Pathogenic mechanisms of human immunodeficiency virus (HIV)-associated pain.

Chronic pain is a prevalent neurological complication among individuals living with human immunodeficiency virus (PLHIV) in the post-combination antiretroviral therapy (cART) era. These individuals experience malfunction in various cellular and molecular pathways involved in pain transmission and modulation, including the neuropathology of the peripheral sensory neurons and neurodegeneration and neuroinflammation in the spinal dorsal horn. However, the underlying etiologies and mechanisms leading to pain pathogenesis are complex and not fully understood. In this review, we aim to summarize recent progress in this field. Specifically, we will begin by examining neuropathology in the pain pathways identified in PLHIV and discussing potential causes, including those directly related to HIV-1 infection and comorbidities, such as antiretroviral drug use. We will also explore findings from animal models that may provide insights into the molecular and cellular processes contributing to neuropathology and chronic pain associated with HIV infection. Emerging evidence suggests that viral proteins and/or antiretroviral drugs trigger a complex pathological cascade involving neurons, glia, and potentially non-neural cells, and that interactions between these cells play a critical role in the pathogenesis of HIV-associated pain.

The antitumor effect of mycelia extract of the medicinal macrofungus Inonotus hispidus on HeLa cells via the mitochondrial-mediated pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Inonotus hispidus (I. hispidus), known as shaggy bracket, has been used extensively in China and some East Asian countries as a traditional medicinal macrofungus to treat difficult diseases, such as diabetes, gout, and arthritis. Modern pharmacological research has shown that I. hispidus has an important application value in antitumor treatment. However, the main anti-cervical cancer activity substances from its mycelia and its mechanisms are still not clear. AIMS OF THE STUDY: To enrich the germplasm resources of I. hispidus, to reveal the antitumor activity of the extract from the mycelium of I. hispidus against cervical cancer, and to preliminarily analyze its action mechanism. MATERIALS AND METHODS: The SH3 strain was isolated from wild fruiting bodies and identified by morphology and molecular biology. The antitumor active component from the mycelium of I. hispidus was isolated and identified with liquid chromatography-tandem mass spectrometry. The cell viability was assessed by MTT assay. The cell cycle distribution, apoptotic cell detection, and mitochondrial membrane potential were detected by flow cytometer. The expression of apoptosis-related proteins was assessed by Western blotting. The inhibition of tumor growth in vivo was assessed by a mouse xenograft model. RESULTS: The SH3 strain was isolated and identified as a new strain of I. hispidus. The antitumor active component containing cyclic peptides from the mycelium of I. hispidus (CCM) was isolated for the first time. In addition, we found that CCM had a strong inhibitory effect on HeLa proliferation in vitro and in vivo. Mechanically, the CCM blocked the cell cycle at the G0/G1 phase, decreased the mitochondrial membrane potential, and eventually promoted apoptosis of HeLa cells through the mitochondria-mediated pathway by upregulating the expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3 and downregulating the expression level of Bcl-2. CONCLUSIONS: Our study not only enriches the strain resources of I. hispidus but also confirms that the mycelium of this strain has active components that can inhibit cervical cancer. This is highly significant for the development of active drugs and drug lead molecules for treating cervical cancer.

Gonadal hormone-dependent nociceptor sensitization maintains nociplastic pain state in female mice.

ABSTRACT: Nociplastic pain conditions develop predominantly in women. We recently established a murine nociplastic pain model by applying postinjury thermal (40°C) stimulation to an injured (capsaicin-injected) area, triggering a transition to a nociplastic pain state manifesting as persistent mechanical hypersensitivity outside of the previously injured area. The nociplastic pain state was centrally maintained by spinal microglia in males but peripherally by ongoing afferent activity at the previously injured area in females. Here, we investigated whether gonadal hormones are critical for the development of this peripherally maintained nociplastic pain state in females. Although the transition to a nociplastic pain state still occurred in ovariectomized females, the pain state was maintained neither by ongoing afferent activity at the previously injured area nor by spinal microglia. Estradiol reconstitution a week before the injury plus postinjury stimulation, but not after the transition had already occurred, restored the development of peripherally maintained nociplastic mechanical hypersensitivity in ovariectomized females. G protein-coupled estrogen receptor antagonism during the transition phase mimicked ovariectomy in gonad-intact females, whereas the receptor antagonism after the transition gradually alleviated the nociplastic mechanical hypersensitivity. At the previously injured area, afferents responsive to allyl isothiocyanate (AITC), a TRPA1 agonist, contributed to the maintenance of nociplastic mechanical hypersensitivity in gonad-intact females. In ex vivo skin-nerve preparations, only AITC-responsive afferents from the nociplastic pain model in gonad-intact females showed ongoing activities greater than control. These results suggest that gonadal hormones are critical for peripherally maintained nociplastic pain state in females by sensitizing AITC-responsive afferents to be persistently active.

Mediators of Neuropathic Pain; Focus on Spinal Microglia, CSF-1, BDNF, CCL21, TNF-α, Wnt Ligands, and Interleukin 1ß.

Intractable neuropathic pain is a frequent consequence of nerve injury or disease. When peripheral nerves are injured, damaged axons undergo Wallerian degeneration. Schwann cells, mast cells, fibroblasts, keratinocytes and epithelial cells are activated leading to the generation of an "inflammatory soup" containing cytokines, chemokines and growth factors. These primary mediators sensitize sensory nerve endings, attract macrophages, neutrophils and lymphocytes, alter gene expression, promote post-translational modification of proteins, and alter ion channel function in primary afferent neurons. This leads to increased excitability and spontaneous activity and the generation of secondary mediators including colony stimulating factor 1 (CSF-1), chemokine C-C motif ligand 21 (CCL-21), Wnt3a, and Wnt5a. Release of these mediators from primary afferent neurons alters the properties of spinal microglial cells causing them to release tertiary mediators, in many situations via ATP-dependent mechanisms. Tertiary mediators such as BDNF, tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and other Wnt ligands facilitate the generation and transmission of nociceptive information by increasing excitatory glutamatergic transmission and attenuating inhibitory GABA and glycinergic transmission in the spinal dorsal horn. This review focusses on activation of microglia by secondary mediators, release of tertiary mediators from microglia and a description of their actions in the spinal dorsal horn. Attention is drawn to the substantial differences in the precise roles of various mediators in males compared to females. At least 25 different mediators have been identified but the similarity of their actions at sensory nerve endings, in the dorsal root ganglia and in the spinal cord means there is considerable redundancy in the available mechanisms. Despite this, behavioral studies show that interruption of the actions of any single mediator can relieve signs of pain in experimental animals. We draw attention this paradox. It is difficult to explain how inactivation of one mediator can relieve pain when so many parallel pathways are available.

HIV-related Neuropathy: Pathophysiology, Treatment and Challenges.

HIV-sensory neuropathy (HIV-SN) is a debilitating complication in HIV patients with or without anti-retroviral treatment (ART). Common symptoms of HIV-SN include pain, decreased sensation, paresthesias, and dysesthesias in a symmetric stocking-glove distribution. While HIV-1 protein such as gp120 is implicated in HIV-SN (e.g. impaired large-diameter fiber), ART itself was recently shown to contribute to HIV-SN in HIV patients and impair thin fiber. Multiple host mechanisms may play roles during the pathogenesis of HIV-SN, including neuron-glia interactions in the spinal dorsal horn (SDH), inflammation, mitochondrial dysfunction and endoplasmic reticulum stress. Concurrent infections, such as tuberculosis, also carry a higher likelihood of HIV-SN as well as environmental or genetic predisposition. Pro-inflammatory cytokines such as IL-1, IL2 receptor-alpha, and tumor necrosis factor (TNF) along with abnormal lactate levels have been identified as potential players within the complex pathophysiology of this condition. In this paper, we review the pathophysiology of HIV neuropathy, focusing on the various treatment options available or under investigation. Although several treatment options are available e.g., the capsaicin patch and spinal cord stimulation, symptomatic control of HIV-SN are often challenging. Alternative approaches such as self-hypnosis, resistance exercise, cannabinoids, and acupuncture have all shown promising results, but need further investigation.

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis.

Plasmin-mediated fibrinolysis at the surface of vascular endothelial cells (SVEC) plays a key role in maintaining vascular hemostasis, in which the cAMP pathway participates. After externalization to the SVEC, annexin A2 (ANXA2) serves as a platform for conversion of plasminogen to plasmin. Here we describe a regulatory role of the exchange protein directly activated by cAMP (EPAC) in ANXA2 externalization and vascular fibrinolysis. Knockout of EPAC1 in mice results in a decreased ANXA2 expression on the SVEC associated with increased fibrin deposition and fibrinolytic dysfunction. Reduced levels of EPAC1 are also found in endocardial tissues beneath atrial mural thrombi in patients. Notably, administration of recombinant ANXA2 ameliorates fibrinolytic dysfunction in the EPAC1-null mice. Mechanistically, EPAC1 regulates the SVEC plasminogen conversion depended on ANXA2. EPAC1 promotes tyrosine-23 phosphorylation of ANXA2, a prerequisite for its recruitment to the SVEC. Our data thus reveal a novel regulatory role for EPAC1 in vascular fibrinolysis.

Peli1 facilitates virus replication and promotes neuroinflammation during West Nile virus infection.

The E3 ubiquitin ligase Pellino 1 (Peli1) is a microglia-specific mediator of autoimmune encephalomyelitis. Its role in neurotropic flavivirus infection is largely unknown. Here, we report that mice deficient in Peli1 (Peli1-/-) were more resistant to lethal West Nile virus (WNV) infection and exhibited reduced viral loads in tissues and attenuated brain inflammation. Peli1 mediates chemokine and proinflammatory cytokine production in microglia and promotes T cell and macrophage infiltration into the CNS. Unexpectedly, Peli1 was required for WNV entry and replication in mouse macrophages and mouse and human neurons and microglia. It was also highly expressed on WNV-infected neurons and adjacent inflammatory cells from postmortem patients who died of acute WNV encephalitis. WNV passaged in Peli1-/- macrophages or neurons induced a lower viral load and impaired activation in WT microglia and thereby reduced lethality in mice. Smaducin-6, which blocks interactions between Peli1 and IRAK1, RIP1, and IKKε, did not inhibit WNV-triggered microglia activation. Collectively, our findings suggest a nonimmune regulatory role for Peli1 in promoting microglia activation during WNV infection and identify a potentially novel host factor for flavivirus cell entry and replication.

Synaptic activity-regulated Wnt signaling in synaptic plasticity, glial function and chronic pain.

Wnt signaling pathways play important roles in various developmental and oncogenic processes. In the nervous system, Wnt signaling regulates neuronal morphogenesis and synaptic differentiation. Disturbance of Wnt signaling is implicated in the pathogenesis of neurological diseases. Recent studies indicate that Wnt signaling in neurons is closely coupled to synaptic activation, and that the activity-regulated Wnt signaling is critical for the expression of synaptic plasticity and the formation of memory. Dysregulation of the activity-regulated Wnt signaling may have a significant impact on the function of the nervous system. In this article, we will review the identified mechanisms by which synaptic activity controls Wnt signaling in neurons and the neurological functions of the activity-regulated Wnt signaling under normal and specific disease conditions. In particular, we will discuss the role of Wnt signaling in the pathogenesis of chronic pain.

Wingless-type mammary tumor virus integration site family, member 5A (Wnt5a) regulates human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein 120 (gp120)-induced expression of pro-inflammatory cytokines via the Ca2+/calmodulin-dependent protein kinase II (CaMKII) and c-Jun N-terminal kinase (JNK) signaling pathways.

BACKGROUND: HIV-1 infection causes chronic neuroinflammation in the central nervous system (CNS). RESULTS: The spinal cytokine up-regulation induced by HIV-1 gp120 protein depends on Wnt5a/CaMKII and/or Wnt5a/JNK pathways. CONCLUSION: gp120 stimulates cytokine expression in the spinal cord dorsal horn by activating Wnt5a signaling. SIGNIFICANCE: The finding reveals Wnt signaling-mediated novel mechanisms by which HIV-1 may cause neuroinflammation. Chronic expression of pro-inflammatory cytokines critically contributes to the pathogenesis of HIV-associated neurological disorders (HANDs), but the host mechanism that regulates the HIV-induced cytokine expression in the CNS remains elusive. Here, we present evidence for a crucial role of Wnt5a signaling in the expression of pro-inflammatory cytokines in the spinal cord induced by a major HIV-envelope protein, gp120. Wnt5a is mainly expressed in spinal neurons, and rapidly up-regulated by intrathecal injection (i.t.) of gp120. We show that inhibition of Wnt5a by specific antagonists blocks gp120-induced up-regulation of IL-1ß, IL-6, and TNF-α in the spinal cord. Conversely, injection (i.t.) of purified recombinant Wnt5a stimulates the expression of these cytokines. To elucidate the role of the Wnt5a-regulated signaling pathways in gp120-induced cytokine expression, we have focused on CaMKII and JNKs, the well characterized down-stream targets of Wnt5a signaling. We find that Wnt5a is required for gp120 to activate CaMKII and JNK signaling. Furthermore, we demonstrate that the Wnt5a/CaMKII pathway is critical for the gp120-induced expression of IL-1ß, whereas the Wnt5a/JNK pathway is for TNF-α expression. Meanwhile, the expression of IL-6 is co-regulated by both pathways. These results collectively suggest that Wnt5a signaling cascades play a crucial role in the regulation of gp120-induced expression of pro-inflammatory cytokines in the CNS.

Chronic-pain-associated astrocytic reaction in the spinal cord dorsal horn of human immunodeficiency virus-infected patients.

Studies with animal models have suggested that reaction of glia, including microglia and astrocytes, critically contributes to the development and maintenance of chronic pain. However, the involvement of glial reaction in human chronic pain is unclear. We performed analyses to compare the glial reaction profiles in the spinal dorsal horn (SDH) from three cohorts of sex- and age-matched human postmortem tissues: (1) HIV-negative patients, (2) HIV-positive patients without chronic pain, and (3) HIV patients with chronic pain. Our results indicate that the expression levels of CD11b and Iba1, commonly used for labeling microglial cells, did not differ in the three patient groups. However, GFAP and S100ß, often used for labeling astrocytes, were specifically upregulated in the SDH of the "pain-positive" HIV patients but not in the "pain-negative" HIV patients. In addition, proinflammatory cytokines, TNFα and IL-1ß, were specifically increased in the SDH of pain-positive HIV patients. Furthermore, proteins in the MAPK signaling pathway, including pERK, pCREB and c-Fos, were also upregulated in the SDH of pain-positive HIV patients. Our findings suggest that reaction of astrocytes in the SDH may play a role during the maintenance phase of HIV-associated chronic pain.

A role of the mammalian target of rapamycin (mTOR) in glutamate-induced down-regulation of tuberous sclerosis complex proteins 2 (TSC2).

Mammalian target of rapamycin (mTOR) signaling plays a critical role in the regulation of activity-dependent protein synthesis in neurons. It is well established that the GTPase-activating protein tuberous sclerosis complex proteins (2TSC2) is an upstream inhibitor of mTOR. In this study, we show that glutamate stimulation down-regulates TSC2 protein in cortical cultures via NMDA receptor (NMDAR) activation. Interestingly, the mTOR-specific inhibitor rapamycin blocks the glutamate-induced TSC2 down-regulation. This finding suggests that NMDAR activation evokes an mTOR-mediated negative regulation of TSC2. In addition, we also show that the glutamate-induced down-regulation of TSC2 protein is blocked by proteasome inhibitor MG132, indicating the involvement of proteasome-mediated protein degradation. We propose that the NMDAR activation stimulates an mTOR-proteasome pathway to degrade TSC2 protein.

WNT5A signaling contributes to Aß-induced neuroinflammation and neurotoxicity.

Neurodegenration is a pathological hallmark of Alzheimer's disease (AD), but the underlying molecular mechanism remains elusive. Here, we present evidence that reveals a crucial role of Wnt5a signaling in this process. We showed that Wnt5a and its receptor Frizzled-5 (Fz5) were up-regulated in the AD mouse brain, and that beta-amyloid peptide (Aß), a major constituent of amyloid plaques, stimulated Wnt5a and Fz5 expression in primary cortical cultures; these observations indicate that Wnt5a signaling could be aberrantly activated during AD pathogenesis. In support of such a possibility, we observed that inhibition of Wnt5a signaling attenuated while activation of Wnt5a signaling enhanced Aß-evoked neurotoxicity, suggesting a role of Wnt5a signaling in AD-related neurodegeneration. Furthermore, we also demonstrated that Aß-induced neurotoxicity depends on inflammatory processes, and that activation of Wnt5a signaling elicited the expression of proinflammatory cytokines IL-1ß and TNF-α whereas inhibition of Wnt5a signaling attenuated the Aß-induced expression of the cytokines in cortical cultures. Our findings collectively suggest that aberrantly up-regulated Wnt5a signaling is a crucial pathological step that contributes to AD-related neurodegeneration by regulating neuroinflammation.

Aberrant expression of synaptic plasticity-related genes in the NF1+/- mouse hippocampus.

Neurofibromatosis 1 (NF1) is a common single-gene disorder that causes learning impairments in patients. Neurofibromin encoded by the NF1 causal gene regulates Ras/MAPK and cAMP signaling pathways. These signaling pathways play critical roles in controlling gene transcription during synaptic plasticity and memory formation. We hypothesized that NF1 mutations disturb the expression of genes important for memory formation. To test this hypothesis, we performed DNA microarray analysis on the hippocampus of NF1(+/-) mice, the mouse model for NF1 learning disabilities. Our results indicated that genes involved in a wide spectrum of biological processes are dysregulated in the NF1(+/-) hippocampus. Many of the NF1-affected genes play critical roles in synaptic plasticity, such as Rabs, synaptotagmins, NMDAR1, CaMKII, and CREB1. Because NF1-associated learning disabilities can be reversed by lovastatin, we also determined the effect of lovastatin treatment on genome-wide expression patterns of the NF1(+/-) hippocampus. We found that lovastatin altered the expression of a large number of genes, including those disturbed by NF1 mutations. Our results reveal a genome-wide overview of the molecular abnormalities in the NF1(+/-) hippocampus and should be useful for further identifying the novel molecular pathways that cause NF1 learning deficits.

Activity-dependent synaptic Wnt release regulates hippocampal long term potentiation.

Wnts are important for various developmental and oncogenic processes. Here we show that Wnt signaling functions at synapses in hippocampal neurons. Tetanic stimulations induce N-methyl-d-aspartate receptor-dependent synaptic Wnt3a release, nuclear beta-catenin accumulations, and the activation of Wnt target genes. Suppression of Wnt signaling impairs long term potentiation. Conversely, activation of Wnt signaling facilitates long term potentiation. These findings suggest that Wnt signaling plays a critical role in regulating synaptic plasticity.

A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus.

Many forms of long-lasting behavioral and synaptic plasticity require the synthesis of new proteins. For example, long-term potentiation (LTP) that endures for more than an hour requires both transcription and translation. The signal-transduction mechanisms that couple synaptic events to protein translational machinery during long-lasting synaptic plasticity, however, are not well understood. One signaling pathway that is stimulated by growth factors and results in the translation of specific mRNAs includes the rapamycin-sensitive kinase mammalian target of rapamycin (mTOR, also known as FRAP and RAFT-1). Several components of this translational signaling pathway, including mTOR, eukaryotic initiation factor-4E-binding proteins 1 and 2, and eukaryotic initiation factor-4E, are present in the rat hippocampus as shown by Western blot analysis, and these proteins are detected in the cell bodies and dendrites in the hippocampal slices by immunostaining studies. In cultured hippocampal neurons, these proteins are present in dendrites and are often found near the presynaptic protein, synapsin I. At synaptic sites, their distribution completely overlaps with a postsynaptic protein, PSD-95. These observations suggest the postsynaptic localization of these proteins. Disruption of mTOR signaling by rapamycin results in a reduction of late-phase LTP expression induced by high-frequency stimulation; the early phase of LTP is unaffected. Rapamycin also blocks the synaptic potentiation induced by brain-derived neurotrophic factor in hippocampal slices. These results demonstrate an essential role for rapamycin-sensitive signaling in the expression of two forms of synaptic plasticity that require new protein synthesis. The localization of this translational signaling pathway at postsynaptic sites may provide a mechanism that controls local protein synthesis at potentiated synapses.