Experimental Type 2 diabetes and lipotoxicity-associated neuroinflammation involve mitochondrial DNA-mediated cGAS/STING axis: implication of Type-1 interferon response in cognitive impairment.

Type-1 IFN (interferon)-associated innate immune response is increasingly getting attention in neurodegenerative and metabolic diseases like type 2 diabetes (T2DM). However, its significance in T2DM/lipotoxicity-induced neuroglia changes and cognitive impairment is missing. The present study aims to evaluate the involvement of cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon gene), IRF3 (interferon regulatory factor-3), TBK (TANK binding kinase)-mediated Type-1 IFN response in the diabetic brain, and lipotoxicity (palmitate-bovine serum albumin conjugate/PA-BSA)-induced changes in cells (neuro2a and BV2). T2DM was induced in C57/BL6 mice by feeding on a high-fat diet (HFD, 60% Kcal) for 16 weeks and injecting a single dose of streptozotocin (100 mg/kg, i.p) in the 12th week. Plasma biochemical parameter analysis, neurobehavioral assessment, protein expression, and quantitative polymerase chain reaction study were carried out to decipher the hypothesis. T2DM-associated metabolic and lipotoxic stress led to mitochondrial impairment causing leakage of mtDNA to the cytoplasm further commencing cGAS activation and its downstream signaling. The diseased hippocampus and cortex showed decreased expression of synaptophysin (p < 0.01) and PSD-95 (p < 0.01, p < 0.05) with increased expression of cGAS (p < 0.001), p-STING (p < 0.001), p-STAT1 (signal transducer and activator of transcription) (p < 0.01), and IFN-ß (p < 0.001) compared to normal control. The IFN-ß/p-STAT1-mediated microglia activation was executed employing a conditioned media approach. C-176, a selective STING inhibitor, alleviated cGAS/p-STING/IFN-ß expression and proinflammatory microglia/M1-associated markers (CD16 expression, CXCL10, TNF-α, IL-1ß mRNA fold change) in the diabetic brain. The present study suggests Type-1IFN response may result in neuroglia dyshomeostasis affecting normal brain function. Alleviating STING signaling has the potential to protect T2DM-associated central ailment.

Quality by design endorsed atorvastatin-loaded nanostructured lipid carriers embedded in pH-responsive gel for melanoma.

AIM: Our aim was to repurpose atorvastatin for melanoma by encapsulating in a nanostructured lipid carrier matrix to promote tumour cell internalisation and skin permeation. pH-responsive chitosan gel was employed to restrict At-NLCs in upper dermal layers. METHODS: We utilised a quality by design approach for encapsulating At within the NLC matrix. Further, cellular uptake and cytotoxicity was evaluated along with pH-responsive release and ex vivo skin permeation. RESULTS: Cytotoxicity assay showed 3.13-fold enhanced cytotoxicity on melanoma cells compared to plain drug with nuclear staining showing apoptotic markers. In vitro, release studies showed 5.9-fold rapid release in chitosan gel matrix at pH 5.5 compared to neutral pH. CONCLUSIONS: At-NLCs prevented precipitation, promoted skin permeation, and SK-MEL 28 cell internalisation. The localisation of NLCs on the upper dermal layer due to electrostatic interactions of skin with chitosan gel diminished the incidence of untoward systemic effects.

S1PR2 inhibition mitigates cognitive deficit in diabetic mice by modulating microglial activation via Akt-p53-TIGAR pathway.

Cognitive deficit is one of the challenging complications of type 2 diabetes. Sphingosine 1- phosphate receptors (S1PRs) have been implicated in various neurodegenerative and metabolic disorders. The association of S1PRs and cognition in type 2 diabetes remains elusive. Microglia-mediated neuronal damage could be the thread propagating cognitive deficit. The effects of S1PR2 inhibition on cognition in high-fat diet and streptozotocin-induced diabetic mice were examined in this work. We further assessed microglial activation and putative microglial polarisation routes. Cognitive function loss was observed after four months of diabetes induction in Type 2 diabetes animal model. JTE013, an S1PR2 inhibitor, was used to assess neuroprotection against cognitive decline and neuroinflammation in vitro and in vivo diabetes model. JTE013 (10 mg/kg) improved synaptic plasticity by upregulating psd95 and synaptophysin while reducing cognitive decline and neuroinflammation. It further enhanced anti-inflammatory microglia in the hippocampus and prefrontal cortex (PFC), as evidenced by increased Arg-1, CD206, and YM-1 levels and decreased iNOS, CD16, and MHCII levels. TIGAR, TP53-induced glycolysis and apoptosis regulator, might facilitate the anti-inflammatory microglial phenotype by promoting oxidative phosphorylation and decreasing apoptosis. However, since p53 is a TIGAR suppressor, inhibiting p53 could be beneficial. S1PR2 inhibition increased p-Akt and TIGAR levels and reduced the levels of p53 in the PFC and hippocampus of type 2 diabetic mice, thereby decreasing apoptosis. In vitro, palmitate was used to imitate sphingolipid dysregulation in BV2 cells, followed by conditioned media exposure to Neuro2A cells. JTE013 rescued the palmitate-induced neuronal apoptosis by promoting the anti-inflammatory microglia. In the present study, we demonstrate that the inhibition of S1PR2 improves cognitive function and skews microglia toward anti-inflammatory phenotype in type 2 diabetic mice, thereby promising to be a potential therapy for neuroinflammation.

Necrostatin-1S mitigates type-2 diabetes-associated cognitive decrement and lipotoxicity-induced neuro-microglia changes through p-RIPK-RIPK3-p-MLKL axis.

Type-2 diabetes mellitus (T2DM) is associated with neuroinflammation and cognitive decrement. Necroptosis programmed necrosis is emerging as the major contributing factor to central changes. It is best characterized by the upregulation of p-RIPK(Receptor Interacting Kinase), p-RIPK3, and the phosphorylated-MLKL (mixed-lineage kinase domain-like protein). The present study aims to evaluate the neuroprotective effect of Necrostatin (Nec-1S), a p-RIPK inhibitor, on cognitive changes in the experimental T2DM model in C57BL/6 mice and lipotoxicity-induced neuro-microglia changes in neuro2A and BV2 cells. Further, the study also explores whether Nec-1S would restore mitochondrial and autophago-lysosomal function.T2DM was developed in mice by feeding them a high-fat diet (HFD) for 16 weeks and injecting a single dose of streptozotocin (100 mg/kg, i.p) on the 12th week. Nec-1S was administered for 3 weeks at (10 mg/kg, i.p) once every 3 days. Lipotoxicity was induced in neuro2A, and BV2 cells using 200 µM palmitate/bovine serum albumin conjugate. Nec-1S (50 µM), and GSK-872(10 µM) were further used to explore their relative effect. The neurobehavioral performance was assessed using mazes and task-assisted performance tests. To decipher the hypothesis plasma parameters, western blot, immunofluorescence, microscopy, and quantitative reverse transcription-PCR studies were carried out. The Nec-1S treatment restored cognitive performance and reduced the p-RIPK-p-RIPK3-p-MLKL mediated neuro-microglia changes in the brain and in cells as well, under lipotoxic stress. Nec-1S reduced tau, and amyloid oligomer load. Moreover, Nec-1S restored mitochondrial function and autophago-lysosome clearance. The findings highlight the central impact of metabolic syndrome and how Nes-1S, by acting as a multifaceted agent, improved central functioning.

Sphingosine 1 phosphate lyase inhibition rescues cognition in diabetic mice by promoting anti-inflammatory microglia.

Sphingosine-1-phosphate (S1P) is emerging as a crucial sphingolipid modulating neuroinflammation and cognition. S1P levels in the brain have been found to be decreased in cognitive impairment. S1P lyase (S1PL) is the key enzyme in metabolizing S1P and has been implicated in neuroinflammation. This study evaluated the effect of S1PL inhibition on cognition in type 2 diabetic mice. Fingolimod (0.5 mg/kg and 1 mg/kg) rescued cognition in high-fat diet and streptozotocin-induced diabetic mice, as evident in the Y maze and passive avoidance test. We further evaluated the effect of fingolimod on the activation of microglia in the pre-frontal cortex (PFC) and hippocampus of diabetic mice. Our study revealed that fingolimod inhibited S1PL and promoted anti-inflammatory microglia in both PFC and hippocampus of diabetic mice as it increased Ym-1 and arginase-1. The levels of p53 and apoptotic proteins (Bax and caspase-3) were elevated in the PFC and hippocampus of type 2 diabetic mice which fingolimod reversed. The underlying mechanism promoting anti-inflammatory microglial phenotype was also explored in this study. TIGAR, TP53-associated glycolysis and apoptosis regulator, is known to foster anti-inflammatory microglia and was found to be downregulated in the brain of type 2 diabetic mice. S1PL inhibition decreased the levels of p53 and promoted TIGAR, thereby increasing anti-inflammatory microglial phenotype and inhibiting apoptosis in the brain of diabetic mice. Our study reveals that S1PL inhibition could be beneficial in mitigating cognitive deficits in diabetic mice.

Fingolimod Alleviates Cognitive Deficit in Type 2 Diabetes by Promoting Microglial M2 Polarization via the pSTAT3-jmjd3 Axis.

Sphingosine receptors (S1PRs) are implicated in the progression of neurodegenerative diseases and metabolic disorders like obesity and type 2 diabetes (T2D). The link between S1PRs and cognition in type 2 diabetes, as well as the mechanisms that underpin it, are yet unknown. Neuroinflammation is the common pathology shared among T2D and cognitive impairment. However, the interplay between the M1 and M2 polarization state of microglia, a primary driver of neuroinflammation, could be the driving factor for impaired learning and memory in diabetes. In the present study, we investigated the effects of fingolimod (S1PR1 modulator) on cognition in high-fat diet and streptozotocin-induced diabetic mice. We further assessed the potential pathways linking microglial polarization and cognition in T2D. Fingolimod (0.5 mg/kg and 1 mg/kg) improved M2 polarization and synaptic plasticity while ameliorating cognitive decline and neuroinflammation. Sphingolipid dysregulation was mimicked in vitro using palmitate in BV2 cells, followed by conditioned media exposure to Neuro2A cells. Mechanistically, type 2 diabetes induced microglial activation, priming microglia towards the M1 phenotype. In the hippocampus and cortex of type 2 diabetic mice, there was a substantial drop in pSTAT3, which was reversed by fingolimod. This protective effect of fingolimod on microglial M2 polarization was primarily suppressed by selective jmjd3 blockade in vitro using GSK-J4, revealing that jmjd3 was involved downstream of STAT3 in the fingolimod-enabled shift of microglia from M1 to M2 polarization state. This study suggested that fingolimod might effectively improve cognition in type 2 diabetes by promoting M2 polarization.

Neuroepigenetic Changes in DNA Methylation Affecting Diabetes-Induced Cognitive Impairment.

Chronic diabetic conditions have been associated with certain cerebral complications, that include neurobehavioral dysfunctional patterns and morphological alterations of neurons, especially the hippocampus. Neuroanatomical studies done by the authors have shown decreased total dendritic length, intersections, dendritic length per branch order and nodes in the CA1 hippocampal region of the diabetic brain as compared to its normal control group, indicating reduced dendritic arborization of the hippocampal CA1 neurons. Epigenetic alterations in the brain are well known to affect age-associated disorders, however its association with the evolving diabetes-induced damage in the brain is still not fully understood. DNA hypermethylation within the neurons, tend to silent the gene expression of several regulatory proteins. The findings in the study have shown an increase in global DNA methylation in palmitic acid-induced lipotoxic Neuro-2a cells as well as within the diabetic mice brain. Inhibiting DNA methylation, restored the levels of HSF1 and certain HSPs, suggesting plausible effect of DNMTs in maintaining the proteostasis and synaptic fidelity. Neuroinflammation, as exhibited by the astrocyte activation (GFAP), were further significantly decreased in the 5-azadeoxycytidine group (DNMT inhibitor). This was further evidenced by decrease in proinflammatory cytokines TNF⍺, IL-6, and mediators iNOS and Phospho-NFkB. Our results suggest that changes in DNA methylation advocate epigenetic dysregulation and its involvement in disrupting the synaptic exactitude in the hippocampus of diabetic mice model, providing an insight into the pathophysiology of diabetes-induced neuroepigenetic changes.

Quality by design steered development of Niclosamide loaded liposomal thermogel for Melanoma: In vitro and Ex vivo evaluation.

Melanoma is the most malignant form of skin cancer across the globe. Conventional therapies are currently ineffective which could be attributed to the rampant chemo-resistance, metastasis, inability to cross the skin barriers and accumulate within the tumor microenvironment. This advent brings in the principles of drug repurposing by repositioning Niclosamide (NIC), an anthelmintic drug for skin cancer. Incorporation into the liposomes facilitated enhanced melanoma cell uptake and apoptosis. Cytotoxicity studies revealed 1.756-fold enhancement in SK-MEL-28 cytotoxicity by NIC-loaded liposomes compared to free drug. Qualitative and quantitative cell internalization indicated greater drug uptake within the melanoma cells illustrating the efficacy of liposomes as efficient carrier systems. Nuclear staining showed blebbing and membrane shrinkage. Elevated ROS levels and apoptosis shown by DCFDA and acridine orange-ethidium bromide staining revealed greater melanoma cell death by liposomes compared to free drug. Incorporating NIC liposomes into the thermogel system restricted the liposomes as a depot onto the upper skin layers. Sustained zero order release up to 48 h with liposomes and 23.58-fold increase in viscosity led to the sol-to-gel transition at 33℃ was observed with liposomal thermogel. Ex vivo gel permeation studies revealed that C-6 loaded liposomes incorporated within the thermogel successfully formed a depot over the upper skin layer for 6 h to prevent transdermal delivery and systemic adverse effects. Thus, it could be concluded that NIC loaded liposomal thermogel system could be an efficacious therapeutic alternative for the management of melanoma.

Quality by design (QbD) assisted Fabrication & evaluation of Simvastatin loaded Nano-Enabled thermogel for melanoma therapy.

Melanoma is a form of skin cancer that starts in melanocytes. Rampant chemo-resistance, metastasis, and inability to cross the skin barriers and accumulate within the tumor microenvironment render the conventional chemotherapeutic approaches ineffective. Simvastatin (SIM), a cholesterol synthesis inhibitor, has shown tremendous anticancer potential. Due to the lack of therapeutic alternatives, repositioning SIM in melanoma could be beneficial. Incorporating SIM within the nanoparticles promoted increased melanoma cell internalization, apoptosis, and sustained release profile. Further, the incorporation of nanoparticles into the thermogel facilitated depot formation over the upper dermal layers. Sol-to-gel transition at 34 °C was observed with a 14.03-fold increase in viscosity. This could be fruitful in limiting systemic exposure and preventing adverse effects. Entrapment of SIM in the PLGA NPs enhanced the cytotoxicity by 9.38-fold (p less than 0.05). Nuclear staining with DAPI showed blebbing, membrane shrinkage, and apoptosis confirmed by DCFDA and acridine orange/ethidium bromide staining. Ex vivo diffusion studies revealed the accumulation of C-6 loaded nanoparticles incorporated within the thermogel onto the upper dermal layer and depot formation up to 6 h. Thus, we conclude that SIM-loaded nanoparticulate thermogel could be an efficacious therapeutic alternative for melanoma.

Neuroepigenetic alterations in the prefrontal cortex of type 2 diabetic mice through DNA hypermethylation.

BACKGROUND: DNA methylation changes have known to downregulate several regulatory proteins epigenetically during various neurodegenerative disorders. Our study aims to understand the effect of this global DNA methylation on the cerebral complications of type 2 diabetes mice, and its notable effect on maintaining the synaptic fidelity. METHODS AND RESULTS: Chronic high fat diet and streptozotocin-induced diabetic mice were studied for the neurobehavioral and neuroanatomic parameters pertaining to prefrontal cortex, subsequently elucidating the associated changes in DNA methylation within these diabetic brains. Further, the impact of this epigenetic dysregulation on HSF1, BDNF and PSD95 were studied by assessing the binding affinity and level of % methylation within the promoter site of their respective genes. Our study suggest increased DNMT aberrations within the prefrontal cortex, with increased MeCP2 levels, confirming DNA hypermethylation. This was in accordance with the altered neurobehavioral changes. Further, the hypermethylation was found to participate in gene silencing of HSF1, BDNF and PSD95 proteins, responsible for maintaining the synaptic fidelity. CONCLUSION: Overall, our study concludes the plausible involvement of neuroepigenetic alterations in the prefrontal cortex (PFC) of the type 2 diabetes mice, specifically DNA hypermethylation. PFC plays a central role in modulating cognitive and other executive functions through its connection with several brain regions, and thus therapeutic strategies targeting epigenetic modulations in it, can pave a way in controlling several neurological alterations in the brain.

Metabolic Regulation of Glia and Their Neuroinflammatory Role in Alzheimer's Disease.

Alzheimer's disease (AD) is an aging-related neurodegenerative disorder. It is characterized clinically by progressive memory loss and impaired cognitive function. Its progression occurs from neuronal synapse loss to amyloid pathology and Tau deposit which eventually leads to the compromised neuronal function. Neurons in central nervous tissue work in a composite and intricate network with the glia and vascular cells. Microglia and astrocytes are becoming the prime focus due to their involvement in various aspects of neurophysiology, such as trophic support to neurons, synaptic modulation, and brain surveillance. AD is also often considered as the sequela of prolonged metabolic dyshomeostasis. The neuron and glia have different metabolic profiles as cytosolic glycolysis and mitochondrial-dependent oxidative phosphorylation (OXPHOS), especially under dyshomeostasis or with aging pertaining to their unique genetic built-up. Various efforts are being put in to decipher the role of mitochondrial dynamics regarding their trafficking, fission/fusion imbalance, and mitophagy spanning over both neurons and glia to improve aging-related brain health. The mitochondrial dysfunction may lead to activation in various signaling mechanisms causing metabolic reprogramming in glia cells, further accelerating AD-related pathogenic events. The glycolytic-dominant astrocytes switch to the neurotoxic phenotype, i.e., disease-associated astrocyte under metabolic stress. The microglia also transform from resting to reactive phenotype, i.e., disease-associated microglia. It may also exist in otherwise a misconception an M1, glycolytic, or M2, an OXPHOS-dependent phenotype. Further, glial transformation plays a vital role in regulating hallmarks of AD pathologies like synapse maintenance, amyloid, and Tau clearance. In this updated review, we have tried to emphasize the metabolic regulation of glial reactivity, mitochondrial quality control mechanisms, and their neuroinflammatory response in Alzheimer's progression.

Role of MicroRNAs, Aptamers in Neuroinflammation and Neurodegenerative Disorders.

Exploring the microRNAs and aptamers for their therapeutic role as biological drugs has expanded the horizon of its applicability against various human diseases, explicitly targeting the genetic materials. RNA-based therapeutics are widely being explored for the treatment and diagnosis of multiple diseases, including neurodegenerative disorders (NDD). Latter includes microRNA, aptamers, ribozymes, and small interfering RNAs (siRNAs), which control the gene expression mainly at the transcriptional strata. One RNA transcript translates into different protein types; hence, therapies targeted at the transcriptional sphere may have prominent and more extensive effects than alternative therapeutics. Unlike conventional gene therapy, RNAs, upon delivery, can either altogether abolish or alter the synthesis of the protein of interest, therefore, regulating their activities in a controlled and diverse manner. NDDs like Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, Prion disease, and others are characterized by deposition of misfolded protein such as amyloid-ß, tau, α-synuclein, huntingtin and prion proteins. Neuroinflammation, one of the perquisites for neurodegeneration, is induced during neurodegenerative pathogenesis. In this review, we discuss microRNAs and aptamers' role as two different RNA-based approaches for their unique ability to regulate protein production at the transcription level, hence offering many advantages over other biologicals. The microRNA acts either by alleviating the malfunctioning RNA expression or by working as a replacement to lost microRNA. On the contrary, aptamer act as a chemical antibody and forms an aptamer-target complex.

Glia: A major player in glutamate-GABA dysregulation-mediated neurodegeneration.

The imbalance between glutamate and γ-aminobutyric acid (GABA) results in the loss of synaptic strength leading to neurodegeneration. The dogma on the field considered neurons as the main players in this excitation-inhibition (E/I) balance. However, current strategies focusing only on neurons have failed to completely understand this condition, bringing up the importance of glia as an alternative modulator for neuroinflammation as glia alter the activity of neurons and is a source of both neurotrophic and neurotoxic factors. This review's primary goal is to illustrate the role of glia over E/I balance in the central nervous system and its interaction with neurons. Rather than focusing only on the neuronal targets, we take a deeper look at glial receptors and proteins that could also be explored as drug targets, as they are early responders to neurotoxic insults. This review summarizes the neuron-glia interaction concerning GABA and glutamate, possible targets, and its involvement in the E/I imbalance in neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, Huntington's disease, and multiple sclerosis.

Mitochondrial quality control: Epigenetic signatures and therapeutic strategies.

Mitochondria are semi-autonomous organelle staging a crucial role in cellular stress response, energy metabolism and cell survival. Maintaining mitochondrial quality control is very important for its homeostasis. Pathological conditions such as oxidative stress and neurodegeneration, disrupt this quality control, and involvement of genetic and epigenetic materials in this disruption have been reported. These regulatory factors trigger mitochondrial imbalance, as seen in many neurodegenerative diseases like Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, and Huntington's disease. The dynamic regulatory pathways i.e. mitophagy, biogenesis, permeability pore transitioning, fusion-fission are affected as a consequence and have been reviewed in this article. Moreover, several epigenetic mechanisms such as DNA methylation and histone modulation participating in such neurological disorders have also been discussed. Apart from it, therapeutic approaches targeting mitochondrial quality control have been tremendously explored showing ameliorative effects for these diseases, and have been discussed here with a novel perspective.

Proteotoxicity and mitochondrial dynamics in aging diabetic brain.

Impaired neuronal proteostasis is a salient feature of both aging and protein misfolding disorders. Amyloidosis, a consequence of this phenomena is observed in the brains of diabetic patients over the chronic time period. These toxic aggregates not only cause age-related decline in proteostasis, but also dwindle its ability to increase or restore the chaperones in response to any stressful condition. Mitochondria acts as the main source of energy regulation and many metabolic disorders such as diabetes have been associated with altered oxidative phosphorylation (OxPhos) and redox imbalance in the mitochondria. The mitochondrial unfolded protein response (UPRmt) acts as a mediator for maintaining the mitochondrial protein homeostasis and quality control during such conditions. Over a long time period, these responses start shutting off leading to proteotoxic stress in the neurons. This reduces the buffering capacity of protein network signalling during aging, thereby increasing the risk of neurodegeneration in the brain. In this review, we focus on the proteotoxic stress that occurs as an amalgamation of diabetes and aging, as well as the impact of mitochondrial dysfunction on the neuronal survival affecting the diabetic brain and its long term consequences on the memory changes.

Neuroprotective effects of silibinin: an in silico and in vitro study.

AIM OF THE STUDY: Astrogliosis is a key contributor for many neurological disorders involving apoptosis, neuroinflammation and subsequent neuronal death. Silibinin, a polyphenol isolated from milk thistle (Silybum marianum), has been shown to suppress the astrocyte activation in various neurodegenerative disorders and also exhibit a neuroprotective role in neuroinflammation-driven oxidative damage. The present study was designed with an aim to investigate the neuroprotective effects of Silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation. MATERIALS AND METHODS: We have used in-silico molecular modelling techniques to study the interaction and binding affinity of silibinin with chemokine receptors associated with neuroinflammation. We have also tested silibinin against LPS induced oxido-inflammatory cascade and astrocyte activation in C6 glia cell lines. RESULTS: In the present study, we found that treatment with silibinin significantly attenuates LPS-oxidative-nitrosative stress in C6 astrocytoma cells. We also observed the significant inhibition of induced astrocyte activity after treatment with silibinin. Moreover, molecular modelling studies have proposed a binding pose of silibinin with binding sites of p38 MAPK, CX3CR1 and P2X4 which is an important downstream cascade involved in glia cell activation and neuroinflammation. CONCLUSIONS: Overall, the findings from the current study suggests that silibinin exhibits neuroprotective activity by attenuating oxidative damage and astrocytes activation.

Cellular and molecular mechanisms driving neuropathic pain: recent advancements and challenges.

INTRODUCTION: Current pharmacotherapeutics for neuropathic pain offer only symptomatic relief without treating the underlying pathophysiology. Additionally, they are associated with various dose-limiting side effects. Pain research in the past few decades has revolved around the role of oxidative-nitrosative stress, protein kinases, glial cell activation, and inflammatory signaling cascades but has failed to produce specific and effective therapies. Areas covered: This review focuses on recent advances in cellular and molecular mechanisms of neuropathic pain that may be translated into future therapies. We discuss emerging targets such as WNT signaling mechanisms, the tetrahydrobiopterin pathway, Mrg receptors, endogenous lipid mediators, micro-RNAs and their roles in pain regulation. Recent evidence is also presented regarding genetic and epigenetic mechanisms of pain modulation. Expert opinion: During chronic neuropathic pain, maladaptation occurs in the peripheral and central nervous systems, including a shift in microglial phenotype from a surveillance state to an activated state. Microglial activation leads to an altered expression of cell surface proteins, growth factors, and intracellular signaling molecules that contribute to development of a neuroinflammatory cascade and chronic pain sensitization. Specific targeting of these cellular and molecular mechanisms may provide the key to development of effective neuropathic pain therapies that have minimal side effects.