Intra-Tumoral Nerve-Tracing in a Novel Syngeneic Model of High-Grade Serous Ovarian Carcinoma

Dense tumor innervation is associated with enhanced cancer progression and poor prognosis. We observed innervation in breast, prostate, pancreatic, lung, liver, ovarian, and colon cancers. Defining innervation in high-grade serous ovarian carcinoma (HGSOC) was a focus since sensory innervation was observed whereas the normal tissue contains predominantly sympathetic input. The origin, specific nerve type, and the mechanisms promoting innervation and driving nerve-cancer cell communications in ovarian cancer remain largely unknown. The technique of neuro-tracing enhances the study of tumor innervation by offering a means for identification and mapping of nerve sources that may directly and indirectly affect the tumor microenvironment. Here, we establish a murine model of HGSOC and utilize image-guided microinjections of retrograde neuro-tracer to label tumor-infiltrating peripheral neurons, mapping their source and circuitry. We show that regional sensory neurons innervate HGSOC tumors. Interestingly, the axons within the tumor trace back to local dorsal root ganglia as well as jugular–nodose ganglia. Further manipulations of these tumor projecting neurons may define the neuronal contributions in tumor growth, invasion, metastasis, and responses to therapeutics.

Sexually dimorphic mechanosensory neurons regulate copulation duration and persistence in male Drosophila

Peripheral sensory neurons are the gateway to the environment across species. In Drosophila, olfactory and gustatory senses are required to initiate courtship, as well as for the escalation of courtship patterns that lead to copulation. To be successful, copulation must last long enough to ensure the transfer of sperm and seminal fluid that ultimately leads to fertilization. The fly genitalia contain sex-specific bristle hairs innervated by mechanosensory neurons. To date, the role of the sensory information collected by these peripheral neurons in male copulatory behavior is unknown. Here, we employed genetic manipulations that allow driving gene expression in the male genitalia as a tool to uncover the role of these genitalia specific neurons in copulation. We found that the sensory information received by the mechanosensory neurons (MSNs) at the male genitalia plays a key role in copulation duration. We confirmed that these MSNs are cholinergic and co-express both fru and dsx. Moreover, our results show that the function of these fru/dsx cholinergic MSNs is required for copulation persistence, which ensures copulation is undisrupted in the presence of environmental stress before sperm transfer is complete.

Predictive Assay for Chemotherapy-Induced Peripheral Neuropathy Associated with Vinca Alkaloids in Lymphoid Malignancies

Introduction: Vinca alkaloids, such as vincristine, are important anticancer agents mainly employed in the treatment of hematological cancers. The principal mechanism for the antineoplastic activity is microtubule disruption. However, these agents also cause damage to mitochondria which leads to oxidative stress and production of reactive oxygen species (ROS). Other chemotherapeutic drugs that are suggested to cause peripheral neuropathy by this mechanism, i.e., increased oxidative stress, include taxanes and platinum compounds. Oxidative damage to peripheral neurons can cause damage to myelin sheath, mitochondrial proteins, and other antioxidant enzymes, resulting in hyperexcitability of peripheral neurons. This nerve damage results in the commonly seen dose-limiting neurological side effect chemotherapy-induced peripheral neuropathy (CIPN). Hence, assessment of biomarkers for peroxidation, myelin repair/maintenance, and red-ox balance (glutathione recycling) can be helpful in monitoring the on-set and course of peripheral neuropathy. We hypothesize that chemotherapeutic agents with similar effects on mitochondria (vinca alkaloids, taxanes and platinum compounds) will produce a specific pattern of glutathione recycling in patients prone to CIPN. Methods: Patients who had given written consent to participate in this exploratory single-centered, prospective IRB approved study contributed a blood sample prior to each treatment cycle. At each visit, the Rotterdam Symptoms Check-List (RSCL) was filled out and reported symptoms confirmed by comparison to notes in medical records. Whole blood was analyzed for glutathione recycling capacity using the bioactive probe hydroxyethyldisulfide (HEDS) and incubated at room temperature for 2 hours with gentle mixing. Prior to spectrophotometric determination, blood cells and proteinaceous thiols will be removed from the sample by acid precipitation and centrifugation. The final spectrophotometric reading will be converted into ME produced using the conversion factor provided in the assay kit Rockland Inc). Recycling capacity was compared to self-reported grade of CIPN. Results: Thus far we have enrolled 276 patients with an average age of 63.69 years (±12.85 STDEV; Range; 25-91). Patients are predominantly of Caucasian heritage (80.8%) along with 18.1 % African American and 1.1% Asian heritage. Females constitute 53.26 % of the cohort. To date we have 150 patients with more than 24 months of follow-up and among these patients, 5.5% had no reported symptoms of CIPN. Of those with severe CIPN (NCCN grade 3) neuropathy, 71% had a reduction in GSH recycling of greater than or equal to 40% from pre-treatment level. The reduced glutathione recycling capacity preceded on-set of symptoms by approximately 4 weeks. The majority of patients demonstrating this pattern of glutathione recycling had persistence CIPN that lasted for 6-18 months before an improvement was noted in medical records. We are currently assessing the value of adding biomarkers for lipid peroxidation and/or myelin formation/maintenance to further improve the likelihood ratio and AUROC values for the test. Conclusions: This data suggests the importance of GSH recycling in the ability to predict risk of CIPIN. Patients whose pre-treatment baseline was less than 1 or progressively dropped by at least 40%, seem to be at most risk for CIPN. Further, this may predict persistence of CIPN even after cessation of chemotherapy. Most patients in this cohort received taxanes or platinum therapy. However, given the similarity in mechanism and results from an early assessment of lymphoma patients treated with vinca alkaloids that resulted in similar outcomes with reduced glutathione recycling capacity suggest the possible use of this test to predict CIPN among lymphoma patients. Disclosures No relevant conflicts of interest to declare.

Systematic expression profiling of dprs and DIPs reveals cell surface codes in Drosophila larval peripheral neurons

In complex nervous systems, neurons must identify their correct partners to form synaptic connections. The prevailing model to ensure correct recognition posits that cell surface proteins (CSPs) in individual neurons act as identification tags. Thus, knowing what cells express which CSPs would provide insights into neural development, synaptic connectivity, and nervous system evolution. Here, we investigated expression of dprs and DIPs, two CSP subfamilies belonging to the immunoglobulin superfamily (IgSF), in Drosophila larval motor neurons (MNs), sensory neurons (SNs), peripheral glia and muscles using a collection of GAL4 driver lines. We found that dprs are more broadly expressed than DIPs in MNs and SNs, and each examined neuron expresses a unique combination of dprs and DIPs. Interestingly, many dprs and DIPs are not robustly expressed, but instead, are found in gradient and temporal expression patterns. Hierarchical clustering showed a similar expression pattern of dprs and DIPs in neurons from the same type and with shared synaptic partners, suggesting these CSPs may facilitate synaptic wiring. In addition, the unique expression patterns of dprs and DIPs revealed three uncharacterized MNs - MN23-Ib, MN6-Ib (A2) and MN7-Ib (A2). This study sets the stage for exploring the functions of dprs and DIPs in Drosophila MNs and SNs and provides genetic access to subsets of neurons.

Quercus acuta Thunb. (Fagaceae) and Its Component, Isoquercitrin, Inhibit HSV-1 Replication by Suppressing Virus-Induced ROS Production and NF-κB Activation

HSV-1 is a neurotropic virus that replicates lytically during acute infection and establishes latency in peripheral neurons. Currently, the clinically approved compounds for the prevention of HSV-1 infection include acyclovir and penciclovir; however, long-term use of the drug is associated with serious side effects, and drug-resistant strains often appear. Therefore, it is important to find a safe and novel antiviral agent for HSV-1 infection. Quercus acuta Thunb. (Fagaceae) (QA) is widely distributed as an ornamental and dietary plant in Korea, Taiwan, China, and Japan. Thus far, the effects of QA extract and its active ingredients are known to have antioxidant, antibacterial, and anti-inflammatory activity, but studies of possible antiviral effects have not been reported. We studied the antiviral effects and molecular mechanism of QA after HSV-1 infection at the cellular level. We confirmed that QA suppresses ROS expression after HSV-1 infection and also suppresses inflammatory cytokine expression through inhibition of NF-кB activity. In addition, we found that QA increases the phosphorylation activity of IRF3 through induction of TBK1 activity during HSV-1 infection. QA exhibits an antiviral effect, and we confirmed through UPLC-DAD-mass spectrometer (MS)/MS analysis that it contains five main components: catechin, chlorogenic acid, fraxin, isoquercitrin, and taxifolin. Of these, isoquercitrin was confirmed to exhibit an antiviral effect on SK-N-SH cells through ICP27 inhibition. Overall, our results suggest that QA is a novel inhibitor with antiviral effects against HSV-1 infection and may be used specifically to prevent and treat of herpes simplex virus encephalitis infection.

PRDM12 Is Transcriptionally Active and Required for Nociceptor Function Throughout Life

PR domain-containing member 12 (PRDM12) is a key developmental transcription factor in sensory neuronal specification and survival. Patients with rare deleterious variants in PRDM12 are born with congenital insensitivity to pain (CIP) due to the complete absence of a subtype of peripheral neurons that detect pain. In this paper, we report two additional CIP cases with a novel homozygous PRDM12 variant. To elucidate the function of PRDM12 during mammalian development and adulthood, we generated temporal and spatial conditional mouse models. We find that PRDM12 is expressed throughout the adult nervous system. We observed that loss of PRDM12 during mid-sensory neurogenesis but not in the adult leads to reduced survival. Comparing cellular biophysical nociceptive properties in developmental and adult-onset PRDM12 deletion mouse models, we find that PRDM12 is necessary for proper nociceptive responses throughout life. However, we find that PRDM12 regulates distinct age-dependent transcriptional programs. Together, our results implicate PRDM12 as a viable therapeutic target for specific pain therapies even in adults.

Synovial single-cell heterogeneity, zonation, and interactions: a patchwork of effectors in arthritis

Despite extensive research, there is still no treatment that would lead to remission in all patients with rheumatoid arthritis as our understanding of the affected site, the synovium, is still incomplete. Recently, single-cell technologies helped to decipher the cellular heterogeneity of the synovium; however, certain synovial cell populations, such as endothelial cells or peripheral neurons, remain to be profiled on a single-cell level. Furthermore, associations between certain cellular states and inflammation were found; whether these cells cause the inflammation remains to be answered. Similarly, cellular zonation and interactions between individual effectors in the synovium are yet to be fully determined. A deeper understanding of cell signalling and interactions in the synovium is crucial for a better design of therapeutics with the goal of complete remission in all patients.

AAV-PHP.S-mediated delivery of reporters to cranial ganglion sensory neurons

ABSTRACTBecause of their ease of use and low risk containment, Adeno-Associated Virus vectors are indispensable tools for much of neuroscience. Yet AAVs have been used relatively little to study the identities and connectivity of peripheral sensory neurons because methods to selectively target particular receptive fields or neuron types have been limited. The introduction of the AAV-PHP.S capsid with selective tropism for peripheral neurons (Chan et al., 2017) offered a solution, which we further elaborate here. We demonstrate using AAV-PHP.S with GFP or mScarlet reporters, that all cranial sensory ganglia, i.e. for cranial nerves V, VII, IX and X, are targeted. Pseudounipolar neurons of both somatic and visceral origin, but not satellite glia, are efficiently transduced rapidly and express the gene of interest within 1 week of injection. Fluorescent reporter proteins are transported into the central and peripheral axons of these sensory neurons, permitting visualization of terminals at high resolution, and/or in intact, cleared brain using light sheet microscopy. By combining a Cre-dependent reporter with the AAV-PHP.S capsid, we confirmed expression in a cell-type dependent manner for both anatomical and targeted functional analyses. The AAV-PHP.S capsid will be a powerful tool for mapping the receptive fields and circuits of molecular subtypes of many somatosensory, gustatory and visceral sensory neurons.SIGNIFICANCE STATEMENTAAV vectors have become an essential tool for visualizing, manipulating, and recoding from neurons of the central nervous system. However, the technology is not widely used for peripheral neurons because of several technical limitations. The AAV-PHP.S synthetic capsid, which targets peripheral neurons, was recently introduced (Chan et al., 2017). Here, we establish key parameters for using this virus, including which cells are transduced, the timing of expression in central and peripheral terminals, distant from neuronal somata, and the effectiveness of Cre-dependent constructs for cell type selective expression. This permits the use of AAV for constructing detailed anatomic and functional maps of the projections of molecular subtypes of peripheral sensory neurons.

Dermal macrophages set pain sensitivity by modulating tissue NGF levels through SNX25–Nrf2 signaling

Crosstalk between peripheral neurons and immune cells plays important roles in pain sensation. We identified sorting nexin 25 (Snx25) as a pain-modulating gene in a transgenic mouse line with reduced pain behavior. Snx25 conditional-KO (cKO) in monocyte/macrophage-lineage cells but not in the peripheral sensory neurons reduced pain responses in both normal and neuropathic conditions. Cross transplantation experiments of bone marrows between cKO and wild type (WT) mice revealed that cKO macrophages caused dull phenotype in WT mice and WT macrophages in turn increased pain behavior in cKO mice. SNX25 in dermal macrophages enhances NGF (one of the key factors in pain sensation) production by inhibiting ubiquitin-mediated degradation of Nrf2, a transcription factor that activates Ngf mRNA synthesis. We conclude that dermal macrophages set pain sensitivity by producing and secreting NGF into the dermis in addition to their host defense functions.

Circadian Clock-Controlled Checkpoints in the Pathogenesis of Complex Disease

The circadian clock coordinates physiology, metabolism, and behavior with the 24-h cycles of environmental light. Fundamental mechanisms of how the circadian clock regulates organ physiology and metabolism have been elucidated at a rapid speed in the past two decades. Here we review circadian networks in more than six organ systems associated with complex disease, which cluster around metabolic disorders, and seek to propose critical regulatory molecules controlled by the circadian clock (named clock-controlled checkpoints) in the pathogenesis of complex disease. These include clock-controlled checkpoints such as circadian nuclear receptors in liver and muscle tissues, chemokines and adhesion molecules in the vasculature. Although the progress is encouraging, many gaps in the mechanisms remain unaddressed. Future studies should focus on devising time-dependent strategies for drug delivery and engagement in well-characterized organs such as the liver, and elucidating fundamental circadian biology in so far less characterized organ systems, including the heart, blood, peripheral neurons, and reproductive systems.