Seena K. Ajit

Circulating microRNAs as Biomarkers, Therapeutic Targets, and Signaling Molecules

Small noncoding microRNAs (miRNAs) are important regulators of post-transcriptional gene regulation and have altered the prevailing view of a linear relationship between gene and protein expression. Aberrant miRNA expression is an emerging theme for a wide variety of diseases, highlighting the fundamental role played by miRNAs in both physiological and pathological states. The identification of stable miRNAs in bodily fluids paved the way for their use as novel biomarkers amenable to clinical diagnosis in translational medicine. Identification of miRNAs in exosomes that are functional upon delivery to the recipient cells has highlighted a novel method of intercellular communication. Delivery of miRNAs to recipient cells via blood, with functional gene regulatory consequences, opens up novel avenues for target intervention. Exosomes thus offer a novel strategy for delivering drugs or RNA therapeutic agents. Though much work lies ahead, circulating miRNAs are unequivocally ushering in a new era of novel biomarker discovery, intercellular communication mechanisms, and therapeutic intervention strategies.

Dynamic Changes in the MicroRNA Expression Profile Reveal Multiple Regulatory Mechanisms in the Spinal Nerve Ligation Model of Neuropathic Pain

Neuropathic pain resulting from nerve lesions or dysfunction represents one of the most challenging neurological diseases to treat. A better understanding of the molecular mechanisms responsible for causing these maladaptive responses can help develop novel therapeutic strategies and biomarkers for neuropathic pain. We performed a miRNA expression profiling study of dorsal root ganglion (DRG) tissue from rats four weeks post spinal nerve ligation (SNL), a model of neuropathic pain. TaqMan low density arrays identified 63 miRNAs whose level of expression was significantly altered following SNL surgery. Of these, 59 were downregulated and the ipsilateral L4 DRG, not the injured L5 DRG, showed the most significant downregulation suggesting that miRNA changes in the uninjured afferents may underlie the development and maintenance of neuropathic pain. TargetScan was used to predict mRNA targets for these miRNAs and it was found that the transcripts with multiple predicted target sites belong to neurologically important pathways. By employing different bioinformatic approaches we identified neurite remodeling as a significantly regulated biological pathway, and some of these predictions were confirmed by siRNA knockdown for genes that regulate neurite growth in differentiated Neuro2A cells. In vitro validation for predicted target sites in the 3′-UTR of voltage-gated sodium channel Scn11a, alpha 2/delta1 subunit of voltage-dependent Ca-channel, and purinergic receptor P2rx ligand-gated ion channel 4 using luciferase reporter assays showed that identified miRNAs modulated gene expression significantly. Our results suggest the potential for miRNAs to play a direct role in neuropathic pain.

MicroRNA modulation in complex regional pain syndrome

BackgroundAberrant expression of small noncoding RNAs called microRNAs (miRNAs) is a common feature of several human diseases. The objective of the study was to identify miRNA modulation in patients with complex regional pain syndrome (CRPS) a chronic pain condition resulting from dysfunction in the central and/or peripheral nervous systems. Due to a multitude of inciting pathologies, symptoms and treatment conditions, the CRPS patient population is very heterogeneous. Our goal was to identify differentially expressed miRNAs in blood and explore their utility in patient stratification.MethodsWe profiled miRNAs in whole blood from 41 patients with CRPS and 20 controls using TaqMan low density array cards. Since neurogenic inflammation is known to play a significant role in CRPS we measured inflammatory markers including chemokines, cytokines, and their soluble receptors in blood from the same individuals. Correlation analyses were performed for miRNAs, inflammatory markers and other parameters including disease symptoms, medication, and comorbid conditions.ResultsThree different groups emerged from miRNA profiling. One group was comprised of 60% of CRPS patients and contained no control subjects. miRNA profiles from the remaining patients were interspersed among control samples in the other two groups. We identified differential expression of 18 miRNAs in CRPS patients. Analysis of inflammatory markers showed that vascular endothelial growth factor (VEGF), interleukin1 receptor antagonist (IL1Ra) and monocyte chemotactic protein-1 (MCP1) were significantly elevated in CRPS patients. VEGF and IL1Ra showed significant correlation with the patients reported pain levels. Analysis of the patients who were clustered according to their miRNA profile revealed correlations that were not significant in the total patient population. Correlation analysis of miRNAs detected in blood with additional parameters identified miRNAs associated with comorbidities such as headache, thyroid disorder and use of narcotics and antiepileptic drugs.ConclusionsmiRNA profiles can be useful in patient stratification and have utility as potential biomarkers for pain. Differentially expressed miRNAs can provide molecular insights into gene regulation and could lead to new therapeutic intervention strategies for CRPS.

MrgD Activation Inhibits KCNQ/M-Currents and Contributes to Enhanced Neuronal Excitability

The recently identified Mas-related gene (Mrg) family of G-protein-coupled receptors is expressed almost exclusively in dorsal root ganglion (DRG) neurons. The expression of one family member, MrgD, is even further confined to IB4+, nonpeptidergic, small-diameter nociceptors. Although the functional consequences of MrgD activation are not known, this expression profile provides intriguing potential for a role in pain sensation or modulation. In a recombinant cell line, we first assessed the functional significance of MrgD activation by coexpressing MrgD with the KCNQ2/3 potassium channel, a channel implicated in pain. Whole-cell voltage-clamp recordings revealed that bath application of the ligand for MrgD, β-alanine, resulted in robust inhibition of KCNQ2/3 activity. Pharmacological blockade of Gi/o and phospholipase C signaling revealed a partial and complete block of the response, respectively. We extended these observations to dissociated DRG neuron cultures by examining MrgD modulation of M-currents (carried primarily by KCNQ2/3). Here too, β-alanine-induced activation of endogenous MrgD inhibited M-currents, but primarily via a pertussis toxin-sensitive pathway. Finally, we assessed the consequence of β-alanine-induced activation of MrgD in phasic neurons. Phasic neurons that fired a single action potential (AP) before β-alanine application fired multiple APs during β-alanine exposure. In sum, we provide evidence for a novel interaction between MrgD and KCNQ/M-type potassium channels that contributes to an increase in excitability of DRG neurons and thus may enhance the signaling of primary afferent nociceptive neurons.

Functional significance of macrophage-derived exosomes in inflammation and pain

Summary Macrophage‐derived exosomes attenuated complete Freunds adjuvant‐induced thermal hyperalgesia in mice. Exosomal microRNA signature from patients with complex regional pain syndrome suggests a potential therapeutic and biomarker utility for exosomes. ABSTRACT Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome‐mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide‐stimulated cells were sufficient to cause nuclear factor‐&kgr;B activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage‐derived exosomes. Macrophage‐derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility.

The molecular basis of pain and its clinical implications in rheumatology

Nociceptive pain in response to peripheral noxious stimuli, and inflammatory pain resulting from tissue damage, serve as warnings that normal bodily function cannot resume until the stimulus abates or the tissue repairs. Stimuli cause numerous receptors, ion channels and other cellular machinery to respond, and propagate signals to the central nervous system, where this information is processed and perceived as pain. In healthy individuals, tissue damage results in physiologic—generally reparative—changes that lead to heightened sensory perception and, often, pain. In rheumatic diseases, the joint pain bears much in common with chronic inflammatory pain, but the underlying disease state is typically much more intricate and no reparative function is evident. Addressing the complex pains of rheumatic disease remains an ongoing challenge. Pain signaling pathways involve many molecular components that could potentially be targets for pharmacotherapeutic intervention, but the complexity of this system might also mean that multiple sites must be affected simultaneously to disrupt propagation of pain signals. In addition, to be therapeutically viable, pain drugs must be safe and not alter tactile sensory function, alertness or cognitive function. In this article we review the molecular functions in nociceptive, inflammatory and rheumatic pain pathways, and the therapeutic options they might offer.

Development of a FLIPR Assay for the Simultaneous Identification of MrgD Agonists and Antagonists from a Single Screen

MrgD, a member of the Mas-related gene family, is expressed exclusively in small diameter IB4+ neurons in the dorsal root ganglion. This unique expression pattern, the presence of a single copy of MrgD in rodents and humans, and the identification of a putative ligand, beta-alanine, make it an experimentally attractive therapeutic target for pain with limited likelihood of side effects. We have devised a high throughput calcium mobilization assay that enables identification of both agonists and antagonists from a single screen for MrgD. Screening of the Library of Pharmacologically Active Compounds (LOPAC) validated this assay approach, and we identified both agonists and antagonists active at micromolar concentrations in MrgD expressing but not in parental CHO-DUKX cell line. Further characterization was performed using a subset of these screening hits. Our results demonstrated that the dual agonist/antagonist assay format is feasible and likely can be extended to most GPCRs with known agonist.

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.

Analgesic Response to Intravenous Ketamine Is Linked to a Circulating microRNA Signature in Female Patients With Complex Regional Pain Syndrome

Although ketamine is beneficial in treating complex regional pain syndrome (CRPS), a subset of patients respond poorly to therapy. We investigated treatment-induced microRNA (miRNA) changes and their predictive validity in determining treatment outcome by assessing miRNA changes in whole blood from patients with CRPS. Blood samples from female patients were collected before and after 5 days of intravenous ketamine administration. Seven patients were responders and 6 were poor responders. Differential miRNA expression was observed in whole blood before and after treatment. In addition, 33 miRNAs differed between responders and poor responders before therapy, suggesting the predictive utility of miRNAs as biomarkers. Investigation of the mechanistic significance of hsa-miR-548d-5p downregulation in poor responders showed that this miRNA can downregulate UDP-glucuronosyltransferase UGT1A1 mRNA. Poor responders had a higher conjugated/unconjugated bilirubin ratio, indicating increased UGT1A1 activity. We propose that lower pretreatment levels of miR-548d-5p may result in higher UDP-GT activity, leading to higher levels of inactive glucuronide conjugates, thereby minimizing the therapeutic efficacy of ketamine in poor responders. Differences in miRNA signatures can provide molecular insights distinguishing responders from poor responders. Extending this approach to other treatment and outcome assessments might permit stratification of patients for maximal therapeutic outcome. Perspective: This study suggests the usefulness of circulating miRNAs as potential biomarkers. Assessing miRNA signatures before and after treatment demonstrated miRNA alterations from therapy; differences in miRNA signature in responders and poor responders before therapy indicate prognostic value. Mechanistic studies on altered miRNAs can provide new insights into disease.

Effect of Histone Deacetylase Inhibitor JNJ-26481585 in Pain

Recent studies have shown that histone deacetylase (HDAC) inhibitors can alleviate inflammatory and neuropathic pain. We investigated the effects of JNJ-26481585, a pan-HDAC inhibitor on basal mechanical sensitivity. Unlike previous reports for HDAC inhibitors, JNJ-26481585 induced mechanical hypersensitivity in mice. This effect was reversible with gabapentin. Voltage-dependent calcium channel subunit alpha-2/delta-1, one of the putative targets for gabapentin, was upregulated in the spinal cord from JNJ-26481585-treated mice. Transcriptional profiling of spinal cord from JNJ-26481585-treated mice showed significant alterations in pathways involved in axon guidance, suggesting overlap in mechanisms underlying neurotoxicity caused by other known chemotherapeutic agents. To investigate the mechanisms underlying the development of pain, RAW 264.7 mouse macrophage cells were treated with JNJ-26481585. There was a dose- and time-dependent activation of nuclear factor-kappaB and interleukin-1β increase. Thus, alterations in the axon guidance pathway, increase in voltage-dependent calcium channel alpha(2)delta-1 subunit, and the induction of proinflammatory mediators by JNJ-26481585 could all contribute to increased mechanical sensitivity. Our data indicate that the effect of HDAC inhibitors may be unique to the compound studied and highlights the potential to develop chemotherapy-induced peripheral neuropathy with the use of a pan-HDAC inhibitor for cancer treatment, and this pain may be alleviated by gabapentin.