RESUMO
Exosomes and other extracellular vesicles (EVs) participate in cell-cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α2-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α2-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrPC) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor-related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R-LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrPC. Finally, EVs harvested from rat astrocytes carried PrPC and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrPC with the NMDA-R-LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.
Assuntos
Vesículas Extracelulares , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Crescimento Neuronal , Proteínas Priônicas , Receptores de Lipoproteínas , Receptores de N-Metil-D-Aspartato , Animais , Vesículas Extracelulares/metabolismo , Humanos , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , N-Metilaspartato , Células PC12 , Proteínas Priônicas/metabolismo , Ratos , Receptores de Lipoproteínas/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMO
Schwann cells (SCs) are known to produce extracellular vesicles (EV) that participate in cell-cell communication by transferring cargo to target cells, including mRNAs, microRNAs, and biologically active proteins. Herein, we report a novel mechanism whereby SC EVs may regulate PNS physiology, especially in injury, by controlling the activity of TNFα. SCs actively sequester tumor necrosis factor receptor-1 (TNFR1) into EVs at high density, accounting for about 2% of the total protein in SC EVs (~1000 copies TNFR1/EV). Although TNFR2 was robustly expressed by SCs in culture, TNFR2 was excluded from SC EVs. SC EV TNFR1 bound TNFα, decreasing the concentration of free TNFα available to bind to cells and thus served as a TNFα decoy. SC EV TNFR1 significantly inhibited TNFα-induced p38 MAPK phosphorylation in cultured SCs. When TNFR1 was proteolytically removed from SC EVs using tumor necrosis factor-α converting enzyme (TACE) or neutralized with antibody, the ability of TNFα to activate p38 MAPK in the presence of these EVs was restored. As further evidence of its decoy activity, SC EV TNFR1 modified TNFα activities in vitro including: (1) regulation of expression of other cytokines; (2) effects on SC morphology; and (3) effects on SC viability. SC EVs also modified the effects of TNFα on sciatic nerve morphology and neuropathic pain-related behavior in vivo. By sequestering TNFR1 in EVs, SCs may buffer against the potentially toxic effects of TNFα. SC EVs provide a novel mechanism for the spatial and temporal regulation of neuro-inflammation.
Assuntos
Vesículas Extracelulares , Receptores Tipo I de Fatores de Necrose Tumoral , Células de Schwann , Fator de Necrose Tumoral alfa , Células Cultivadas , Vesículas Extracelulares/metabolismo , Receptores Tipo I de Fatores de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral/metabolismo , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Células de Schwann/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Abnormalities in interactions between sensory neurons and Schwann cells (SCs) may result in heightened pain processing and chronic pain states. We previously reported that SCs express the NMDA receptor (NMDA-R), which activates cell signaling in response to glutamate and specific protein ligands, such as tissue-type plasminogen activator. Herein, we genetically targeted grin1 encoding the essential GluN1 NMDA-R subunit, conditionally in SCs, to create a novel mouse model in which SCs are NMDA-R-deficient (GluN1- mice). These mice demonstrated increased sensitivity to light touch, pinprick, and thermal hyperalgesia in the absence of injury, without associated changes in motor function. Ultrastructural analysis of adult sciatic nerve in GluN1- mice revealed increases in the density of Aδ fibers and Remak bundles and a decrease in the density of Aß fibers, without altered g-ratios. Abnormalities in adult Remak bundle ultrastructure were also present including aberrant C-fiber ensheathment, distances between axons, and increased poly-axonal pockets. Developmental and post radial sorting defects contributed to altered nerve fiber densities in adult. Uninjured sciatic nerves in GluN1- mice did not demonstrate an increase in neuroinflammatory infiltrates. Transcriptome profiling of dorsal root ganglia (DRGs) revealed 138 differentially regulated genes in GluN1- mice. One third of the regulated genes are known to be involved in pain processing, including sprr1a, npy, fgf3, atf3, and cckbr, which were significantly increased. The intraepidermal nerve fiber density (IENFD) was significantly decreased in the skin of GluN1- mice. Collectively, these findings demonstrate that SC NMDA-R is essential for normal PNS development and for preventing development of pain states.SIGNIFICANCE STATEMENT Chronic unremitting pain is a prevalent medical condition; however, the molecular mechanisms that underlie heightened pain processing remain incompletely understood. Emerging data suggest that abnormalities in Schwann cells (SCs) may cause neuropathic pain. We established a novel mouse model for small fiber neuropathy (SFN) in which grin1, the gene that encodes the NMDA receptor (NMDA-R) GluN1 subunit, is deleted in SCs. These mice demonstrate hypersensitivity in pain processing in the absence of nerve injury. Changes in the density of intraepidermal small fibers, the ultrastructure of Remak bundles, and the transcriptome of dorsal root ganglia (DRGs) provide possible explanations for the increase in pain processing. Our results support the hypothesis that abnormalities in communication between sensory nerve fibers and SCs may result in pain states.
Assuntos
Hiperalgesia/genética , Proteínas do Tecido Nervoso/genética , Dor/genética , Dor/fisiopatologia , Receptores de N-Metil-D-Aspartato/genética , Células de Schwann/ultraestrutura , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Perfilação da Expressão Gênica , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Fibras Nervosas/fisiologia , Proteínas do Tecido Nervoso/deficiência , Estimulação Física , Cultura Primária de Células , Receptores de N-Metil-D-Aspartato/deficiência , Nervo Isquiático/ultraestrutura , Transdução de SinaisRESUMO
Opioids are first-line drugs for moderate to severe acute pain and cancer pain. However, these medications are associated with severe side effects, and whether they are efficacious in treatment of chronic nonmalignant pain remains controversial. Medications that act through alternative molecular mechanisms are critically needed. Antagonists of α9α10 nicotinic acetylcholine receptors (nAChRs) have been proposed as an important nonopioid mechanism based on studies demonstrating prevention of neuropathology after trauma-induced nerve injury. However, the key α9α10 ligands characterized to date are at least two orders of magnitude less potent on human vs. rodent nAChRs, limiting their translational application. Furthermore, an alternative proposal that these ligands achieve their beneficial effects by acting as agonists of GABAB receptors has caused confusion over whether blockade of α9α10 nAChRs is the fundamental underlying mechanism. To address these issues definitively, we developed RgIA4, a peptide that exhibits high potency for both human and rodent α9α10 nAChRs, and was at least 1,000-fold more selective for α9α10 nAChRs vs. all other molecular targets tested, including opioid and GABAB receptors. A daily s.c. dose of RgIA4 prevented chemotherapy-induced neuropathic pain in rats. In wild-type mice, oxaliplatin treatment produced cold allodynia that could be prevented by RgIA4. Additionally, in α9 KO mice, chemotherapy-induced development of cold allodynia was attenuated and the milder, temporary cold allodynia was not relieved by RgIA4. These findings establish blockade of α9-containing nAChRs as the basis for the efficacy of RgIA4, and that α9-containing nAChRs are a critical target for prevention of chronic cancer chemotherapy-induced neuropathic pain.
Assuntos
Dor do Câncer/tratamento farmacológico , Hiperalgesia/tratamento farmacológico , Peptídeos/administração & dosagem , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Analgésicos Opioides/efeitos adversos , Animais , Dor do Câncer/induzido quimicamente , Dor do Câncer/genética , Dor do Câncer/patologia , Humanos , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hiperalgesia/patologia , Ligantes , Camundongos , Camundongos Knockout , Neuralgia/induzido quimicamente , Neuralgia/tratamento farmacológico , Neuralgia/genética , Neuralgia/patologia , Antagonistas Nicotínicos/administração & dosagem , Compostos Organoplatínicos/efeitos adversos , Oxaliplatina , Receptores de GABA-B/genéticaRESUMO
We identified a previously unidentified conotoxin gene from Conus generalis whose precursor signal sequence has high similarity to the O1-gene conotoxin superfamily. The predicted mature peptide, αO-conotoxin GeXIVA (GeXIVA), has four Cys residues, and its three disulfide isomers were synthesized. Previously pharmacologically characterized O1-superfamily peptides, exemplified by the US Food and Drug Administration-approved pain medication, ziconotide, contain six Cys residues and are calcium, sodium, or potassium channel antagonists. However, GeXIVA did not inhibit calcium channels but antagonized nicotinic AChRs (nAChRs), most potently on the α9α10 nAChR subtype (IC50 = 4.6 nM). Toxin blockade was voltage-dependent, and kinetic analysis of toxin dissociation indicated that the binding site of GeXIVA does not overlap with the binding site of the competitive antagonist α-conotoxin RgIA. Surprisingly, the most active disulfide isomer of GeXIVA is the bead isomer, comprising, according to NMR analysis, two well-resolved but uncoupled disulfide-restrained loops. The ribbon isomer is almost as potent but has a more rigid structure built around a short 310-helix. In contrast to most α-conotoxins, the globular isomer is the least potent and has a flexible, multiconformational nature. GeXIVA reduced mechanical hyperalgesia in the rat chronic constriction injury model of neuropathic pain but had no effect on motor performance, warranting its further investigation as a possible therapeutic agent.
Assuntos
Conotoxinas/química , Caramujo Conus/química , Antagonistas Nicotínicos/química , Receptores Nicotínicos/química , Amidas/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Canais de Cálcio/química , Clonagem Molecular , Retículo Endoplasmático/metabolismo , Hiperalgesia/tratamento farmacológico , Concentração Inibidora 50 , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Neuralgia/terapia , Oócitos/citologia , Conformação Proteica , Sinais Direcionadores de Proteínas , Ratos , Ratos Sprague-Dawley , Xenopus laevisRESUMO
Animals perform innate behaviors that are stereotyped responses to specific evolutionarily relevant stimuli in the absence of prior learning or experience. These behaviors can be reduced to an axis of valence, whereby specific odors evoke approach or avoidance. The cortical amygdala (plCoA) mediates innate attraction and aversion to odor. However, little is known about how this brain area gives rise to behaviors of opposing motivational valence. Here, we sought to define the circuit features of plCoA that give rise to innate olfactory behaviors of valence. We characterized the physiology, gene expression, and projections of this structure, identifying a divergent, topographic organization that selectively controls innate attraction and avoidance to odor. First, we examined odor-evoked responses in these areas and found sparse encoding of odor identity, but not valence. We next considered a topographic organization and found that optogenetic stimulation of the anterior and posterior domains of plCoA elicits attraction and avoidance, respectively, suggesting a functional axis for valence. Using single cell and spatial RNA sequencing, we identified the molecular cell types in plCoA, revealing an anteroposterior gradient in cell types, whereby anterior glutamatergic neurons preferentially express Slc17a6 and posterior neurons express Slc17a7. Activation of these respective cell types recapitulates appetitive and aversive valence behaviors, and chemogenetic inhibition reveals partial necessity for valence responses to innate appetitive or aversive odors. Finally, we identified topographically organized circuits defined by projections, whereby anterior neurons preferentially project to medial amygdala, and posterior neurons preferentially project to nucleus accumbens, which are respectively sufficient and necessary for innate negative and positive olfactory valence. Together, these data advance our understanding of how the olfactory system generates stereotypic, hardwired attraction and avoidance, and supports a model whereby distinct, topographically distributed plCoA populations direct innate olfactory valence responses by signaling to divergent valence-specific targets, linking upstream olfactory identity to downstream valence behaviors, through a population code. This represents a novel circuit motif in which valence encoding is represented not by the firing properties of individual neurons, but by population level identity encoding that is routed through divergent targets to mediate distinct valence.
RESUMO
Circadian disruptions impact nearly all people with Alzheimer's disease (AD), emphasizing both their potential role in pathology and the critical need to investigate the therapeutic potential of circadian-modulating interventions. Here, we show that time-restricted feeding (TRF) without caloric restriction improved key disease components including behavioral timing, disease pathology, hippocampal transcription, and memory in two transgenic (TG) mouse models of AD. We found that TRF had the remarkable capability of simultaneously reducing amyloid deposition, increasing Aß42 clearance, improving sleep and memory, and normalizing daily transcription patterns of multiple genes, including those associated with AD and neuroinflammation. Thus, our study unveils for the first time the pleiotropic nature of timed feeding on AD, which has far-reaching effects beyond metabolism, ameliorating neurodegeneration and the misalignment of circadian rhythmicity. Since TRF can substantially modify disease trajectory, this intervention has immediate translational potential, addressing the urgent demand for accessible approaches to reduce or halt AD progression.
Assuntos
Doença de Alzheimer , Camundongos , Animais , Humanos , Doença de Alzheimer/terapia , Doença de Alzheimer/genética , Camundongos Transgênicos , Modelos Animais de Doenças , Ritmo Circadiano , Encéfalo/metabolismo , Peptídeos beta-AmiloidesRESUMO
Mild traumatic brain injury (mTBI) disproportionately affects military service members and is very difficult to diagnose. To-date, there is currently no blood-based, diagnostic biomarker for mTBI cases with persistent post concussive symptoms. To examine the potential of neuronally-derived (NDE) and astrocytic-derived (ADE) exosome cargo proteins as biomarkers of chronic mTBI in younger adults, we examined plasma exosomes from a prospective longitudinal study of combat-related risk and resilience, marine resiliency study II (MRSII). After return from a combat-deployment participants were interviewed to assess TBI exposure while on deployment. Plasma exosomes from military service members with mTBI (mean age, 21.7 years, n = 19, avg. days since injury 151), and age-matched, controls (deployed service members who did not endorse a deployment-related TBI or a pre-deployment history of TBI; mean age, 21.95 years, n = 20) were precipitated and enriched against a neuronal adhesion protein, L1-CAM, and an astrocyte marker, glutamine aspartate transporter (GLAST) using magnetic beads to immunocapture the proteins and subsequently selected by fluorescent activated cell sorting (FACS). Extracted protein cargo from NDE and ADE preparations were quantified for protein levels implicated in TBI neuropathology by standard ELISAs and on the ultra-sensitive single molecule assay (Simoa) platform. Plasma NDE and ADE levels of Aß42 were significantly higher while plasma NDE and ADE levels of the postsynaptic protein, neurogranin (NRGN) were significantly lower in participants endorsing mTBI exposure compared to controls with no TBI history. Plasma NDE and ADE levels of Aß40, total tau, and neurofilament light (NFL), P-T181-tau, P-S396-tau were either undetectable or not significantly different between the two groups. In an effort to understand the pathogenetic potential of NDE and ADE cargo proteins, neuron-like cultures were treated with NDE and ADE preparations from TBI and non-TBI groups. Lastly, we determined that plasma NDE but not ADE cargo proteins from mTBI samples were found to be toxic to neuron-like recipient cells in vitro. These data support the presence of markers of neurodegeneration in NDEs of mTBI and suggest that these NDEs can be used as tools to identify pathogenic mechanisms of TBI.