Probes for INS

Neuropathic pain is amongst the most common non-communicable disorders and the poor effectiveness of current treatment is an unmet need. Although pain is a universal experience, there are significant inter-individual phenotypic differences. Developing models that can accurately recapitulate the clinical pain features is crucial to better understand underlying pathophysiological mechanisms and find innovative treatments. Current data from heterologous expression systems that investigate properties of specific molecules involved in pain signaling, and from animal models, show limited success with their translation into the development of novel treatments for pain. This is in part because they do not recapitulate the native environment in which a particular molecule functions, and due to species-specific differences in the properties of several key molecules that are involved in pain signaling. The limited availability of post-mortem tissue, in particular dorsal root ganglia (DRG), has hampered research using human cells in pre-clinical studies. Human induced-pluripotent stem cells (iPSCs) have emerged as an exciting alternative platform to study patient-specific diseases. Sensory neurons that are derived from iPSCs (iPSC-SNs) have provided new avenues towards elucidating peripheral pathophysiological mechanisms, the potential for development of personalized treatments, and as a cell-based system for high-throughput screening for discovering novel analgesics. Nevertheless, reprogramming and differentiation protocols to obtain nociceptors have mostly yielded immature homogenous cell populations that do not recapitulate the heterogeneity of native sensory neurons. To close the gap between native human tissue and iPSCs, alternative strategies have been developed. We will review here recent developments in differentiating iPSC-SNs and their use in pre-clinical translational studies. Direct conversion of stem cells into the cells of interest has provided a more cost- and time-saving method to improve reproducibility and diversity of sensory cell types. Furthermore, multi-cellular strategies that mimic in vivo microenvironments for cell maturation, by improving cell contact and communication (co-cultures), reproducing the organ complexity and architecture (three-dimensional organoid), and providing iPSCs with the full spatiotemporal context and nutrients needed for acquiring a mature phenotype (xenotransplantation), have led to functional sensory neuron-like systems. Finally, this review touches on novel prospective strategies, including fluorescent-tracking to select the differentiated neurons of relevance, and dynamic clamp, an electrophysiological method that allows direct manipulation of ionic conductances that are missing in iPSC-SNs.

The milestone achievement of reprogramming a human somatic cell into a pluripotent stem cell by Yamanaka and Takahashi in 2007 has changed the stem cell research landscape tremendously. Their discovery opened the unprecedented opportunity to work with human-induced pluripotent stem cells and the differentiated progeny thereof, without major ethical restrictions. Additionally, the new method offers the possibility to generate pluripotent stem cells from patients with various genetic diseases which is of great importance (a) to understand the basic mechanisms of a specific disease in a human cellular context and (b) to help find suitable therapies for the persons concerned. In individual cases, this can even help to develop personalized treatment options. Chronic pain is a disease that affects roughly one in five people worldwide, but its onset is rarely based upon genetic alterations. Nevertheless, the work with sensory-like neurons derived from human pluripotent stem cells has become a more widely used tool also in the field of pain research, as during the past years several differentiation procedures have been published that describe the generation of different types of sensory-like neurons and their useful contribution to studying mechanisms of sensitization. Especially also to complement and verify cellular and molecular mechanisms identified in rodent model systems, the model of choice for decades. Although a sole cellular system is not able to mimic a disease as complex as pain, it is a valid tool to understand basic mechanisms of sensitization in specific subsets of human neurons that might be at the onset of the disease. In addition, the creativity of basic researchers and the more and more advanced available technologies will most likely find ways to implement the derived human cells in more complex networks. In this chapter, I want to introduce a selection of published differentiation strategies that result in the generation of human sensory-like neurons. Additionally, I will point out some studies whose results helped to further understand pain-related mechanisms and which were conducted using the aforementioned differentiation procedures.

Activity in the dorsal vagal complex (DVC) is essential to gastric motility regulation. We and others have previously shown that this activity is greatly influenced by local GABAergic signaling primarily due to somatostatin-expressing GABAergic neurons (SST). To further understand the network dynamics associated with gastric motility control in the DVC, we focused on another neuron prominently distributed in this complex, neuropeptide-Y (NPY) neurons. However, the effect of these neurons on gastric motility remains unknown. Here we investigate the anatomical and functional characteristics of the NPY neurons in the nucleus tractus solitarius (NTS) and their interactions with SST neurons using transgenic mice of both sexes. We sought to determine if NPY neurons influence the activity of gastric projecting neurons, synaptically interact with SST neurons, and affect end-organ function. Our results using combined neuroanatomy and optogenetic in vitro and in vivo show that NPY neurons: are part of the gastric vagal circuit as they are trans-synaptically labeled by a viral tracer from the gastric antrum; are primarily excitatory as optogenetic activation of these neurons evoke EPSCs in gastric-antrum projecting neurons; are functionally coupled to each other and reciprocally connected to SST neurons, whose stimulation has a potent inhibitory effect on the action potential firing of the NPY neurons; and affect gastric tone and motility as reflected by their robust optogenetic response in vivo. These findings indicate that interacting NPY and SST neurons are integral to the network that controls vagal transmission to the stomach.Significance StatementThe brainstem neurons in the dorsal nuclear complex are essential for regulating vagus nerve activity that affects the stomach via tone and motility. Two distinct non-overlapping populations of predominantly excitatory neuropeptide Y (NPY) neurons and predominantly inhibitory somatostatin (SST) neurons form reciprocal connections with each other in the nucleus of the tractus solitarius (NTS) and with premotor neurons in the dorsal motor nucleus of the vagus to control gastric mechanics. Light activation and inhibition of NTS. NPY neurons increased and decreased gastric motility, respectively, while both activation and inhibition of NTS SST neurons enhanced gastric motility.

The lateral hypothalamic area (LHA) is a highly conserved brain region critical for maintaining physiological homeostasis and goal-directed behavior. LHA neurons that express melanin-concentrating hormone (MCH) are key regulators of arousal, energy balance and motivated behavior. However, cellular and functional diversity among LHAMCH neurons is not well understood. Previous anatomical and molecular data suggest that LHAMCH neurons may be parsed into at least two distinct subpopulations, one of which is enriched in neurokinin-3 receptor (NK3R), the receptor for neurokinin B (NKB), encoded by the Tac2 gene. This tachykininergic ligand-receptor system has been implicated in reproduction, fear memory and stress in other brain regions, but NKB interactions with LHAMCH neurons is poorly understood. We first identified how LHAMCH subpopulations may be distinguished anatomically and electrophysiologically. To dissect functional connectivity between NKB-expressing neurons and LHAMCH neurons, we used Cre-dependent retrograde and anterograde viral tracing in male Tac2-Cre mice and identified Tac2/EYFP+ neurons in the bed nucleus of the stria terminalis (BNST) and central nucleus of the amygdala (CeA), the central extended amygdala, as major sources of NKB input onto LHAMCH neurons. In addition to innervating the LHA, these limbic forebrain NKB neurons also project to midbrain and brainstem targets. Finally, using a dual virus approach, we found that optogenetic activation of these inputs in slices evokes GABA release onto a subset of LHAMCH neurons, but lacked specificity for the NK3R+ subpopulation. Overall, these data define parallel tachykininergic/GABAergic limbic forebrain projections that are positioned to modulate multiple nodes of homeostatic and behavioral control.SIGNIFICANCE STATEMENTThe lateral hypothalamic area (LHA) orchestrates fundamental behavioral states in the mammalian hypothalamus, including arousal, energy balance, memory, stress and motivated behavior. The neuropeptide melanin-concentrating hormone (MCH) defines one prominent population of LHA neurons, with multiple roles in the regulation of homeostatic behavior. Outstanding questions remain concerning the upstream inputs that control MCH neurons. We sought to define neurochemically distinct pathways in the mouse brain that may communicate with specific MCH neuron subpopulations using viral-based retrograde and anterograde neural pathway tracing and optogenetics in brain slices. Here we identify a specific neuropeptide-defined forebrain circuit that makes functional synaptic connections with MCH neuron subpopulations. This work lays the foundation for further manipulating molecularly distinct neural circuits that modulate innate behavioral states.

Background: Pseudomonas aeruginosa, the predominant pathogen in chronic lung infection of adults with cystic fibrosis (CF), possesses a number of mechanisms that contribute to antimicrobial resistance (AMR). Even aggressive antibiotic treatment is unable to effectively clear chronic P. aeruginosa, which may partially be due to the rapid diversification displayed by P. aeruginosa during long-term CF infection, but our knowledge of the role of population heterogeneity on AMR is limited, because prior studies have undersampled P. aeruginosa isolates in CF lungs. Specifically, the role of evolutionary trade-offs on AMR in these patients has been overlooked. We propose that P. aeruginosa trades off between AMR, surface attachment, and growth rate to sustain diverse populations in the CF lung. Methods: We sampled 75 P. aeruginosa isolates from expectorated sputum samples of 4 adults with CF chronically infected with P. aeruginosa (n = 300) and tested each for growth rate in lysogeny broth and synthetic CF sputum media, susceptibility profiles to 6 antibiotics commonly prescribed to CF patients, and surface attachment to assess the role of population heterogeneity on AMR. Results: We found significant within-patient heterogeneity in AMR across all patients and antibiotics. The majority of isolates were well within the range of susceptibility for the tested antibiotics, despite ineffective clearing of P. aeruginosa infection for each of these patients. One patient harbored isolates that grew better in the presence of tobramycin. This patient showed evidence of trade-offs between surface attachment and AMR, whereas the other 3 did not. There was some evidence of within-patient trade-offs between AMR and growth rate, but these relationships were not found to be consistent across patients. Conclusion: Overall, our results demonstrate that in vitro susceptibility testing is not representative of in situ AMR levels; further work is needed to address this. Furthermore, we found weak evidence of evolutionary tradeoffs as a driver of heterogeneity in AMR in diverse P. aeruginosa populations sourced from the CF lung, although this may mean that these trade-offs exist at below detectable levels.

Background: Culture-independent microbiota analysis has permitted comprehensive investigation of bacterial diversity in cystic fibrosis (CF) lung infections and is being increasingly used to examine fungal communities. The prevalence and clinical impact of fungi in CF is relatively poorly understood, with studies largely focused on adults. We investigated fungal diversity in children with CF aged 1 to 18 years using bronchoalveolar lavage (BAL) and induced-sputum (IS) samples to capture multiple niches within the lung. Methods: Sequencing and analysis of the fungal ITS2 region was performed on 22 matched sets of BAL-IS samples collected as part of the CF-SpIT study (UKCRN14615; ISRCTNR12473810). Each set comprised 4 samples: BAL1 (right middle lobe), BAL2 (left lingular lobe), BAL3 (pooled right and left lower and upper lobes), and IS. Bioinformatic analysis was performed in QIIME2, with downstream analysis using R statistical software (R packages phyloseq and vegan). Fungal community diversity and composition were evaluated at the genus level for each individual and the different sampling types. Results: All 88 samples (22 individuals) had evidence of fungi, and 370 fungal genera were identified across the dataset. The fungal diversity (Shannon index) captured in BAL was not significantly different from that captured in IS, and all 4 sampling types overlapped in mycobiome composition. A core group of 29 genera were identified across all BAL and IS samples, with Candida, Aspergillus, Dipodascus, Simplicillium, and Lecanicillium being the most prevalent and abundant. Candida was found at a higher average relative abundance in IS samples (30%) than in BAL samples (10%). Co-occurrence network analysis showed variable interactions between fungal genera, with positive and negative interactions identified irrespective of sample type. At the individual patient level, there was evidence of both concordance and dissimilarity between the fungal community profiles captured by BAL1, BAL2, BAL3, and IS, indicating that compartmentalization of the lung mycobiome can occur. Although this cross-sectional dataset was limited, there were also trends for greater Candida, Aspergillus, and Exophiala relative abundance and decreasing fungal diversity with increasing age. Conclusion: This study has shown that the mycobiome in pediatric CF samples is diverse and complex. There was overlap between the fungal communities identified in BAL and IS samples, suggesting that IS can capture fungal genera associated with the lower airway. The data show that IS is suitable for large-scale studies to relate clinical outcome to individual mycobiome heterogeneity

Peters plus syndrome, characterized by defects in eye and skeletal development with isolated cases of ventriculomegaly/hydrocephalus, is caused by mutations in the β3-glucosyltransferase (B3GLCT) gene. In the endoplasmic reticulum, B3GLCT adds glucose to O-linked fucose on properly folded Thrombospondin Type 1 Repeats (TSRs). The resulting glucose-fucose disaccharide is proposed to stabilize the TSR fold and promote secretion of B3GLCT substrates, with some substrates more sensitive than others to loss of glucose. Mouse B3glct mutants develop hydrocephalus at high frequency. In this study, we demonstrated that B3glct mutant ependymal cells had fewer cilia basal bodies and altered translational polarity compared to controls. Localization of mRNA encoding A Disintegrin and Metalloproteinase with ThromboSpondin type 1 repeat 20 (ADAMTS20) and ADAMTS9, suggested that reduced function of these B3GLCT substrates contributed to ependymal cell abnormalities. In addition, we showed that multiple B3GLCT substrates (Adamts3, Adamts9, and Adamts20) are expressed by the subcommissural organ, that subcommissural organ-spondin (SSPO) TSRs were modified with O-linked glucose-fucose, and that loss of B3GLCT reduced secretion of SSPO in cultured cells. In the B3glct mutant subcommissural organ intracellular SSPO levels were reduced and BiP levels increased, suggesting a folding defect. Secreted SSPO colocalized with BiP, raising the possibility that abnormal extracellular assembly of SSPO into Reissner's fiber also contributed to impaired CSF flow in mutants. Combined, these studies underscore the complexity of the B3glct mutant hydrocephalus phenotype and demonstrate that impaired cerebrospinal fluid (CSF) flow likely stems from the collective effects of the mutation on multiple processes.

Hepatic innate immune function plays an important role in the pathogenesis of many diseases. Importantly, a growing body of literature has firmly established the spatial heterogeneity of hepatocyte metabolic function; however, whether innate immune function is zonated remains unknown. To test this question, we exposed adult C57BL/6 mice to endotoxemia, and hepatic tissue was assessed for the acute phase response (APR). The zone-specific APR was evaluated in periportal and pericentral/centrilobular hepatocytes isolated using digitonin perfusion and on hepatic tissue using RNAscope and immunohistochemistry. Western blot, EMSA, chromatin immunoprecipitation, and immunohistochemistry were used to determine the role of the transcription factor NF-κB in mediating hepatic C-reactive protein (CRP) expression. Finally, the ability of mice lacking the NF-κB subunit p50 (p50-/-) to raise a hepatic APR was evaluated. We found that endotoxemia induces a hepatocyte transcriptional APR in both male and female mice, with Crp, Apcs, Fga, Hp, and Lbp expression being enriched in pericentral/centrilobular hepatocytes. Focusing our work on CRP expression, we determined that NF-κB transcription factor subunit p50 binds to consensus sequence elements present in the murine CRP promoter. Furthermore, pericentral/centrilobular hepatocyte p50 nuclear translocation is temporally associated with zone-specific APR during endotoxemia. Lastly, the APR and CRP expression is blunted in endotoxemic p50-/- mice. These results demonstrate that the murine hepatocyte innate immune response to endotoxemia includes zone-specific activation of transcription factors and target gene expression. These results support further study of zone-specific hepatocyte innate immunity and its role in the development of various disease states.

Contrary to public perception, hypertension remains one of the most important public health problems in the United States, affecting 46% of adults with increased risk for heart attack, stroke, and kidney diseases. The mechanisms underlying poorly controlled hypertension remain incompletely understood. Recent development in the Cre/LoxP approach to study gain or loss of function of a particular gene has significantly helped advance our new insights into the role of proximal tubule angiotensin II (Ang II) and its AT1 (AT1a) receptors in basal blood pressure control and the development of Ang II-induced hypertension. This novel approach has provided us and others with an important tool to generate novel mouse models with proximal tubule-specific loss (deletion) or gain of the function (overexpression). The objective of this invited review article is to review and discuss recent findings using novel genetically modifying proximal tubule-specific mouse models. These new studies have consistently demonstrated that deletion of AT1 (AT1a) receptors or its direct downstream target Na+/H+ exchanger 3 (NHE3) selectively in the proximal tubules of the kidney lowers basal blood pressure, increases the pressure-natriuresis response, and induces natriuretic responses, whereas overexpression of an intracellular Ang II fusion protein or AT1 (AT1a) receptors selectively in the proximal tubules increases proximal tubule Na+ reabsorption, impairs the pressure-natriuresis response, and elevates blood pressure. Furthermore, the development of Ang II-induced hypertension by systemic Ang II infusion or by proximal tubule-specific overexpression of an intracellular Ang II fusion protein was attenuated in mutant mice with proximal tubule-specific deletion of AT1 (AT1a) receptors or NHE3. Thus, these recent studies provide evidence for and new insights into the important roles of intratubular Ang II via AT1 (AT1a) receptors and NHE3 in the proximal tubules in maintaining basal blood pressure homeostasis and the development of Ang II-induced hypertension.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer with unmet medical needs. Several studies have proved that high levels of tumor infiltrating lymphocytes (TILs) at diagnosis of TNBC confer better prognosis and patients respond better to specific chemotherapies. Nonetheless, current evidence suggests that only 15% of TNBC patients have very high levels of TILs, and another 15% lacks TILs. One possible reason to explain why patients have low TILs at diagnosis is that lymphocytes might be deactivated by an immune checkpoint in local lymph nodes, provoking their retention in there as they are unresponsive to other immune stimuli. We have identified 15 high TILs (≥50%) and 20 low TILs (≤5%) TNBC patients with localised tumour (T1c-T2N0M0) and compared the protein expression of five immune checkpoints in lymph nodes. We have also performed a customised 50-immune gene NanoString expression panel, the NanoString 360 Breast Cancer panel, and whole exome sequencing for mutation and neoantigen load analyses. In low TILs, we observed higher expression of CTLA-4 in local lymph nodes, which could explain why lymphocytes get retained in there and do not migrate to tumour. These patients have also higher neoantigen load and higher expression of B7.H3 and B7.H4 in the tumour. In high TILs, we observed more PD-L1+ tumour cells and more expanded humoral response. These results could provide a strategy to revert low tumour immune infiltration at diagnosis of TNBC, improving their prognosis.

The ventral tegmental area (VTA) is a heterogeneous midbrain area that plays a significant role in diverse neural processes such as reward, aversion, and motivation. The VTA contains three main neuronal populations, namely, dopamine (DA), γ-aminobutyric acid (GABA), and glutamate neurons, but some neurons exhibit combinatorial molecular characteristics of dopaminergic, GABAergic, or glutamatergic neurons. However, little information is available regarding detailed distribution of neurons with single, double, and triple molecular characteristics of glutamatergic, dopaminergic, or GABAergic neurons in mice. We present a topographical distribution map of three main neuronal populations expressing a single molecular characteristic of dopaminergic, GABAergic, or glutamatergic neurons, and four neuronal populations co-expressing double or triple molecular characteristics in combinatorial manners, in the mouse VTA, following analysis of triple fluorescent in situ hybridization for the simultaneous detection of tyrosine hydroxylase (TH, marker for dopaminergic neurons), vesicular glutamate transporter 2 (VGLUT2, marker for glutamatergic neurons), and glutamic acid decarboxylase 2 (GAD2, marker for GABAergic neurons) mRNA. We found that the vast majority of neurons expressed a single type of mRNA, and these neurons were intermingled with neurons co-expressing double or triple combinations of VGLUT2, TH, or GAD2 in the VTA. These seven neuronal populations were differentially distributed in the VTA sub-nuclei across the rostro-caudal and latero-medial axes. This histochemical study will lead to a deeper understanding of the complexity of neuronal molecular characteristics in different VTA sub-nuclei, and potentially facilitate clarification of diverse functions of the VTA.

Severe debilitating pain is the most common complication and reason for hospitalization for individuals with sickle cell disease (SCD), a genetic blood disorder that affects 100,000 people in the US and over 3 million worldwide. Despite this, the biological basis of chronic SCD pain is not fully understood. Using transgenic SCD mice and fecal material transplant paradigms, we determined that gastrointestinal tract contents drive persistent SCD pain. Mechanical allodynia was temporarily alleviated in SCD mice following fecal transplant from wildtype animals. In contrast, wildtype mice developed mechanical and cold allodynia following fecal transplant from SCD animals. To identify gut bacterial species and metabolites responsible for SCD pain, we completed 16s rRNA sequencing and metabolomic screening respectively on transplant recipient feces. Bilirubin, a product of heme breakdown, was significantly elevated in the feces of SCD mice and mice that received SCD fecal transplants, as well as in the plasma of individuals with SCD. Oral administration of bilirubin induced mechanical allodynia in wildtype mice that depended on vagus nerve signaling. Using whole cell patch clamp recordings, we demonstrated that bilirubin directly activates vagal afferents and increases afferent excitability. Ongoing experiments are investigating the specific receptors through which bilirubin alters neuronal activity as drugs targeting these proteins may prove effective analgesics for SCD pain. In summary, these experiments are the first to demonstrate that sickle cell gut contents drive chronic widespread pain in this disease, and furthermore, are the first to identify gut metabolites that should be targeted for chronic SCD pain management. National Institutes of Health: K99HL155791(KS), R01HL142657(AB), R01NS070711 CS).