Src homology 2 domain-containing inositol 5-phosphatase 1 (SHIP1), encoded by the gene INPP5D, is a lipid phosphatase that negatively regulates immune receptor signaling in hematopoietic cells and microglia. Here, we describe a pyridyl-pyrazole-piperidine scaffold and the lead compound 3-((2-chlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)pyridine (32), which demonstrates SHIP1 target engagement, brain exposure, and evidence of a central pharmacodynamic response in vivo. Structure-activity relationship studies, guided by biochemical and cellular assays using multiple human and murine protein constructs and cells, identified SHIP1-active ligands. A thermal shift assay using full-length SHIP1 was used to assess compounds for cellular target engagement, while studies in IL-4 conditioned THP-1 cells was used to demonstrate changes in downstream AKT signaling. Targeted lipidomics revealed changes in the overall phosphoinositide pool consistent with SHIP1 target engagement and reduction of phospho-AKT levels. In a protein-lipid overlay assay, compound 32 induced changes in the relative association of SHIP1 with multiple phosphatidylinositols on a membrane surface. In high-content cellular imaging assays, compound 32 enhanced the uptake of myelin/membrane debris and fibrillar amyloid by primary murine microglia, phenocopying a genetic model with reduced SHIP1 expression. Finally, oral administration of compound 32 resulted in brain exposure sufficient to alter gene expression and reduce IL-1β levels as pharmacodynamic markers of microglial activation and neuroinflammation in an amyloidosis mouse model of Alzheimer's disease. Collectively, these results define a scaffold with SHIP1 target engagement, CNS exposure, and in vivo activity, providing a foundation for the optimization of brain-penetrant SHIP1 ligands suitable for further mechanistic studies and therapeutic development for the treatment of Alzheimer's disease.

BACKGROUND:While there is ongoing support to position amyloid-targeting antibody-based therapeutics as standard-of-care treatments for Alzheimer's disease (AD), their benefits and limitations are debated. Despite significant reductions in amyloid with treatment, the disease continues to progress, and clinical trial data have now clearly demonstrated differential effects of sex. Given that females have a higher risk of AD, it is prudent to understand and predict beneficial effects and limitations in both sexes. The present studies aimed to characterize a murinized chimeric Aducanumab (chAducanumab) through the MODEL-AD Preclinical Testing Core (PTC) Drug Screening Pipeline. We evaluated the pharmacokinetic (PK), pharmacodynamic (PD), and behavioral effects of chronic administration to 5XFAD mice in comparison to murine IgG2a kappa control antibody (IgG) and saline. METHODS:5XFAD mice with significant plaque accumulation were chronically dosed IP once weekly chAducanumab and compared to IgG and saline controls. Brain and plasma levels of chAducanumab were analyzed using an attomolar sensitive, targeted mass spectrometry parallel reaction monitoring assay. Pre- and post-treatment plasma Aβ, terminal soluble and insoluble brain Aβ, and in vivo 18F-AV-45 and 18F-FDG PET were quantified and cross-referenced for the presence of anti-drug antibodies. Behaviors and cognitive function were tested in a behavioral battery. RESULTS:Both chAducanumab and IgG were effective in attenuating plasma and brain Aβ with more robust effects on Aβ lowering in males than in females, and dose-dependent levels of chAducanumab were detectable in plasma and brain homogenate. Reduction of Aβ plaques did not correspond to significant improvements in performance on cognitive tasks or healthspan measures. The variability in PD responses within chAducanumab treatment groups was cross-referenced to individual data for the presence of anti-drug antibodies. CONCLUSIONS:Sex-dependent differential clearance of amyloid plaques with monoclonal antibody treatment has been observed translationally across species. The interaction with IgG may contribute to both the amyloid-lowering effects and possibly to the reported adverse events. Results from this study will inform future MODEL-AD PTC evaluations of novel therapeutics, particularly those combined with or following anti-Aβ immunotherapies once amyloid is lowered and the disease continues to progress, and importantly to identify and predict differential effects of sex.

While there is ongoing support to position amyloid-targeting antibody-based therapeutics as standard-of-care treatments for Alzheimer’s disease (AD), their benefits and limitations are debated. Despite significant reductions in amyloid with treatment, the disease continues to progress, and clinical trial data have now clearly demonstrated differential effects of sex. Given that females have a higher risk of AD, it is prudent to understand and predict beneficial effects and limitations in both sexes. The present studies aimed to characterize a murinized chimeric Aducanumab ( ch Aducanumab) through the MODEL-AD Preclinical Testing Core (PTC) Drug Screening Pipeline. We evaluated the pharmacokinetic (PK), pharmacodynamic (PD), and behavioral effects of chronic administration to 5XFAD mice in comparison to murine IgG2a kappa control antibody (IgG) and saline. 5XFAD mice with significant plaque accumulation were chronically dosed IP once weekly ch Aducanumab and compared to IgG and saline controls. Brain and plasma levels of ch Aducanumab were analyzed using an attomolar sensitive, targeted mass spectrometry parallel reaction monitoring assay. Pre- and post-treatment plasma Aβ, terminal soluble and insoluble brain Aβ, and in vivo 18F-AV-45 and 18F-FDG PET were quantified and cross-referenced for the presence of anti-drug antibodies. Behaviors and cognitive function were tested in a behavioral battery. Both ch Aducanumab and IgG were effective in attenuating plasma and brain Aβ with more robust effects on Aβ lowering in males than in females, and dose-dependent levels of ch Aducanumab were detectable in plasma and brain homogenate. Reduction of Aβ plaques did not correspond to significant improvements in performance on cognitive tasks or healthspan measures. The variability in PD responses within ch Aducanumab treatment groups was cross-referenced to individual data for the presence of anti-drug antibodies. Sex-dependent differential clearance of amyloid plaques with monoclonal antibody treatment has been observed translationally across species. The interaction with IgG may contribute to both the amyloid-lowering effects and possibly to the reported adverse events. Results from this study will inform future MODEL-AD PTC evaluations of novel therapeutics, particularly those combined with or following anti-Aβ immunotherapies once amyloid is lowered and the disease continues to progress, and importantly to identify and predict differential effects of sex.

Background A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.

Background: A major goal of the Target Enablement to Accelerate Therapy Development for Alzheimer’s disease (TREAT-AD) program is to develop and identify high-quality tools to test target or mechanistic hypotheses. As part of this initiative, it is important that commercial reagents including research antibodies being used to interrogate drug targets have confirmed validation data in knock-out cell lines. Ideally, these antibodies should also have utility for both in vitro and in vivo studies such that the levels of target proteins in target tissues can be quantified. Methods: We evaluated commercial antibodies against TREAT-AD protein targets Moesin (Uniprot ID: P26038), CD44 (Uniprot ID: P16070), Midkine (Uniprot ID: P21741) and Secreted frizzled-related protein 1, referred to as “sFRP-1” (sFRP-1; Uniprot ID: Q8N474). Moesin, Midkine and sFRP-1, that were confirmed as selective based on data in knock-out cell lines. Western blot analysis was used to compare protein levels in brain homogenates from a mouse model with AD-relevant pathology (5XFAD) versus age-matched C57BL/6J control mice. Results: Anti-Moesin ab52490 reacted in mouse brain homogenate with a predicted molecular weight of 68 kDa. Moesin protein expression was 2.8 times higher in 5xFAD compared to WT. Anti-CD44 ab189524 reacted with a band at the predicted size of 82 kDa. CD44 protein expression was 1.9 times higher in 5xFAD compared to WT. Anti-Midkine AF7769 reacted with a band ~16 kDa and a 17.8 times greater expression in 5xFAD compared to WT. Anti-sFRP-1 ab267466 reacted with a band at 35 kDa as predicted. sFRP-1 protein expression was 11.9 times greater in 5xFAD compared to WT. Conclusions: These data confirm the utility of these selective commercially available antibodies against Moesin, CD44, Midkine, and sFRP-1 for in vivo studies in mice and provide insight into the use of 5XFAD mice for in vivo target engagement studies for these target proteins.

The ability to effectively translate therapeutic efficacy from the bench to clinical success for Alzheimer’s disease (AD) has been hampered in part due to limited recapitulation of the complexity of the disease in animal models. While analogous AD risk mutations have been engineered into animal models and have dominated the research field, these have primarily been familial, early onset risk alleles which do not capture the risk for AD for the majority of patients that present with sporadic late onset AD (LOAD). The IU/JAX/PITT MODEL-AD consortium is focused on developing mouse models with genetic risk variants associated with LOAD, in combination with environmental risk factors and aging to enable improved translation. The present studies aimed to characterize mice expressing humanized Aβ in combination with multiple genetic risk factors (APOE4 and the R47H risk variant in the Trem2 gene; LOAD2) and aged in the presence of a high-fat, high-sugar diet (HFD). LOAD2 mice exposed to HFD from adolescence (LOAD2+HFD) demonstrated aging changes relative to LOAD2 mice in the absence of HFD, including presentation of insoluble Aβ42 in brain and plasma, and increased inflammatory cytokines. 12-month aged LOAD2+HFD mice also demonstrated increased NfL in CSF, as well as vascular and perfusion changes as measured by PET/MRI. By 18 months, LOAD2+HFD mice demonstrated reductions in hippocampal neurons as well as cognitive impairment relative to LOAD2 mice in the absence of HFD on a translational touchscreen task. Intriguingly, gene expression profiles and proteomic signatures of aged LOAD2+HFD mice aligned with ‘omics signatures of AD patients in the absence of core neuritic plaques, which were not detected up to 24 months of age. Mice with genetic risk for LOAD coupled with environmental risk factors demonstrate aging-dependent changes in line with a spectrum and trajectory of features of clinical LOAD. From a precision medicine approach, our MODEL-AD Preclinical Testing Core and our TREAT-AD colleagues are prioritizing LOAD2+HFD mice as an important model system for evaluating the therapeutic potential of non-amyloid targeting therapeutics as well as for prophylactic interventions initiated prior to significant amyloid accumulation.

INTRODUCTION:The risk of developing Alzheimer's disease is associated with genes involved in microglial function. Inositol polyphosphate-5-phosphatase (INPP5D), which encodes Src homology 2 (SH2) domain-containing inositol polyphosphate 5-phosphatase 1 (SHIP1), is a risk gene expressed in microglia. Because SHIP1 binds receptor immunoreceptor tyrosine-based inhibitory motifs (ITIMs), competes with kinases, and converts PI(3,4,5)P3 to PI(3,4)P2, it is a negative regulator of microglia function. Validated inhibitors are needed to evaluate SHIP1 as a potential therapeutic target. METHODS:We identified inhibitors and screened the enzymatic domain of SHIP1. A protein construct containing two domains was used to evaluate enzyme inhibitor potency and selectivity versus SHIP2. Inhibitors were tested against a construct containing all ordered domains of the human and mouse proteins. A cellular thermal shift assay (CETSA) provided evidence of target engagement in cells. Phospho-AKT levels provided further evidence of on-target pharmacology. A high-content imaging assay was used to study the pharmacology of SHIP1 inhibition while monitoring cell health. Physicochemical and absorption, distribution, metabolism, and excretion (ADME) properties were evaluated to select a compound suitable for in vivo studies. RESULTS:SHIP1 inhibitors displayed a remarkable array of activities and cellular pharmacology. Inhibitory potency was dependent on the protein construct used to assess enzymatic activity. Some inhibitors failed to engage the target in cells. Inhibitors that were active in the CETSA consistently destabilized the protein and reduced pAKT levels. Many SHIP1 inhibitors were cytotoxic either at high concentration due to cell stress or they potently induced cell death depending on the compound and cell type. One compound activated microglia, inducing phagocytosis at concentrations that did not result in significant cell death. A pharmacokinetic study demonstrated brain exposures in mice upon oral administration. DISCUSSION:3-((2,4-Dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine activated primary mouse microglia and demonstrated exposures in mouse brain upon oral dosing. Although this compound is our recommended chemical probe for investigating the pharmacology of SHIP1 inhibition at this time, further optimization is required for clinical studies. Highlights:Cellular thermal shift assay (CETSA) and signaling (pAKT) assays were developed to provide evidence of src homology 2 (SH2) domain-contaning inositol phosphatase 1 (SHIP1) target engagement and on-target activity in cellular assays.A phenotypic high-content imaging assay with simultaneous measures of phagocytosis, cell number, and nuclear intensity was developed to explore cellular pharmacology and monitor cell health.SHIP1 inhibitors demonstrate a wide range of activity and cellular pharmacology, and many reported inhibitors are cytotoxic.The chemical probe 3-((2,4-dichlorobenzyl)oxy)-5-(1-(piperidin-4-yl)-1H-pyrazol-4-yl) pyridine is recommended to explore SHIP1 pharmacology.

Recent evidence including GWAS and differential gene expression comparing normal to affected Alzheimer’s brain tissue have identified risk and protective variants in genes such as TREM2, PLCG2 and INPP5D that are essential to microglia function. INPP5D encodes SHIP1, a multi-domain protein with a phosphatase that converts PI(3,4,5)P 3 to PI(3,4)P 2 , a SH2 domain that interacts with receptor ITAMs and competes with SYK, and a proline rich region that binds many other proteins. SHIP1 therefore limits microglia activation in multiple ways. Inhibition of SHIP1 early in disease would increase microglial protective functions and reduce the rate of disease progression and cognitive decline in Alzheimer’s patients. We performed a screen of 50K compounds at the SHIP1 phosphatase, analyzed a publicly available fragment-based screen, and evaluated inhibitors reported in the literature. We utilized the malachite green assay with PtdIns(3,4,5)P 3 -diC8 and SHIP1 Ptase-C2 to measure inhibitory potency. A Cellular Thermal Shift Assay was used to confirm target engagement in cells. A high-content imaging assay measuring phagocytosis, cell number, and nuclear intensity was implemented using the BV2 and HMC3 cell lines to characterize cellular pharmacology and cytotoxicity. Mouse microglia were assayed to demonstrate similar activity in primary cells. Inhibitors predicted to have drug-like properties were subjected to assays measuring solubility, cellular permeability, and mouse microsomal stability. A physiological based pharmacokinetic model was compared to measured exposure in vivo for select compounds upon oral administration in mice. SHIP1 inhibitors have been compared head-to-head in a set of assays relevant to both enzyme inhibition and microglia activation. Structurally distinct, novel, and selective SHIP1 inhibitors have been discovered. The enzyme mode of action, cellular activity and drug-like properties were determined. Pharmacokinetic profiles were determined for inhibitors with sufficient cellular potency and drug-like properties for in vivo studies in mice. Inhibition of SHIP1 is a novel therapeutic strategy for treatment of Alzheimer’s. We identified structurally distinct molecular scaffolds with varying degrees of enzyme inhibition, cellular activity, and exposure in mice. Recommendations for use of probe molecules in target validation studies and the development of lead-like molecules for clinical studies will be made.

Endogenous lipid mediators are proposed to contribute to headache and facial pain by activating trigeminal neurons (TN). We recently identified 11-hydroxy-epoxide- and 11-keto-epoxide derivatives of linoleic acid (LA) that are present in human skin and plasma and potentially contribute to nociception. Here we expand upon initial findings by examining the effects of 11-hydroxy- and 11-keto-epoxide-LA derivatives on TN activation in comparison to LA, the LA derivative [9-hydroxy-octadecadienoic acid (9-HODE)] and prostaglandin E2 (PGE2). 11-hydroxy- and 11-keto-epoxide-LA derivatives elicited Ca2+ transients in TN subpopulations. The proportion of neurons responding to test compounds (5 μM, 5 min) ranged from 16.2 ± 3.8 cells (11 K-9,10E-LA) to 34.1 ± 2.4 cells (11H-12,13E-LA). LA and 9-HODE (5 μM, 5 min) elicited responses in 11.6 ± 3.1% and 9.7 ± 3.4% of neurons, respectively. 11H-12,13E-LA, 11K-12,13E-LA, and 11H-9,10E-LA produced Ca2+ responses in significantly higher proportions of neurons compared to either LA or 9-HODE (F (6, 36) = 5.12, P = 0.0007). 11H-12,13E-LA and 11H-9,10E-LA increased proportions of responsive neurons in a concentration-dependent fashion, similar to PGE2. Most sensitive neurons responded to additional algesic agents (32.9% to capsaicin, 40.1% to PGE2, 58.0% to AITC), however 20.6% did not respond to any other agent. In summary, 11-hydroxy-epoxide derivatives of LA increase trigeminal neuron excitability, suggesting a potential role in headache or facial pain.

A major dose-limiting side effect of docetaxel chemotherapy is peripheral neuropathy. Patients' symptoms include pain, numbness, tingling and burning sensations, and motor weakness in the extremities. The molecular mechanism is currently not understood, and there are no treatments available. Previously, we have shown an association between neuropathy symptoms of patients treated with paclitaxel and the plasma levels of neurotoxic sphingolipids, the 1-deoxysphingolipids (1-deoxySL) (Kramer et al, FASEB J, 2015). 1-DeoxySL are produced when the first enzyme of the sphingolipid biosynthetic pathway, serine palmitoyltransferase (SPT), uses L-alanine as a substrate instead of its canonical amino acid substrate, L-serine. In the current investigation, we tested whether 1-deoxySL accumulate in the nervous system following systemic docetaxel treatment in mice. In dorsal root ganglia (DRG), we observed that docetaxel (45 mg/kg cumulative dose) significantly elevated the levels of 1-deoxySL and L-serine-derived ceramides, but not sphingosine-1-phosphate (S1P). S1P is a bioactive sphingolipid and a ligand for specific G-protein-coupled receptors. In the sciatic nerve, docetaxel decreased 1-deoxySL and ceramides. Moreover, we show that in primary DRG cultures, 1-deoxysphingosine produced neurite swellings that could be reversed with S1P. Our results demonstrate that docetaxel chemotherapy up-regulates sphingolipid metabolism in sensory neurons, leading to the accumulation of neurotoxic 1-deoxySL. We suggest that the neurotoxic effects of 1-deoxySL on axons can be reversed with S1P.

Acute inflammation induces sensitization of nociceptive neurons and triggers the accumulation of calcium permeable (CP) alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) in the dorsal horn of the spinal cord. This coincides with behavioral signs of acute inflammatory pain, but whether CP-AMPARs contribute to chronic pain remains unclear. To evaluate this question, we first constructed current-voltage (I-V) curves of C-fiber stimulus-evoked, AMPAR-mediated EPSCs in lamina II to test for inward rectification, a key characteristic of CP-AMPARs. We found that the intraplantar injection of complete Freund's adjuvant (CFA) induced an inward rectification at 3 d that persisted to 21 d after injury. Furthermore, the CP-AMPAR antagonist IEM-1460 (50 mu M) inhibited AMPAR-evoked Ca2+ transients 21 d after injury but had no effect in uninflamed mice. We then used a model of long-lasting vulnerability for chronic pain that is determined by the balance between latent central sensitization (LCS) and mu opioid receptor constitutive activity (MORCA). When administered 21 d after the intraplantar injection of CFA, intrathecal administration of the MORCA inverse agonist naltrexone (NTX, 1 mu g, i.t) reinstated mechanical hypersensitivity, and superfusion of spinal cord slices with NTX (10 mu M) increased the peak amplitude of AMPAR-evoked Ca2+ transients in lamina II neurons. The CP-AMPAR antagonist naspm (0-10 nmol, i.t.) inhibited these NTX-induced increases in mechanical hypersensitivity. NTX had no effect in uninflamed mice. Subsequent western blot analysis of the postsynaptic density membrane fraction from lumbar dorsal horn revealed that CFA increased GluA1 expression at 2 d and GluA4 expression at both 2 and 21 d post-injury, indicating that not just the GluA1 subunit, but also the GluA4 subunit, contributes to the expression of CP-AMPARs and synaptic strength during hyperalgesia. GluA2 expression increased at 21 d, an unexpected result that requires further study. We conclude that after tissue injury, dorsal horn AMPARs retain a Ca2+ permeability that underlies LCS. Because of their effectiveness in reducing naltrexone-induced reinstatement of hyperalgesia and potentiation of AMPAR-evoked Ca2+ signals, CP-AMPAR inhibitors are a promising class of agents for the treatment of chronic inflammatory pain.

Peripheral inflammation produces a long-lasting latent sensitization of spinal nociceptive neurons, that is, masked by tonic inhibitory controls. We explored mechanisms of latent sensitization with an established four-step approach: (1) induction of inflammation; (2) allow pain hypersensitivity to resolve; (3) interrogate latent sensitization with a channel blocker, mutant mouse, or receptor antagonist; and (4) disrupt compensatory inhibition with a receptor antagonist so as to reinstate pain hypersensitivity. We found that the neuropeptide Y Y1 receptor antagonist BIBO3304 reinstated pain hypersensitivity, indicative of an unmasking of latent sensitization. BIBO3304-evoked reinstatement was not observed in AC1 knockout mice and was prevented with intrathecal co-administration of a pharmacological blocker to the N-methyl-D-aspartate receptor (NMDAR), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), transient receptor potential cation channel A1 (TRPA1), channel V1 (TRPV1), or exchange protein activated by cAMP (Epac1 or Epac2). A PKA activator evoked both pain reinstatement and touch-evoked pERK expression in dorsal horn; the former was prevented with intrathecal co-administration of a TRPA1 or TRPV1 blocker. An Epac activator also evoked pain reinstatement and pERK expression. We conclude that PKA and Epac are sufficient to maintain long-lasting latent sensitization of dorsal horn neurons that is kept in remission by the NPY-Y1 receptor system. Furthermore, we have identified and characterized 2 novel molecular signaling pathways in the dorsal horn that drive latent sensitization in the setting of chronic inflammatory pain: NMDAR→AC1→PKA→TRPA1/V1 and NMDAR→AC1→Epac1/2. New treatments for chronic inflammatory pain might either increase endogenous NPY analgesia or inhibit AC1, PKA, or Epac.

Painful diabetic neuropathy (PDN) is a devastating neurological complication of diabetes. Methylglyoxal (MG) is a reactive metabolite whose elevation in the plasma corresponds to PDN in patients and pain-like behavior in rodent models of type 1 and type 2 diabetes. Here, we addressed the MG-related spinal mechanisms of PDN in type 2 diabetes using db/db mice, an established model of type 2 diabetes, and intrathecal injection of MG in conventional C57BL/6J mice. Administration of either a MG scavenger (GERP10) or a vector overexpressing glyoxalase 1, the catabolic enzyme for MG, attenuated heat hypersensitivity in db/db mice. In C57BL/6J mice, intrathecal administration of MG produced signs of both evoked (heat and mechanical hypersensitivity) and affective (conditioned place avoidance) pain. MG-induced Ca2+ mobilization in lamina II dorsal horn neurons of C57BL/6J mice was exacerbated in db/db, suggestive of MG-evoked central sensitization. Pharmacological and/or genetic inhibition of transient receptor potential ankyrin subtype 1 (TRPA1), adenylyl cyclase type 1 (AC1), protein kinase A (PKA), or exchange protein directly activated by cyclic adenosine monophosphate (Epac) blocked MG-evoked hypersensitivity in C57BL/6J mice. Similarly, intrathecal administration of GERP10, or inhibitors of TRPA1 (HC030031), AC1 (NB001), or Epac (HJC-0197) attenuated hypersensitivity in db/db mice. We conclude that MG and sensitization of a spinal TRPA1-AC1-Epac signaling cascade facilitate PDN in db/db mice. Our results warrant clinical investigation of MG scavengers, glyoxalase inducers, and spinally-directed pharmacological inhibitors of a MG-TRPA1-AC1-Epac pathway for the treatment of PDN in type 2 diabetes.

Multiple sclerosis (MS) is an autoimmune-inflammatory neurodegenerative disease that is often accompanied by a debilitating neuropathic pain. Disease-modifying agents slow down the progression of multiple sclerosis and prevent relapses, yet it remains unclear if they yield analgesia. We explored the analgesic potential of fingolimod (FTY720), an agonist and/or functional antagonist at the sphingosine-1-phosphate receptor 1 (S1PR1), because it reduces hyperalgesia in models of peripheral inflammatory and neuropathic pain. We used a myelin oligodendrocyte glycoprotein 35 to 55 (MOG(35-55) ) mouse model of experimental autoimmune encephalomyelitis, modified to avoid frank paralysis, and thus, allow for assessment of withdrawal behaviors to somatosensory stimuli. Daily intraperitoneal fingolimod reduced behavioral signs of central neuropathic pain (mechanical and cold hypersensitivity) in a dose-dependent and reversible manner. Both autoimmune encephalomyelitis and fingolimod changed hyperalgesia before modifying motor function, suggesting that pain-related effects and clinical neurological deficits were modulated independently. Fingolimod also reduced cellular markers of central sensitization of neurons in the dorsal horn of the spinal cord: glutamate-evoked Ca2+ signaling and stimulus-evoked phospho-extracellular signal-related kinase ERK (pERK) expression, as well as upregulation of astrocytes (GFAP) and macrophage/microglia (Iba1) immunoreactivity. The antihyperalgesic effects of fingolimod were prevented or reversed by the S1PR1 antagonist W146 (1 mg/kg daily, i.p.) and could be mimicked by either repeated or single injection of the S1PR1-selective agonist SEW2871. Fingolimod did not change spinal membrane S1PR1 content, arguing against a functional antagonist mechanism. We conclude that fingolimod behaves as an S1PR1 agonist to reduce pain in multiple sclerosis by reversing central sensitization of spinal nociceptive neurons.

The putative strong anti-nociceptive properties of the antidepressant phenelzine (PLZ) have not been widely explored as a treatment for pain. Antinociceptive effects of PLZ were identified in the formalin model of tonic pain (Mifflin et al., 2016) and in allodynia associated with experimental autoimmune encephalomyelitis, (EAE) a mouse model of multiple sclerosis (Potter et al., 2016). Here, we further clarify the specific types of stimuli and contexts in which PLZ modulates nociceptive sensitivity. Our findings indicate that PLZ selectively inhibits ongoing inflammatory pain while sparing transient reflexive and acute nociception. We also investigated the cellular mechanisms of action of PLZ in the dorsal horn, and as expected of a monoamine-oxidase inhibitor, PLZ increased serotonin (5HT) immunoreactivity. We next used two approaches to test the hypothesis that PLZ inhibits the activation of spinal nociresponsive neurons. First, we evaluated the formalin-evoked protein expression of the immediate early gene, c-fos. PLZ reduced Fos expression in the superficial dorsal horn. Second, we evaluated the effects of PLZ on intracellular calcium responses to superfusion of glutamate (0.3-1.0 mM) in an ex vivo lumbar spinal cord slice preparation. Superfusion with PLZ (100-300 μM) reduced 1 mM glutamate-evoked calcium responses. This was blocked by pretreatment with the 5HT1A-receptor antagonist WAY-100,635, but not the alpha-2 adrenergic antagonist idazoxan. We conclude that PLZ exerts antinociceptive effects through a 5-HT/5HT1AR-dependent inhibition of neuronal responses within nociceptive circuits of the dorsal horn.

The complement 3a receptor (C3aR1) participates in microglial signaling under pathological conditions and was recently shown to be activated by the neuropeptide TLQP-21. We previously demonstrated that TLQP-21 elicits hyperalgesia and contributes to nerve injury-induced hypersensitivity through an unknown mechanism in the spinal cord. Here we determined that this mechanism requires C3aR1 and that microglia are the cellular target for TLQP-21. We propose a novel neuroimmune signaling pathway involving TLQP-21-induced activation of microglial C3aR1 that then contributes to spinal neuroplasticity and neuropathic pain. This unique dual-ligand activation of C3aR1 by a neuropeptide (TLQP-21) and an immune mediator (C3a) represents a potential broad-spectrum mechanism throughout the CNS for integration of neuroimmune crosstalk at the molecular level.

Multiple sclerosis (MS) is a human neuroinflammatory disease, affecting millions of individuals worldwide; it leads to autonomic dysregulation, paresis or paralysis, and chronic pain. Despite its high prevalence (about 50%, many indicating this is the most debilitating symptom) and non-responsiveness to disease-modifying therapies (DMTs), neither clinical nor basic science studies have determined the mechanism of pain. Our premise comes from recent studies indicating that fingolimod—a new-generation, sphingosine-1-phosphate receptor (S1PR)-modulating DMT—reduces behavioral signs of pain in multiple animal models of peripheral inflammation or nerve injury. To extend this to a model of MS chronic pain, we used a myelin oligodendrocyte glycoprotein, 35-55 (MOG35-55)-induced murine experimental autoimmune encephalomyelitis (EAE) model of MS, optimized to avoid frank paralysis while producing robust mechanical (von Frey) and cold (plantar acetone) hypersensitivity. We found that daily systemic administration of fingolimod dose-dependently (0.001, 0.01, 0.03, 0.1, 1 mg/kg) reduced von Frey and acetone hypersensitivity within 7 days. The S1PR1-selective antagonist W146 (1 mg/kg daily i.p.) either prevented (naïve: vF 2.740 g ± 0.163, cold 2.2 s ± 0.3; fingolimod: vF 0.198 g ± 0.019, cold 4.0 s ± 0.1; fingolimod+W146: vF 0.023 g ± 0.005, cold 5.1 s ± 0.2) or reversed the antihyperalgesic effects of fingolimod (1 mg/kg daily i.p. for 14 days) and could be mimicked by the S1PR1-selective agonist SEW2871 (20 mg/kg daily i.p.) either by repeated (SEW2871, 14 days: vF 1.869 g ± 0.592, cold 5.5 s ± 0.8; vehicle: vF 0.301 g ± 0.034, cold 7.8 s ± 0.8) or single injection. Our working hypothesis is that fingolimod acts as a lymphopenia-independent, central S1PR1 agonist to reduce chronic neuropathic pain in EAE. To determine whether fingolimod acts centrally, we will measure the blood-brain barrier permeability of W146 and determine the effects of direct intrathecal infusion of fingolimod, SEW2871, or W146.

NPY reduces behavioral signs of acute and chronic pain, in part through activation of the NPY Y1-receptor (Y1R). However, the cellular mechanism of Y1R-mediated analgesia remains unclear. One outstanding question is whether they are expressed in inhibitory and/or excitatory neurons, and where they fit within the dorsal horn microcircuity of pain transmission and pain control. Behavioral pharmacology and targeted neurotoxin studies from our laboratory suggest that Y1R-expressing neurons are excitatory. Furthermore, we report that Y1Rs co-exist with multiple markers of excitatory neurons including Tlx3, but not with inhibitory marker of spinal interneurons, PAX2. Preliminary single-cell PCR experiments in Y1-expressing neurons using GAD67 and vGlut2 probes suggest that Y1-neurons are GABAergic neurons. To further characterize Y1R-expressing neurons we used patch-clamp electrophysiology to obtained firing pattern of spinal cord lamina II Y1-neurons from lumbar L4/L5 segment of adult Y1R-eGFP mice. In Y1R-positive cells, the majority of cells exhibited initial bursting/phasic firing (87%). However, 76 % of these neurons exhibited delayed firing when current injection were applied from hyperpolarizing potentials below -80 mV. These numbers did not significantly change in the spared nerve injury mice models. The delayed firing pattern in of the Y1-neurons coincided with rapid A-type potassium currents. Firing property and single-cell PCR experiments of Gad67 and vGluT2 expressing neurons are currently underway to demonstrate once for all whether Y1R-eGFP neurons express glutamate and/or GABA. On administration of NPY we observed that NPY induced outward current in voltage clamp mode and hyperpolarizing shift of the resting membrane potential by ∼10 mV. DRS evoked action potentials were abolished by application of NPY suggesting that NPY inhibits the excitability of Y1R-expressing interneurons in dorsal horn. Taken together, our data suggest that endogenous spinal NPY produces analgesia by altering a complex dorsal horn microcircuit in the dorsal horn that includes interneurons that express Y1R.