis (GSVA) was used to quantitatively assess changes in specific biologic modules in the context of endoscopic and histologic-endoscopic mucosal improvement (HEMI) responder and non-responder analyses. Nichenet, which predicts ligand–target gene links, was used with single cell UC data to identify ligands that are predicted to be associated with our cell type-specific modules. Results By week 8, both UST treatment arms induced a greater magnitude of transcriptional changes associated with Th17, Th1 cytotoxicity, myeloid inflammatory, epithelial inflammatory, and inflammatory fibroblast modules compared with placebo. Ligand-target gene predictions indicate IL-12 and IL-23 are associated with changes in the Th1 cytotoxicity and Th17 module expression, respectively. Population level molecular changes in both UST treatment arms are consistent with endoscopic and HEMI response at week 8. When evaluating sub-groups stratified by endoscopic improvement or HEMI status, significant changes were observed in transcriptional modules in both UST responders and UST non-responders identifying mechanistic processes associated with dual blockade of IL-12 and IL-23 following exposure to UST. Placebo non-responders showed no significant changes. Conclusion Molecular analysis of colon biopsies from UC patients after UST induction revealed IL-12 and IL-23-related transcriptional changes associated with Th1 cytotoxicity and Th17 biology. By leveraging UC single cell derived transcriptional modules, we have identified key disease-relevant processes that significantly changed with UST treatment, regardless of response status and have extended the mechanistic understanding of p40 blockade in UC.

Oxygen Sensing in Innate Immune Cells: How Inflammation Broadens Classical Hypoxia-Inducible Factor Regulation in Myeloid Cells.

Significance: Oxygen deprivation (hypoxia) is a common feature at sites of inflammation. Immune cells and all other cells present at the inflamed site have to adapt to these conditions. They do so by stabilization and activation of hypoxia-inducible factor subunit α (HIF-1α and HIF-2α, respectively), enabling constant generation of adenosine triphosphate (ATP) under these austere conditions by the induction of, for example, glycolytic pathways. Recent Advances: During recent years, it has become evident that HIFs play a far more important role than initially believed because they shape the inflammatory phenotype of immune cells. They are indispensable for migration, phagocytosis, and the induction of inflammatory cytokines by innate immune cells and thereby enable a crosstalk between innate and adaptive immunity. In short, they ensure the survival and function of immune cells under critical conditions. Critical Issues: Up to now, there are still open questions regarding the individual roles of HIF-1 and HIF-2 for the different cell types. In particular, the loss of both HIF-1 and HIF-2 in myeloid cells led to unexpected and contradictory results in the mouse models analyzed so far. Similarly, the role of HIF-1 in dendritic cell maturation is unclear due to inconsistent results from in vitro experiments. Future Directions: The HIFs are indispensable for immune cell survival and action under inflammatory conditions, but they might also trigger over-activation of immune cells. Therefore, they might be excellent setscrews to adjust the inflammatory response by pharmaceuticals. China and Japan and very recently (August 2021) Europe have approved prolyl hydroxylase inhibitors (PHIs) to stabilize HIF such as roxadustat for clinical use to treat anemia by increasing the production of erythropoietin, the classical HIF target gene. Nonetheless, we need further work regarding the use of PHIs under inflammatory conditions, because HIFs show specific activation and distinct expression patterns in innate immune cells. The extent to which HIF-1 or HIF-2 as a transcription factor regulates the adaptation of immune cells to inflammatory hypoxia differs not only by the cell type but also with the inflammatory challenge and the surrounding tissue. Therefore, we urgently need isoform- and cell type-specific modulators of the HIF pathway. Antioxid. Redox Signal. 37, 956-971.

Dual Akt and Bcl-2 inhibition induces cell-type specific modulation of apoptotic and autophagic signaling in castration resistant prostate cancer cell lines.

Cancer cell survival depends on the cross-regulation between apoptosis and autophagy which share common signaling pathways including PI3K/Akt/mTOR and Bcl-2. The aim of this study was to elucidate the modulation patterns between apoptosis and autophagy following dual inhibition by Akt inhibitor erufosine and Bcl-2 inhibitor ABT-737 in castration-resistant prostate cancer (CRPC) cell lines, PC-3 (Bax+) and DU-145 (Bax-). Cell cycle progression, apoptotic and autophagic signaling were examined by flow cytometry, multi-caspase assay, Hoechst staining, acridine orange staining of acidic vesicular organelles (AVOs), qRT-PCR and Western Blot. Dual inhibition increased G2/M arrest in PC-3 and DU-145, but not in the healthy prostate epithelium cells, PNT-1A. Only in PC-3, dual inhibition induced synergistic apoptotic and additive autophagic effects. In DU-145 and PNT-1A cells, ABT-737 did not display any remarkable effect on multicaspase activity and erufosine and ABT-737, neither alone nor in combination induced AVOs. By dual inhibition, AKT, BCL-2 and NF-κB gene expressions were downregulated in PC-3, both ATG-5 and BECLIN-1 gene expressions were upregulated in DU-145 but Beclin-1 protein expression was substantially reduced in both CRPC cells. Dual inhibition-induced synergistic multicaspase activation in PC-3 degrades and disrupts autophagic activity of Beclin-1, enhancing caspase-dependent apoptosis. However, in DU-145, following dual inhibition, rate of multicaspase induction and apoptosis are lower but autophagy is completely abolished despite markedly increased BECLIN-1 gene expression. In conclusion, antineoplastic drug combinations may display cell-type specific modulation of apoptotic and autophagic signaling and lack of protective autophagy may not necessarily indicate increased chemotherapeutic sensitivity in heterogenous tumor subpopulations.

Cell type-specific modulation of healthspan by Forkhead family transcription factors in the nervous system

Reduced activity of insulin/insulin-like growth factor signaling (IIS) increases healthy lifespan among diverse animal species. Downstream of IIS, multiple evolutionarily conserved transcription factors (TFs) are required; however, distinct TFs are likely responsible for these effects in different tissues. Here we have asked which TFs can extend healthy lifespan within distinct cell types of the adult nervous system in Drosophila Starting from published single-cell transcriptomic data, we report that forkhead (FKH) is endogenously expressed in neurons, whereas forkhead-box-O (FOXO) is expressed in glial cells. Accordingly, we find that neuronal FKH and glial FOXO exert independent prolongevity effects. We have further explored the role of neuronal FKH in a model of Alzheimer's disease-associated neuronal dysfunction, where we find that increased neuronal FKH preserves behavioral function and reduces ubiquitinated protein aggregation. Finally, using transcriptomic profiling, we identify Atg17, a member of the Atg1 autophagy initiation family, as one FKH-dependent target whose neuronal overexpression is sufficient to extend healthy lifespan. Taken together, our results underscore the importance of cell type-specific mapping of TF activity to preserve healthy function with age.

Effects of innate immune receptor stimulation on extracellular \u03b1-synuclein uptake and degradation by brain resident cells

Synucleinopathies are age-related neurological disorders characterized by the progressive deposition of α-synuclein (α-syn) aggregates and include Parkinson’s disease (PD) and dementia with Lewy bodies (DLB). Although cell-to-cell α-syn transmission is thought to play a key role in the spread of α-syn pathology, the detailed mechanism is still unknown. Neuroinflammation is another key pathological feature of synucleinopathies. Previous studies have identified several immune receptors that mediate neuroinflammation in synucleinopathies, such as Toll-like receptor 2 (TLR2). However, the species of α-syn aggregates varies from study to study, and how different α-syn aggregate species interact with innate immune receptors has yet to be addressed. Therefore, we investigated whether innate immune receptors can facilitate the uptake of different species of α-syn aggregates. Here, we examined whether stimulation of TLRs could modulate the cellular uptake and degradation of α-syn fibrils despite a lack of direct interaction. We observed that stimulation of TLR2 in vitro accelerated α-syn fibril uptake in neurons and glia while delaying the degradation of α-syn in neurons and astrocytes. Internalized α-syn was rapidly degraded in microglia regardless of whether TLR2 was stimulated. However, cellular α-syn uptake and degradation kinetics were not altered by TLR4 stimulation. In addition, upregulation of TLR2 expression in a synucleinopathy mouse model increased the density of Lewy-body-like inclusions and induced morphological changes in microglia. Together, these results suggest that cell type-specific modulation of TLR2 may be a multifaceted and promising therapeutic strategy for synucleinopathies; inhibition of neuronal and astroglial TLR2 decreases pathogenic α-syn transmission, but activation of microglial TLR2 enhances microglial extracellular α-syn clearance.

Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y

A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

From Orai to E-Cadherin: Subversion of Calcium Trafficking in Cancer to Drive Proliferation, Anoikis-Resistance, and Metastasis.

The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy.

Cell-type specific modulation of NMDA receptors triggers antidepressant actions.

Both the NMDA receptor (NMDAR) positive allosteric modulator (PAM), and antagonist, can exert rapid antidepressant effects as shown in several animal and human studies. However, how this bidirectional modulation of NMDARs causes similar antidepressant effects remains unknown. Notably, the initial cellular trigger, specific cell-type(s), and subunit(s) of NMDARs mediating the antidepressant-like effects of a PAM or an antagonist have not been identified. Here, we used electrophysiology, microdialysis, and NMR spectroscopy to evaluate the effect of a NMDAR PAM (rapastinel) or NMDAR antagonist, ketamine on NMDAR function and disinhibition-mediated glutamate release. Further, we used cell-type specific knockdown (KD), pharmacological, and behavioral approaches to dissect the cell-type specific role of GluN2B, GluN2A, and dopamine receptor subunits in the actions of NMDAR PAM vs. antagonists. We demonstrate that rapastinel directly enhances NMDAR activity on principal glutamatergic neurons in medial prefrontal cortex (mPFC) without any effect on glutamate efflux, while ketamine blocks NMDAR on GABA interneurons to cause glutamate efflux and indirect activation of excitatory synapses. Behavioral studies using cell-type-specific KD in mPFC demonstrate that NMDAR-GluN2B KD on Camk2a- but not Gad1-expressing neurons blocks the antidepressant effects of rapastinel. In contrast, GluN2B KD on Gad1- but not Camk2a-expressing neurons blocks the actions of ketamine. The results also demonstrate that Drd1-expressing pyramidal neurons in mPFC mediate the rapid antidepressant actions of ketamine and rapastinel. Together, these results demonstrate unique initial cellular triggers as well as converging effects on Drd1-pyramidal cell signaling that underlie the antidepressant actions of NMDAR-positive modulation vs. NMDAR blockade.

(Invited) Engineering Single Wall Carbon Nanotubes for Sub-Cellular Drug Delivery

Single wall carbon nanotubes (SWCNTs) have been rigorously studied and engineered for a myriad of applications including drug delivery due to their unique, desirable properties. Since SWCNTs bundle in aqueous media and the bundling substantially diminishes their desirable properties along with inducing cytotoxicity, strategies to disperse individually SWCNTs with therapeutic molecules and biologic excipients have been heavily investigated. However, a handful of studies has been performed to understand the interactions between therapeutic molecules and excipients with SWCNT interface as well as the mechanism of intracellular release for therapeutic effects. Additionally, little attention has been paid to how cell type and cell metabolic activity level influence internalization of SWCNTs with different lengths. In this talk, I will discuss cell type, cell metabolic activity, and SWCNT length dependent cellular processing of SWCNTs dispersed with diverse biocompatible dispersing agents. Our results demonstrate that proper choice of dispersing agent can allow for interaction with F-actin, accumulation in the endoplasmic reticulum for eventual expulsion, or accumulation in cell nucleus. Further, macrophages with relatively high cell metabolic activity level compared to other cell types such as fibroblasts have shown to internalize significantly higher amount of SWCNTs per cell. The total mass of SWCNTs internalized per cell increases with a decrease in SWCNT length. Lastly, I will discuss our recent effort to create a ternary complex of SWCNTs, drug, and protein for intracellular delivery and release of model drugs. To overcome the limitation of the existing methods being only plausible for model drugs that are soluble in aqueous solutions, we developed a facile schema to assemble stepwise various combinations of therapeutic molecules and excipients on SWCNTs in different solvents using a preformed SWCNT network. These ternary complexes allow dialing in delivery of therapeutic molecules and excipients to achieve desired cell viability reduction. These findings allow for the development of new SWCNT-based biological applications as they establish important parameters of biomolecule adsorption on SWCNTs, SWCNT internalization and subcellular processing, which are crucial for applications ranging from drug delivery to cell type-specific modulation.

Cell type-specific modulation of sensory and affective components of itch in the periaqueductal gray

Itch is a distinct aversive sensation that elicits a strong urge to scratch. Despite recent advances in our understanding of the peripheral basis of itch, we know very little regarding how central neural circuits modulate acute and chronic itch processing. Here we establish the causal contributions of defined periaqueductal gray (PAG) neuronal populations in itch modulation in mice. Chemogenetic manipulations demonstrate bidirectional modulation of scratching by neurons in the PAG. Fiber photometry studies show that activity of GABAergic and glutamatergic neurons in the PAG is modulated in an opposing manner during chloroquine-evoked scratching. Furthermore, activation of PAG GABAergic neurons or inhibition of glutamatergic neurons resulted in attenuation of scratching in both acute and chronic pruritis. Surprisingly, PAG GABAergic neurons, but not glutamatergic neurons, may encode the aversive component of itch. Thus, the PAG represents a neuromodulatory hub that regulates both the sensory and affective aspects of acute and chronic itch.

The Integrator complex regulates differential snRNA processing and fate of adult stem cells in the highly regenerative planarian Schmidtea mediterranea.

In multicellular organisms, cell type diversity and fate depend on specific sets of transcript isoforms generated by post-transcriptional RNA processing. Here, we used Schmidtea mediterranea, a flatworm with extraordinary regenerative abilities and a large pool of adult stem cells, as an in vivo model to study the role of Uridyl-rich small nuclear RNAs (UsnRNAs), which participate in multiple RNA processing reactions including splicing, in stem cell regulation. We characterized the planarian UsnRNA repertoire, identified stem cell-enriched variants and obtained strong evidence for an increased rate of UsnRNA 3’-processing in stem cells compared to their differentiated counterparts. Consistently, components of the Integrator complex showed stem cell-enriched expression and their depletion by RNAi disrupted UsnRNA processing resulting in global changes of splicing patterns and reduced processing of histone mRNAs. Interestingly, loss of Integrator complex function disrupted both stem cell maintenance and regeneration of tissues. Our data show that the function of the Integrator complex in UsnRNA 3’-processing is conserved in planarians and essential for maintaining their stem cell pool. We propose that cell type-specific modulation of UsnRNA composition and maturation contributes to in vivo cell fate choices, such as stem cell self-renewal in planarians.

Coordinated Hedgehog Signaling Induces De Novo Hair Follicles in Primed Epithelial Structures

Mammalian organ development is orchestrated by coordinated molecular signals from epithelial and mesenchymal cells. In the skin, such anatomically adjacent signals cooperate to induce hair follicles (HF). However, this process only occurs during embryonic development, and viable strategies to induce new HFs in adult skin are lacking. Here, we reveal that supra-physiological activation of Hedgehog signaling in both epithelial and adjacent stromal cells induces new HFs in adult mouse skin. Importantly, the cells from which the new HFs derived were lineage traced from their originating touch-dome structure, and demonstrated similar molecular characteristics to embryonic HF development. Therefore, reactivation of productive, embryonic HF programs in adult skin has been achieved. In stark contrast, Hh-pathway activation in either epithelial or stromal cells alone resulted in tumor formation and stromal cell condensation, respectively, without induction of new HFs. Altogether, we demonstrate that cell-type specific modulation of a single pathway was sufficient to induce development of complex epithelial structures in the adult body, an important discovery with widespread implications for regenerative medicine.

Layer- and Cell Type-Specific Modulation of Excitatory Neuronal Activity in the Neocortex.

From an anatomical point of view the neocortex is subdivided into up to six layers depending on the cortical area. This subdivision has been described already by Meynert and Brodmann in the late 19/early 20. century and is mainly based on cytoarchitectonic features such as the size and location of the pyramidal cell bodies. Hence, cortical lamination is originally an anatomical concept based on the distribution of excitatory neuron. However, it has become apparent in recent years that apart from the layer-specific differences in morphological features, many functional properties of neurons are also dependent on cortical layer or cell type. Such functional differences include changes in neuronal excitability and synaptic activity by neuromodulatory transmitters. Many of these neuromodulators are released from axonal afferents from subcortical brain regions while others are released intrinsically. In this review we aim to describe layer- and cell-type specific differences in the effects of neuromodulator receptors in excitatory neurons in layers 2–6 of different cortical areas. We will focus on the neuromodulator systems using adenosine, acetylcholine, dopamine, and orexin/hypocretin as examples because these neuromodulator systems show important differences in receptor type and distribution, mode of release and functional mechanisms and effects. We try to summarize how layer- and cell type-specific neuromodulation may affect synaptic signaling in cortical microcircuits.

Nav1.7 is phosphorylated by Fyn tyrosine kinase which modulates channel expression and gating in a cell type-dependent manner.

Voltage-gated sodium channel Nav1.7 is a key molecule in nociception, and its dysfunction has been associated with various pain disorders. Here, we investigated the regulation of Nav1.7 biophysical properties by Fyn, an Src family tyrosine kinase. Nav1.7 was coexpressed with either constitutively active (FynCA) or dominant negative (FynDN) variants of Fyn kinase. FynCA elevated protein expression and tyrosine phosphorylation of Nav1.7 channels. Site-directed mutagenesis analysis identified two tyrosine residues (Y1470 and Y1471) located within the Nav1.7 DIII-DIV linker (L3) as phosphorylation sites of Fyn. Whole-cell recordings revealed that FynCA evoked larger changes in Nav1.7 biophysical properties when expressed in ND7/23 cells than in Human Embryonic Kidney (HEK) 293 cells, suggesting a cell type-specific modulation of Nav1.7 by Fyn kinase. In HEK 293 cells, substitution of both tyrosine residues with phenylalanine dramatically reduced current amplitude of mutant channels, which was partially rescued by expressing mutant channels in ND7/23 cells. Phenylalanine substitution showed little effect on FynCA-induced changes in Nav1.7 activation and inactivation, suggesting additional modifications in the channel or modulation by interaction with extrinsic factor(s). Our study demonstrates that Nav1.7 is a substrate for Fyn kinase, and the effect of the channel phosphorylation depends on the cell background. Fyn-mediated modulation of Nav1.7 may regulate DRG neuron excitability and contribute to pain perception. Whether this interaction could serve as a target for developing new pain therapeutics requires future study.