Chronic pain is under-managed in individuals over 65 years of age due to a dearth of knowledge regarding the impact of age on opioid efficacy in the elderly. We have previously shown that advanced age and sex alter morphine modulation of persistent inflammatory pain (induced by intraplantar administration of Complete Freund's adjuvant (CFA)), such that morphine potency is highest in adult male rats (2mos), with EC50 values 2-fold higher in aged males (18mos) and females regardless of age. Age-induced reductions in morphine potency were accompanied by reduced mu opioid receptor (MOR) expression in the ventrolateral periaqueductal gray (vlPAG), a CNS region critical in pain modulation. The present studies further explore the impact of age on opioid signaling within the PAG. MOR affinity, availability, and G-protein activation were assessed using radioligand binding assays and GTPγS assays in vlPAG tissue from adult and aged, male and female rats collected 72h following CFA administration. Regulation of opioid induced G-protein signaling was assessed using RNAscope to analyze mRNA expression of Regulator of G-Protein Signaling (RGS) proteins RGS4 and RGS9-2. We find that aged males and females (adult and aged) exhibit reduced vlPAG MOR binding potential and reduced G-protein activation efficiency compared to adult males, suggesting age- and sex- differences in MOR machinery drive reduced opioid potency. RNAscope revealed increased expression of RGS4 and RGS9-2 in the vlPAG of aged animals compared to adults, indicating that MOR signaling is subject to greater negative regulation in the aged vlPAG. The observed age-related reductions in vlPAG MOR agonist binding and opioid induced G-protein activation, along with the observed increase in vlPAG RGS expression have significant implications in pain management in the aged population. Our novel findings elucidate several mechanisms mediating reduced morphine potency in aged animals, and identify potential targets to improve pain management in the elderly. R01DA041529-04.

Chronic low back pain (cLBP) prevalence increases with advancing age and is a leading contributor to mobility disability among older adults. Opioids are commonly prescribed treatments to reduce pain related symptoms. The rise in opioid use and misuse can enhance a variety of issues in the adult population; such as, lack of mobility and decrease in overall health and wellbeing. Few studies have examined the impact of opioid use on mobility in older adults with cLBP. Therefore, we sought to examine the relationship between self-reported opioid use and self-reported mobility. cLBP participants (n = 140) completed a series of questionnaires regarding pain intensity, interference, and disability including demographics, clinical pain assessment, and the Brief Pain Inventory-Short form. Pearson's chi-square tests, and regression-based analyses were conducted using SPSS version 26.0. Among cLBP participants, those who self-reported opioid use were more likely to have greater self-reported difficulty climbing stairs (χ2 = 16.6, p < .05), walking for fifteen minutes (χ2 = 17.7, p < .05), performing chores (χ2 = 15.4, p < .05), and running errands (χ2 = 10.7, p < .05). Among the older cLBP participants above the age of 54 (n = 38), half used an opioid (n = 19) at some point of time as a form of cLBP pain treatment. Among older adults, opioid use was significantly associated with poorer self-reported outcomes for climbing stairs (Wald χ2(1) = 5.9, p < .05), walking (Wald χ2(1) = 7.4, p < .05), and performing chores (Wald χ2(1) = 7.5, p < .05). Opioid use predicts poorer self-reported mobility among adults and older adults with cLBP. Results inform associations between pain treatment and mobility in aging populations. Future research should seek to understand the influence of opioids on objective performance measures in cLBP. This work was supported by Examining Racial And SocioEconomic Disparities in cLBP; ERASED; R01MD010441.

Painful diabetic neuropathy (PDN) is an intractable and debilitating disease characterized by neuropathic pain and small-fiber degeneration. Given the prevalence of the disease, there is a dire need to identify new targets for the development of disease-modifying therapeutics for PDN. In patients with PDN, dorsal root ganglion (DRG) nociceptors become hyperexcitable and eventually degenerate, but the molecular mechanism underlying the phenomenon is unknown. We aim to identify the gene expression profile of the DRG neurons in PDN pathology to facilitate the discovery of novel druggable targets. Using a well-established mouse model of PDN, mice were either fed a regular diet (RD) or a high-fat diet (HFD) for 10 weeks. We used a single-cell RNA (scRNA-seq) sequencing approach to capture the changes in the DRG in an unbiased fashion. As expected, analysis of the scRNA-seq identified both neuronal and non-neuronal clusters and several differentially expressed genes. Interestingly, we saw two closely related non-peptidergic clusters expressing a Mas-related G protein-coupled receptor (Mrgprd). While there were no differences in the expression of Mrgprd in one of the clusters (NP1 Type1), there seemed to be a significant increase in the expression of Mrgprd in a cluster we refer to as the NP1 Type2. To determine the functional relevance of the overexpression of Mrgprd, we used in vivo 2-photon calcium imaging with Nav1.8 Cre-GCaMP6 animals fed an RD or HFD and examined whether administration of β-alanine (a known agonist of Mrgprd) in the hind paw would directly activate Mrgprd positive DRG neurons. We observed an increase in the number, as well as an increase in the magnitude of response in the HFD indicating the hyperexcitability of neurons expressing Mrgprd. Taken together, our data highlights an important role of the Mrgprd receptor in the generation and maintenance of hyperexcitability in a mouse model of PDN. NS104295-01.

Background: The NIH Helping to End Addiction Long-term (HEAL) Initiative aims to focus efforts on advancing scientific solutions to stem the opioid crisis, improving prevention and treatment of opioid misuse/addiction, and enhancing pain management. Goal: NINDS is charged with accelerating the discovery and development of new non-addictive pharmacologic and non-pharmacologic pain therapeutics as part of the HEAL Initiative. NINDS established the Preclinical Screening Platform for Pain (PSPP) to accelerate and enhance testing of novel, non-addictive pain therapeutics. This program will evaluate new, as well as repurposed, small molecules, biologics, devices, and natural products across a range of pain conditions. PSPP is accepting assets from academic and industry sponsors, worldwide. Here we describe efforts within the PSPP program. The overall goal of the PSPP program is to provide an efficient, rigorous, one-stop in vivo screening resource to accelerate identification and efficacy profiling of non-opioid therapeutics for the treatment of pain. Under NINDS direction, preclinical testing of submitted agents is performed by contract facilities on a blinded and confidential basis at no cost to the PSPP participants. Test candidates are evaluated in a suite of in vivo pain-related endpoints and models, following in vitro receptor profiling, pharmacokinetic and safety assessment. Importantly, test candidates are also evaluated in models of abuse liability. We will describe the advances made to date towards establishing program goals of evaluating assets in a rigorous and reproducible manner.

Background Bulk tissue RNA-sequencing studies in human post-mortem brains have reported differentially expressed genes between patients with Schizophrenia (SCZ) and controls. Single-nucleus RNA-sequencing data indicate that genes downregulated in SCZ were particularly enriched in a sub-population of excitatory neurons (Ex21) indexed by SMYD1 gene. POSTN and NR4A2 are two genes highly specific to Ex21 and significantly downregulated in SCZ. We hypothesized that either the number of Ex21 cells or the expression of POSTN and NR4A2 within them would be reduced in SCZ vs. controls. Methods We conducted a RNAscope in situ hybridization study in 20 SCZ and 20 controls. Three sections of the DLPFC per individual were stained for SMYD1 and NR4A2, and another three for SMYD1 and POSTN, using the colorimetric version of RNAscope (240 sections). Each RNAscope slide was counterstained using hematoxylin/eosin. Additional sections were stained with NeuN to assess neuronal numbers. Results ANCOVA revealed that there were no significant differences between SCZ and controls in the proportion of Ex21 (SMYD1+) cells (p = 0.40 for POSTN dataset, 0.36 for NR4A2 dataset), or the expression of marker and target genes within SMYD1+ cells in the DLPFC (p = 0.86 for POSTN dataset, p = 0.15 for NR4A2 dataset). Conclusions These results contradict prior conclusions that both POSTN and NR4A2 are downregulated in SCZ, but the modest sample size and several methodological issues suggest caution in interpreting these results.

Background Aberrant prefrontal cortex (PFC) activity occurs in patients with neuropsychiatric disorders during sustained attention tasks, suggesting that PFC dysfunction underlies attention deficits in these patients. However, the mechanisms by which the PFC regulates sustained attention remain unclear. Methods Behavioral testing and c-Fos immunohistochemistry during performance of a touchscreen sustained attention task (rCPT) in mice. In vivo calcium and norepinephrine imaging to assess patterns of activity during the rCPT. In vivo electrophysiology to detect how the medial PFC (mPFC) and locus coeruleus (LC) communicate during the rCPT. For assessment of molecular function in subsets of mPFC neurons that receive contact from the LC, we used RNAscope and bulk RNA-sequencing. Results We found that the LC and mPFC synchronized their activity during the rCPT, and imaging of neuronal activity in the mPFC revealed that mPFC neurons have heterogeneous response patterns during rCPT performance, with some neurons increasing their calcium activity during stimulus orientation and some neurons increasing their calcium activity during behavioral responses. To determine the molecular identities of mPFC neurons that connect with the LC, we used RNAscope to find that mPFC neurons receiving LC contact are primarily GABAergic, while mPFC neurons projecting to the LC are primarily excitatory. Using bulk RNA-sequencing, we further found that depolarization of LC inputs to the mPFC caused enrichment of a host of transcripts in mPFC tissue. Conclusions We uncover unique biomarkers of neuronal function in the LC-mPFC circuit, providing insight into potential therapeutic targets for attentional regulation in disorders such as ADHD, major depressive disorder, and schizophrenia.

Background PTSD is a multigenic and multifactorial disorder occurring in the aftermath of significant trauma exposure. Recent GWAS have identified many high confidence loci as risk factors for PTSD, which have shed some light on impaired mechanisms. However, there are still fundamental gaps in our understanding of how these risk genes and pathways are interrelated in causing PTSD but are likely reflected in cell type-specific transcriptomic and epigenetic changes in the brain. Therefore, it is necessary to uncover the individual cell type contribution to the molecular pathology of PTSD. Methods We isolated nuclei from human postmortem dorsolateral prefrontal cortex (BA 9/46) from n=50 PTSD, MDD, and controls for single nucleus sequencing. We sequenced RNA from 10,000 nuclei per sample and used RNAscope fluorescence in situ hybridization to validate cell type specific gene expression changes. We performed snATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) on 5000 nuclei to generate disease and control open chromatin maps to compare DNA accessibility. Results These results implicate 19 cell types, in particular inhibitory interneurons and microglia as dysregulated in PTSD brain. Open chromatin profiles matched transcript levels and provided new genomic information and possible functional roles for PTSD risk loci identified by GWAS. Conclusions -Omics technologies have been instrumental in our understanding of the connection between the disruption of particular loci and final molecular pathology of neuropsychiatric disorders. Applying functional genomics approaches to characterize findings from multiple layers of single cell-type studies of postmortem brain tissue may therefore help determine which neurotypical processes are most impacted by PTSD.

Although stressful events predispose individuals to psychiatric disorders, such as depression, not all people who undergo a stressful life experience become depressed, suggesting that gene-environment interactions (GxE) determine depression risk. The ventral hippocampus (vHPC) plays key roles in motivation, sociability, anhedonia, despair-like behaviors, anxiety, sleep, and feeding, pointing to the involvement of this brain region in depression. However, the molecular mechanisms underlying the crosstalk between the vHPC and GxE in shaping behavioral susceptibility and resilience to chronic stress remain elusive. Here, we show that Ca2+/calmodulin-dependent protein kinase IIβ (CaMKIIβ) activity in the vHPC is differentially modulated in GxE mouse models of depression susceptibility and resilience, and that CaMKIIβ–mediated TARPγ-8 phosphorylation enhances the expression of AMPA receptor subunit GluA1 in the post-synaptic sites to enable stress resilience. We present previously missing molecular mechanisms underlying chronic stress-elicited behavioral changes, providing strategies for preventing and treating stress-related psychiatric disorders.

Bile acids (BAs) are signalling molecules that mediate various cellular responses in both physiological and pathological processes. Several studies report that BAs can be detected in the brain1, yet their physiological role in the central nervous system is still largely unknown. Here we show that postprandial BAs can reach the brain and activate a negative-feedback loop controlling satiety in response to physiological feeding via TGR5, a G-protein-coupled receptor activated by multiple conjugated and unconjugated BAs2 and an established regulator of peripheral metabolism3-8. Notably, peripheral or central administration of a BA mix or a TGR5-specific BA mimetic (INT-777) exerted an anorexigenic effect in wild-type mice, while whole-body, neuron-specific or agouti-related peptide neuronal TGR5 deletion caused a significant increase in food intake. Accordingly, orexigenic peptide expression and secretion were reduced after short-term TGR5 activation. In vitro studies demonstrated that activation of the Rho-ROCK-actin-remodelling pathway decreases orexigenic agouti-related peptide/neuropeptide Y (AgRP/NPY) release in a TGR5-dependent manner. Taken together, these data identify a signalling cascade by which BAs exert acute effects at the transition between fasting and feeding and prime the switch towards satiety, unveiling a previously unrecognized role of physiological feedback mediated by BAs in the central nervous system.

Long noncoding RNAs (lncRNAs) are functionally associated with cancer development and progression. Although gene copy number variation (CNV) is common in hepatocellular carcinoma (HCC), it is not known how CNV in lncRNAs affects HCC progression and recurrence. We aimed to identify a CNV-related lncRNA involved in HCC progression and recurrence and illustrate its underlying mechanisms and prognostic value. We analyzed the whole genome sequencing (WGS) data of matched cancerous and noncancerous liver samples from 49 patients with HCC to identify lncRNAs with CNV. The results were validated in another cohort of 238 paired HCC and nontumor samples by TaqMan copy number assay. We preformed Kaplan-Meier analysis and log-rank test to identify lncRNA CNV with prognostic value. We conducted loss- and gain-of-function studies to explore the biological functions of LINC01133 in vitro and in vivo. The competing endogenous RNAs (ceRNAs) mechanism was clarified by microRNA sequencing (miR-seq), quantitative real-time PCR (qRT-PCR), western blot, and dual-luciferase reporter assays. We confirmed the binding mechanism between lncRNA and protein by RNA pull-down, RNA immunoprecipitation, qRT-PCR, and western blot analyses. Genomic copy numbers of LINC01133 were increased in HCC, which were positively related with the elevated expression of LINC01133. Increased copy number of LINC01133 predicted the poor prognosis in HCC patients. LINC01133 overexpression in HCC cells promoted proliferation and aggressive phenotypes in vitro, and facilitated tumor growth and lung metastasis in vivo, whereas LINC01133 knockdown had the opposite effects. LINC01133 sponged miR-199a-5p, resulting in enhanced expression of SNAI1, which induced epithelial-to-mesenchymal transition (EMT) in HCC cells. In addition, LINC01133 interacted with Annexin A2 (ANXA2) to activate the ANXA2/STAT3 signaling pathway. LINC01133 promotes HCC progression by sponging miR-199a-5p and interacting with ANXA2. LINC01133 CNV gain is predictive of poor prognosis in patients with HCC.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Hypoxia-induced miR-210 is a crucial component of the tissue response to ischemia, stimulating angiogenesis and improving tissue regeneration. Previous analysis of miR-210 impact on the transcriptome in a mouse model of hindlimb ischemia showed that miR-210 regulated not only vascular regeneration functions, but also inflammation. To investigate this event, doxycycline-inducible miR-210 transgenic mice (Tg-210) and anti-miR-210 LNA-oligonucleotides were used. It was found that global miR-210 expression decreased inflammatory cells density and macrophages accumulation in the ischemic tissue. To dissect the underpinning cell mechanisms, Tg-210 mice were used in bone marrow (BM) transplantation experiments and chimeric mice underwent hindlimb ischemia. MiR-210 overexpression in the ischemic tissue was sufficient to increase capillary density and tissue repair, and to reduce inflammation in the presence of Wt-BM infiltrating cells. Conversely, when Tg-210-BM cells migrated in a Wt ischemic tissue, dysfunctional angiogenesis, inflammation, and impaired tissue repair, accompanied by fibrosis were observed. The fibrotic regions were positive for α-SMA, Vimentin, and Collagen V fibrotic markers and for phospho-Smad3, highlighting the activation of TGF-β1 pathway. Identification of Tg-210 cells by in situ hybridization showed that BM-derived cells contributed directly to fibrotic areas, where macrophages co-expressing fibrotic markers were observed. Cell cultures of Tg-210 BM-derived macrophages exhibited a pro-fibrotic phenotype and were enriched with myofibroblast-like cells, which expressed canonical fibrosis markers. Interestingly, inhibitors of TGF-β type-1-receptor completely abrogated this pro-fibrotic phenotype. In conclusion, a context-dependent regulation by miR-210 of the inflammatory response was identified. miR-210 expression in infiltrating macrophages is associated to improved angiogenesis and tissue repair when the ischemic recipient tissue also expresses high levels of miR-210. Conversely, when infiltrating an ischemic tissue with mismatched miR-210 levels, macrophages expressing high miR-210 levels display a pro-fibrotic phenotype, leading to impaired tissue repair, fibrosis, and dysfunctional angiogenesis.