RESUMO
Treatment for higher-risk non-muscle invasive bladder cancer (NMIBC) involves intravesical immunotherapy with Bacillus Calmette Guérin (BCG); however, disease recurrence and progression occur frequently. Systemic immunity is critical for successful cancer immunotherapy; thus, recurrence of NMIBC may be due to suboptimal systemic activation of anti-tumor immunity after local immunotherapy. We previously reported that systemically acquired trained immunity (a form of innate immune memory) in circulating monocytes is associated with increased time-to-recurrence in patients with NMIBC treated with BCG. Herein, we used a mouse model of NMIBC to compare the effects of intravesical versus intravenous (systemic) BCG immunotherapy on the local and peripheral immune microenvironments. We also assessed whether BCG-induced trained immunity modulates anti-tumor immune responses. Compared with intravesical BCG, which led to a tumor-promoting immune microenvironment, intravenous BCG resulted in an anti-tumoral bladder microenvironment characterized by increased proportions of cytotoxic T lymphocytes (CTLs), and decreased proportions of myeloid-derived suppressor cells. Polarization toward anti-tumoral immunity occurred in draining lymph nodes, spleen, and bone marrow following intravenous versus intravesical BCG treatment. Pre-treatment with intravesical BCG was associated with increased rate of tumor growth compared with intravenous BCG pre-treatment. Trained immunity contributed to remodeling of the tumor immune microenvironment, as co-instillation of BCG-trained macrophages with ovalbumin-expressing bladder tumor cells increased the proportion of tumor-specific CTLs. Furthermore, BCG-trained dendritic cells exhibited enhanced antigen uptake and presentation and promoted CTL proliferation. Our data support the concept that systemic immune activation promotes anti-tumor responses, and that BCG-induced trained immunity is important in driving anti-tumor adaptive immunity.
Assuntos
Vacina BCG , Imunoterapia , Microambiente Tumoral , Neoplasias da Bexiga Urinária , Neoplasias da Bexiga Urinária/imunologia , Neoplasias da Bexiga Urinária/terapia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Microambiente Tumoral/imunologia , Camundongos , Vacina BCG/imunologia , Vacina BCG/administração & dosagem , Vacina BCG/uso terapêutico , Imunoterapia/métodos , Feminino , Administração Intravesical , Camundongos Endogâmicos C57BL , Linfócitos T Citotóxicos/imunologia , Humanos , Modelos Animais de Doenças , Imunidade Inata/imunologia , Linhagem Celular Tumoral , Memória Imunológica/imunologia , Células Supressoras Mieloides/imunologia , Imunidade TreinadaRESUMO
The synaptic organization of thalamic inputs to motor cortices remains poorly understood in primates. Thus, we compared the regional and synaptic connections of vGluT2-positive thalamocortical glutamatergic terminals in the supplementary motor area (SMA) and the primary motor cortex (M1) between control and MPTP-treated parkinsonian monkeys. In controls, vGluT2-containing fibers and terminal-like profiles invaded layer II-III and Vb of M1 and SMA. A significant reduction of vGluT2 labeling was found in layer Vb, but not in layer II-III, of parkinsonian animals, suggesting a potential thalamic denervation of deep cortical layers in parkinsonism. There was a significant difference in the pattern of synaptic connectivity in layers II-III, but not in layer Vb, between M1 and SMA of control monkeys. However, this difference was abolished in parkinsonian animals. No major difference was found in the proportion of perforated versus macular post-synaptic densities at thalamocortical synapses between control and parkinsonian monkeys in both cortical regions, except for a slight increase in the prevalence of perforated axo-dendritic synapses in the SMA of parkinsonian monkeys. Our findings suggest that disruption of the thalamic innervation of M1 and SMA may underlie pathophysiological changes of the motor thalamocortical loop in the state of parkinsonism.
Assuntos
Córtex Motor/ultraestrutura , Transtornos Parkinsonianos/patologia , Densidade Pós-Sináptica/ultraestrutura , Tálamo/ultraestrutura , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina , Animais , Feminino , Macaca mulatta , Masculino , Vias Neurais/ultraestrutura , Neurotoxinas , Proteína Vesicular 2 de Transporte de Glutamato/metabolismoRESUMO
The primate ventral motor thalamus contains a large number of GABAergic interneurons of poorly understood function and anatomical connectivity. Glutamatergic inputs to these cells arise predominantly from corticothalamic (in both basal ganglia- and cerebellar-receiving ventral motor thalamic territories; BGMT and CBMT, respectively) and cerebellothalamic terminals (in CBMT). In Parkinson's disease patients and animal models, neuronal activity is abnormal within both BGMT and CBMT. Historically, such motor thalamic dysregulation has been largely attributed to changes in inhibitory tone from the basal ganglia output nuclei, ignoring the potential role of other thalamic inputs in such processes, particularly within the CBMT, which is largely devoid of direct basal ganglia afferents. We have recently reported changes in the abundance and structural morphology of corticothalamic terminals in BGMT of parkinsonian monkeys. In this study, we assessed potential changes in the prevalence of cortical (vesicular glutamate transporter 1-positive, vGluT1-positive) and subcortical (vGluT2-positive) glutamatergic inputs in contact with GABAergic interneurons in BGMT and CBMT of MPTP-treated parkinsonian monkeys. Our findings revealed that interneurons represent a major target of both sets of glutamatergic terminals. In both BGMT and CBMT of control and parkinsonian monkeys, 29%-38% of total asymmetric axodendritic synapses (putative glutamatergic) were formed by vGluT1-positive terminals and 11%-17% of total vGluT1-positive terminals targeted dendrites of GABAergic interneurons. In CBMT, 16%-18% of asymmetric synaptic inputs on interneurons involved vGluT2-containing terminals. No major differences in the extent of glutamatergic innervation of thalamic GABAergic interneurons were found between control and parkinsonian monkeys.
Assuntos
Interneurônios , Tálamo , Animais , Haplorrinos , Humanos , Neurônios , SinapsesRESUMO
Designer receptors exclusively activated by designer drugs (DREADDs) are extensively used to modulate neuronal activity in rodents, but their use in primates remains limited. An essential need that remains is the demonstration that DREADDs are efficiently expressed on the plasma membrane of primate neurons. To address this issue, electron microscopy immunogold was used to determine the subcellular localization of the AAV vector-induced DREADDs hM4Di and hM3Dq fused to different tags in various brain areas of rhesus monkeys and mice. When hM4Di was fused to mCherry, the immunogold labelling was mostly confined to the intracellular space, and poorly expressed at the plasma membrane in monkey dendrites. In contrast, the hM4Di-mCherry labelling was mostly localized to the dendritic plasma membrane in mouse neurons, suggesting species differences in the plasma membrane expression of these exogenous proteins. The lack of hM4Di plasma membrane expression may limit the functional effects of systemic administration of DREADD-actuators in monkey neurons. Removing the mCherry and fusing of hM4Di with the haemagglutinin (HA) tag resulted in strong neuronal plasma membrane immunogold labelling in both monkeys and mice neurons. Finally, hM3Dq-mCherry was expressed mostly at the plasma membrane in monkey neurons, indicating that the fusion of mCherry with hM3Dq does not hamper membrane incorporation of this specific DREADD. Our results suggest that the pattern of ultrastructural expression of DREADDs in monkey neurons depends on the DREADD/tag combination. Therefore, a preliminary characterization of plasma membrane expression of specific DREADD/tag combinations is recommended when using chemogenetic approaches in primates.
Assuntos
Encéfalo/metabolismo , Membrana Celular/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Dendritos/metabolismo , Feminino , Macaca mulatta , Masculino , CamundongosRESUMO
The GluN2D subunit of the NMDA receptor is prominently expressed in the basal ganglia and associated brainstem nuclei, including the subthalamic nucleus (STN), globus pallidus, striatum, and substantia nigra. However, little is known about how GluN2D-containing NMDA receptors contribute to synaptic activity in these regions. Using Western blotting of STN tissue punches, we demonstrated that GluN2D is expressed in the rat STN throughout development [age postnatal day 7 (P7)-P60] and in the adult (age P120). Immunoelectron microscopy of the adult rat brain showed that GluN2D is predominantly expressed in dendrites, unmyelinated axons, and axon terminals within the STN. Using subunit-selective allosteric modulators of NMDA receptors (TCN-201, ifenprodil, CIQ, and DQP-1105), we provide evidence that receptors containing the GluN2B and GluN2D subunits mediate responses to exogenously applied NMDA and glycine, as well as synaptic NMDA receptor activation in the STN of rat brain slices. EPSCs in the STN were mediated primarily by AMPA and NMDA receptors and GluN2D-containing NMDA receptors controlled the slow deactivation time course of EPSCs in the STN. In vivo recordings from the STN of anesthetized adult rats demonstrated that the spike firing rate was increased by the GluN2C/D potentiator CIQ and decreased by the GluN2C/D antagonist DQP-1105, suggesting that NMDA receptor activity can influence STN output. These data indicate that the GluN2B and GluN2D NMDA receptor subunits contribute to synaptic activity in the STN and may represent potential therapeutic targets for modulating subthalamic neuron activity in neurological disorders such as Parkinson's disease.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Núcleo Subtalâmico/citologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Animais , Animais Recém-Nascidos , Dendritos/metabolismo , Dendritos/ultraestrutura , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/ultraestrutura , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/genética , Núcleo Subtalâmico/crescimento & desenvolvimentoRESUMO
Biophysical forces play important roles throughout embryogenesis, but the roles of spatial differences in cellular resting potentials during large-scale brain morphogenesis remain unknown. Here, we implicate endogenous bioelectricity as an instructive factor during brain patterning in Xenopus laevis. Early frog embryos exhibit a characteristic hyperpolarization of cells lining the neural tube; disruption of this spatial gradient of the transmembrane potential (Vmem) diminishes or eliminates the expression of early brain markers, and causes anatomical mispatterning of the brain, including absent or malformed regions. This effect is mediated by voltage-gated calcium signaling and gap-junctional communication. In addition to cell-autonomous effects, we show that hyperpolarization of transmembrane potential (Vmem) in ventral cells outside the brain induces upregulation of neural cell proliferation at long range. Misexpression of the constitutively active form of Notch, a suppressor of neural induction, impairs the normal hyperpolarization pattern and neural patterning; forced hyperpolarization by misexpression of specific ion channels rescues brain defects induced by activated Notch signaling. Strikingly, hyperpolarizing posterior or ventral cells induces the production of ectopic neural tissue considerably outside the neural field. The hyperpolarization signal also synergizes with canonical reprogramming factors (POU and HB4), directing undifferentiated cells toward neural fate in vivo. These data identify a new functional role for bioelectric signaling in brain patterning, reveal interactions between Vmem and key biochemical pathways (Notch and Ca(2+) signaling) as the molecular mechanism by which spatial differences of Vmem regulate organogenesis of the vertebrate brain, and suggest voltage modulation as a tractable strategy for intervention in certain classes of birth defects.
Assuntos
Padronização Corporal/fisiologia , Proliferação de Células/fisiologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Receptores Notch/metabolismo , Transdução de Sinais/fisiologia , Fatores Etários , Animais , Padronização Corporal/genética , Cálcio/metabolismo , Embrião não Mamífero , Corantes Fluorescentes/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Potenciais da Membrana/genética , Microinjeções , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Tubo Neural/citologia , Tubo Neural/embriologia , Receptores Notch/genética , Transdução de Sinais/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transdução Genética , Xenopus laevisRESUMO
KEY POINTS: Xenopus laevis craniofacial development is a good system for the study of Andersen-Tawil Syndrome (ATS)-associated craniofacial anomalies (CFAs) because (1) Kcnj2 is expressed in the nascent face; (2) molecular-genetic and biophysical techniques are available for the study of ion-dependent signalling during craniofacial morphogenesis; (3) as in humans, expression of variant Kcnj2 forms in embryos causes a muscle phenotype; and (4) variant forms of Kcnj2 found in human patients, when injected into frog embryos, cause CFAs in the same cell lineages. Forced expression of WT or variant Kcnj2 changes the normal pattern of Vmem (resting potential) regionalization found in the ectoderm of neurulating embryos, and changes the normal pattern of expression of ten different genetic regulators of craniofacial development, including markers of cranial neural crest and of placodes. Expression of other potassium channels and two different light-activated channels, all of which have an effect on Vmem , causes CFAs like those induced by injection of Kcnj2 variants. In contrast, expression of Slc9A (NHE3), an electroneutral ion channel, and of GlyR, an inactive Cl(-) channel, do not cause CFAs, demonstrating that correct craniofacial development depends on a pattern of bioelectric states, not on ion- or channel-specific signalling. Using optogenetics to control both the location and the timing of ion flux in developing embryos, we show that affecting Vmem of the ectoderm and no other cell layers is sufficient to cause CFAs, but only during early neurula stages. Changes in Vmem induced late in neurulation do not affect craniofacial development. We interpret these data as strong evidence, consistent with our hypothesis, that ATS-associated CFAs are caused by the effect of variant Kcnj2 on the Vmem of ectodermal cells of the developing face. We predict that the critical time is early during neurulation, and the critical cells are the ectodermal cranial neural crest and placode lineages. This points to the potential utility of extant, ion flux-modifying drugs as treatments to prevent CFAs associated with channelopathies such as ATS. ABSTRACT: Variants in potassium channel KCNJ2 cause Andersen-Tawil Syndrome (ATS); the induced craniofacial anomalies (CFAs) are entirely unexplained. We show that KCNJ2 is expressed in Xenopus and mouse during the earliest stages of craniofacial development. Misexpression in Xenopus of KCNJ2 carrying ATS-associated mutations causes CFAs in the same structures affected in humans, changes the normal pattern of membrane voltage potential regionalization in the developing face and disrupts expression of important craniofacial patterning genes, revealing the endogenous control of craniofacial patterning by bioelectric cell states. By altering cells' resting potentials using other ion translocators, we show that a change in ectodermal voltage, not tied to a specific protein or ion, is sufficient to cause CFAs. By adapting optogenetics for use in non-neural cells in embryos, we show that developmentally patterned K(+) flux is required for correct regionalization of the resting potentials and for establishment of endogenous early gene expression domains in the anterior ectoderm, and that variants in KCNJ2 disrupt this regionalization, leading to the CFAs seen in ATS patients.
Assuntos
Síndrome de Andersen/genética , Anormalidades Craniofaciais/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Animais , Embrião de Mamíferos , Larva , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/anormalidades , Optogenética , RNA Mensageiro/genética , Xenopus laevisRESUMO
The striatum and the subthalamic nucleus are the main entry points for cortical information to the basal ganglia. Parkinson's disease affects not only the function, but also the morphological integrity of some of these inputs and their synaptic targets in the basal ganglia. Significant morphological changes in the cortico-striatal system have already been recognized in patients with Parkinson's disease and in animal models of the disease. To find out whether the primate cortico-subthalamic system is also subject to functionally relevant morphological alterations in parkinsonism, we used a combination of light and electron microscopy anatomical approaches and in vivo electrophysiological methods in monkeys rendered parkinsonian following chronic exposure to low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). At the light microscopic level, the density of vesicular glutamate transporter 1-positive (i.e. cortico-subthalamic) profiles in the dorsolateral part of the subthalamic nucleus (i.e. its sensorimotor territory) was 26.1% lower in MPTP-treated parkinsonian monkeys than in controls. These results were confirmed by electron microscopy studies showing that the number of vesicular glutamate transporter 1-positive terminals and of axon terminals forming asymmetric synapses in the dorsolateral subthalamic nucleus was reduced by 55.1% and 27.9%, respectively, compared with controls. These anatomical findings were in line with in vivo electrophysiology data showing a 60% reduction in the proportion of pallidal neurons that responded to electrical stimulation of the cortico-subthalamic system in parkinsonian monkeys. These findings provide strong evidence for a partial loss of the hyperdirect cortico-subthalamic projection in MPTP-treated parkinsonian monkeys.
Assuntos
1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/efeitos adversos , Córtex Cerebral/patologia , Corpo Estriado/patologia , Globo Pálido/patologia , Doença de Parkinson Secundária/patologia , Núcleo Subtalâmico/patologia , Animais , Gânglios da Base/efeitos dos fármacos , Gânglios da Base/patologia , Córtex Cerebral/efeitos dos fármacos , Corpo Estriado/efeitos dos fármacos , Feminino , Globo Pálido/efeitos dos fármacos , Haplorrinos , Macaca mulatta , Masculino , Doença de Parkinson Secundária/induzido quimicamente , Núcleo Subtalâmico/efeitos dos fármacosRESUMO
Abnormal dopamine neurotransmission is associated with many different genetic and acquired dystonic disorders. For instance, mutations in genes critical for the synthesis of dopamine, including GCH1 and TH cause l-DOPA-responsive dystonia. Despite evidence that implicates abnormal dopamine neurotransmission in dystonia, the precise nature of the pre- and postsynaptic defects that result in dystonia are not known. To better understand these defects, we generated a knock-in mouse model of l-DOPA-responsive dystonia (DRD) mice that recapitulates the human p.381Q>K TH mutation (c.1141C>A). Mice homozygous for this mutation displayed the core features of the human disorder, including reduced TH activity, dystonia that worsened throughout the course of the active phase, and improvement in the dystonia in response to both l-DOPA and trihexyphenidyl. Although the gross anatomy of the nigrostriatal dopaminergic neurons was normal in DRD mice, the microstructure of striatal synapses was affected whereby the ratio of axo-spinous to axo-dendritic corticostriatal synaptic contacts was reduced. Microinjection of l-DOPA directly into the striatum ameliorated the dystonic movements but cerebellar microinjections of l-DOPA had no effect. Surprisingly, the striatal dopamine concentration was reduced to â¼1% of normal, a concentration more typically associated with akinesia, suggesting that (mal)adaptive postsynaptic responses may also play a role in the development of dystonia. Administration of D1- or D2-like dopamine receptor agonists to enhance dopamine signalling reduced the dystonic movements, whereas administration of D1- or D2-like dopamine receptor antagonists to further reduce dopamine signalling worsened the dystonia, suggesting that both receptors mediate the abnormal movements. Further, D1-dopamine receptors were supersensitive; adenylate cyclase activity, locomotor activity and stereotypy were exaggerated in DRD mice in response to the D1-dopamine receptor agonist SKF 81297. D2-dopamine receptors exhibited a change in the valence in DRD mice with an increase in adenylate cyclase activity and blunted behavioural responses after challenge with the D2-dopamine receptor agonist quinpirole. Together, our findings suggest that the development of dystonia may depend on a reduction in dopamine in combination with specific abnormal receptor responses.
Assuntos
Dopaminérgicos/uso terapêutico , Distonia/tratamento farmacológico , Levodopa/uso terapêutico , Mutação/genética , Tirosina 3-Mono-Oxigenase/genética , Animais , Benzazepinas/farmacocinética , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Catecolaminas/metabolismo , Modelos Animais de Doenças , Antagonistas de Dopamina/farmacocinética , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Distonia/diagnóstico por imagem , Distonia/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Cintilografia , Comportamento Estereotipado/fisiologia , Trítio/farmacocinética , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
GluN2B subunit containing NMDARs (GluN2B-NMDARs) mediate pathophysiological effects of acutely applied amyloid beta (Aß), including impaired long-term potentiation (LTP). However, in transgenic Alzheimer's disease (AD) mouse models which feature gradual Aß accumulation, the function of GluN2B-NMDARs and their contribution to synaptic plasticity are unknown. Therefore, we examined the role of GluN2B-NMDARs in synaptic function and plasticity in the hippocampus of PS2APP transgenic mice. Although LTP induced by theta burst stimulation (TBS) was normal in PS2APP mice, it was significantly reduced by the selective GluN2B-NMDAR antagonist Ro25-6981 (Ro25) in PS2APP mice, but not wild type (wt) mice. While NMDARs activated by single synaptic stimuli were not blocked by Ro25, NMDARs recruited during burst stimulation showed larger blockade by Ro25 in PS2APP mice. Thus, the unusual dependence of LTP on GluN2B-NMDARs in PS2APP mice suggests that non-synaptic GluN2B-NMDARs are activated by glutamate that spills out of synaptic cleft during the burst stimulation used to induce LTP. While long-term depression (LTD) was normal in PS2APP mice, and Ro25 had no impact on LTD in wt mice, Ro25 impaired LTD in PS2APP mice, again demonstrating aberrant GluN2B-NMDAR function during plasticity. Together these results demonstrate altered GluN2B-NMDAR function in a model of early AD pathology that has implications for the therapeutic targeting of NMDARs in AD.
Assuntos
Doença de Alzheimer/fisiopatologia , Hipocampo/fisiopatologia , Potenciação de Longa Duração/fisiologia , Depressão Sináptica de Longo Prazo/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Doença de Alzheimer/patologia , Animais , Western Blotting , Modelos Animais de Doenças , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Hipocampo/efeitos dos fármacos , Hipocampo/ultraestrutura , Potenciação de Longa Duração/efeitos dos fármacos , Depressão Sináptica de Longo Prazo/efeitos dos fármacos , Masculino , Microscopia Eletrônica de Transmissão , Fenóis/farmacologia , Piperidinas/farmacologia , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Técnicas de Cultura de TecidosRESUMO
The subthalamic nucleus (STN) receives a dopaminergic innervation from the substantia nigra pars compacta, but the role of this projection remains poorly understood, particularly in primates. To address this issue, we used immuno-electron microscopy to localize D1, D2, and D5 dopamine receptors in the STN of rhesus macaques and studied the electrophysiological effects of activating D1-like or D2-like receptors in normal and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian monkeys. Labeling of D1 and D2 receptors was primarily found presynaptically, on preterminal axons and putative glutamatergic and GABAergic terminals, while D5 receptors were more significantly expressed postsynaptically, on dendritic shafts of STN neurons. The electrical spiking activity of STN neurons, recorded with standard extracellular recording methods, was studied before, during, and after intra-STN administration of the dopamine D1-like receptor agonist SKF82958, the D2-like receptor agonist quinpirole, or artificial cerebrospinal fluid (control injections). In normal animals, administration of SKF82958 significantly reduced the spontaneous firing but increased the rate of intraburst firing and the proportion of pause-burst sequences of firing. Quinpirole only increased the proportion of such pause-burst sequences in STN neurons of normal monkeys. In MPTP-treated monkeys, the D1-like receptor agonist also reduced the firing rate and increased the proportion of pause-burst sequences, while the D2-like receptor agonist did not change any of the chosen descriptors of the firing pattern of STN neurons. Our data suggest that dopamine receptor activation can directly modulate the electrical activity of STN neurons by pre- and postsynaptic mechanisms in both normal and parkinsonian states, predominantly via activation of D1 receptors.
Assuntos
Intoxicação por MPTP/metabolismo , Receptores Dopaminérgicos/metabolismo , Núcleo Subtalâmico/metabolismo , Potenciais de Ação , Animais , Axônios/metabolismo , Dendritos/metabolismo , Agonistas de Dopamina/farmacologia , Feminino , Intoxicação por MPTP/fisiopatologia , Macaca mulatta , Masculino , Receptores Dopaminérgicos/genética , Núcleo Subtalâmico/citologia , Núcleo Subtalâmico/fisiopatologia , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/fisiologia , Potenciais SinápticosRESUMO
PROBLEM: Preeclampsia (PE) and fetal growth restriction (FGR) are often associated with maternal inflammation and an increased risk of cardiovascular and metabolic disease in the affected mothers. The mechanism responsible for this increased risk of subsequent disease may involve reprogramming of innate immune cells, characterized by epigenetic modifications. METHOD OF STUDY: Circulating monocytes from women with PE, FGR, or uncomplicated pregnancies (control) were isolated before labor. Cytokine release from monocytes following exposure to lipopolysaccharide (LPS) and the presence of lysine 4-trimethylated histone 3 (H3K4me3) within TNF promoter sequences were evaluated. Single-cell transcriptomic profiles of circulating monocytes from women with PE or uncomplicated pregnancies were assessed. RESULTS: Monocytes from women with PE or FGR exhibited increased IL-10 secretion and decreased IL-1ß and GM-CSF secretion in response to LPS. While TNFα secretion was not significantly different in cultures of control monocytes versus those from complicated pregnancies with or without LPS exposure, monocytes from complicated pregnancies had significantly decreased levels of H3K4me3 associated with TNF promoter sequences. Cluster quantification and pathway analysis of differentially expressed genes revealed an increased proportion of anti-inflammatory myeloid cells and a lower proportion of inflammatory non-classical monocytes among the circulating monocyte population in women with PE. CONCLUSIONS: Monocytes from women with PE and FGR exhibit an immune tolerance phenotype before initiation of labor. Further investigation is required to determine whether this tolerogenic phenotype persists after the affected pregnancy and contributes to increased risk of subsequent disease.
Assuntos
Retardo do Crescimento Fetal , Imunidade Inata , Lipopolissacarídeos , Monócitos , Pré-Eclâmpsia , Humanos , Feminino , Gravidez , Adulto , Monócitos/imunologia , Pré-Eclâmpsia/imunologia , Lipopolissacarídeos/imunologia , Retardo do Crescimento Fetal/imunologia , Histonas/metabolismo , Células Cultivadas , Epigênese Genética , Reprogramação Celular , Fator de Necrose Tumoral alfa/metabolismo , Regiões Promotoras Genéticas/genética , Citocinas/metabolismoRESUMO
RGS14 is a complex multifunctional scaffolding protein that is highly enriched within pyramidal cells (PCs) of hippocampal area CA2. There, RGS14 suppresses glutamate-induced calcium influx and related G protein and ERK signaling in dendritic spines to restrain postsynaptic signaling and plasticity. Previous findings show that, unlike PCs of hippocampal areas CA1 and CA3, CA2 PCs are resistant to a number of neurological insults, including degeneration caused by temporal lobe epilepsy (TLE). While RGS14 is protective against peripheral injury, similar roles for RGS14 during pathological injury in hippocampus remain unexplored. Recent studies show that area CA2 modulates hippocampal excitability, generates epileptiform activity and promotes hippocampal pathology in animal models and patients with TLE. Because RGS14 suppresses CA2 excitability and signaling, we hypothesized that RGS14 would moderate seizure behavior and early hippocampal pathology following seizure activity. Using kainic acid (KA) to induce status epilepticus (KA-SE) in mice, we show loss of RGS14 (RGS14 KO) accelerated onset of limbic motor seizures and mortality compared to wild type (WT) mice, and that KA-SE upregulated RGS14 protein expression in CA2 and CA1 PCs of WT. Utilizing proteomics, we saw loss of RGS14 impacted the expression of a number of proteins at baseline and after KA-SE, many of which associated unexpectedly with mitochondrial function and oxidative stress. RGS14 was shown to localize to the mitochondria in CA2 PCs of mice and reduce mitochondrial respiration in vitro . As a readout of oxidative stress, we found RGS14 KO dramatically increased 3-nitrotyrosine levels in CA2 PCs, which was greatly exacerbated following KA-SE and correlated with a lack of superoxide dismutase 2 (SOD2) induction. Assessing for hallmarks of seizure pathology in RGS14 KO, we observed worse neuronal injury in area CA3 (but none in CA2 or CA1), and a lack of microgliosis in CA1 and CA2 compared to WT. Together, our data demonstrates a newly appreciated neuroprotective role for RGS14 against intense seizure activity in hippocampus. Our findings are consistent with a model where, after seizure, RGS14 is upregulated to support mitochondrial function and prevent oxidative stress in CA2 PCs, limit seizure onset and hippocampal neuronal injury, and promote microglial activation in hippocampus.
RESUMO
The intra-pallidal application of γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) or GABA transporter subtype 3 (GAT-3) transporter blockers [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA(B) heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA(A) receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA(B) receptor blockade by the intra-cellular application of N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (OX314), bath application of SKF 89976A (10 µM) or SNAP 5114 (10 µM) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid, a GABA(B) receptor antagonist. The paired-pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA(B) heteroreceptors following GAT-1 or GAT-3 blockade. In conclusion, our findings demonstrate that presynaptic GABA(B) heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced by GAT inactivation, thereby suggesting that GAT blockade represents a potential mechanism by which overactive subthalamopallidal activity may be reduced in parkinsonism.
Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/fisiologia , Globo Pálido/fisiologia , Receptores de GABA-B/fisiologia , Transmissão Sináptica/fisiologia , Animais , Anisóis/farmacologia , Potenciais Pós-Sinápticos Excitadores , Proteínas da Membrana Plasmática de Transporte de GABA/efeitos dos fármacos , Inibidores da Captação de GABA/farmacologia , Antagonistas de Receptores de GABA-B/farmacologia , Ácido Glutâmico/fisiologia , Ácidos Nipecóticos/farmacologia , Ácidos Fosfínicos/farmacologia , Propanolaminas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de GABA-B/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Women who develop preeclampsia (PE) are at high risk for cardiovascular disease (CVD). Early identification of women with PE who may benefit the most from early cardiovascular risk screening and interventions remains challenging. Our objective was to assess whether cytokine and immune cell profiles after PE are helpful in distinguishing women at low and high CVD risk at 6-months postpartum. Individuals who developed PE were followed for immune cell phenotyping and plasma cytokine quantification at delivery, at 3-months, and at 6-months postpartum. Lifetime CVD risk was assessed at 6-months postpartum, and the immune cell and cytokine profiles were compared between risk groups at each time point. Among 31 participants, 18 (58.1%) exhibited high CVD-risk profiles at 6-months postpartum. The proportion of circulating NK-cells was significantly lower in high-risk participants at delivery (p = 0.04). At 3-months postpartum, high-risk participants exhibited a lower proportion of FoxP3+ regulatory T-cells (p = 0.01), a greater proportion of CD8+ T cells (p = 0.02) and a lower CD4+:CD8+ ratio (p = 0.02). There were no differences in immune cell populations at 6-months postpartum. There were no differences in plasma cytokines levels between risk groups at any time point. Subtle differences in immune cell profiles may help distinguish individuals at low and high CVD risk in the early postpartum period and warrants further investigation.
RESUMO
Current therapeutic strategies against bacterial infections focus on reduction of pathogen load using antibiotics; however, stimulation of host tolerance to infection in the presence of pathogens might offer an alternative approach. Computational transcriptomics and Xenopus laevis embryos are used to discover infection response pathways, identify potential tolerance inducer drugs, and validate their ability to induce broad tolerance. Xenopus exhibits natural tolerance to Acinetobacter baumanii, Klebsiella pneumoniae, Staphylococcus aureus, and Streptococcus pneumoniae bacteria, whereas Aeromonas hydrophila and Pseudomonas aeruginosa produce lethal infections. Transcriptional profiling leads to definition of a 20-gene signature that discriminates between tolerant and susceptible states, as well as identification of a more active tolerance response to gram negative compared to gram positive bacteria. Gene pathways associated with active tolerance in Xenopus, including some involved in metal ion binding and hypoxia, are found to be conserved across species, including mammals, and administration of a metal chelator (deferoxamine) or a HIF-1α agonist (1,4-DPCA) in embryos infected with lethal A. hydrophila increased survival despite high pathogen load. These data demonstrate the value of combining the Xenopus embryo infection model with computational multiomics analyses for mechanistic discovery and drug repurposing to induce host tolerance to bacterial infections.
Assuntos
Bactérias Gram-Positivas , Infecções Estafilocócicas , Animais , Tolerância Imunológica , Klebsiella pneumoniae , Mamíferos , Testes de Sensibilidade Microbiana , Infecções Estafilocócicas/tratamento farmacológicoRESUMO
Endosomal sorting mechanisms mediated by AP-3 and BLOC-1 are perturbed in Hermansky-Pudlak Syndrome, a human genetic condition characterized by albinism and prolonged bleeding (OMIM #203300). Additionally, mouse models defective in either one of these complexes possess defective synaptic vesicle biogenesis (Newell-Litwa et al., 2009). These synaptic vesicle phenotypes were presumed uniform throughout the brain. However, here we report that AP-3 and BLOC-1 differentially regulate the composition of presynaptic terminals in the striatum and dentate gyrus of the hippocampus. Quantitative immunoelectron microscopy demonstrated that the majority of AP-3 immunoreactivity in both wild-type striatum and hippocampus localizes to presynaptic axonal compartments, where it regulates synaptic vesicle size. In the striatum, loss of AP-3 (Ap3d(mh/mh)) resulted in decreased synaptic vesicle size. In contrast, loss of AP-3 in the dentate gyrus increased synaptic vesicle size, thus suggesting anatomically specific AP-3-regulatory mechanisms. Loss-of-function alleles of BLOC-1, Pldn(pa/pa), and Muted(mu/mu) revealed that this complex acts as a brain-region-specific regulator of AP-3. In fact, BLOC-1 deficiencies selectively reduced AP-3 and AP-3 cargo immunoreactivity in presynaptic compartments within the dentate gyrus both at the light and/or electron microscopy level. However, the striatum did not exhibit these BLOC-1-null phenotypes. Our results demonstrate that distinct brain regions differentially regulate AP-3-dependent synaptic vesicle biogenesis. We propose that anatomically restricted mechanisms within the brain diversify the biogenesis and composition of synaptic vesicles.
Assuntos
Proteínas de Transporte/metabolismo , Corpo Estriado/citologia , Hipocampo/citologia , Lectinas/metabolismo , Terminações Pré-Sinápticas/metabolismo , Vesículas Sinápticas/metabolismo , Fatores de Transcrição/metabolismo , Complexo 3 de Proteínas Adaptadoras , Subunidades beta do Complexo de Proteínas Adaptadoras , Animais , Corpo Estriado/metabolismo , Embrião de Mamíferos , Regulação da Expressão Gênica/genética , Hipocampo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Lectinas/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Imunoeletrônica/métodos , Neurônios/citologia , Neurônios/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Proteínas R-SNARE/metabolismo , Ratos , Ratos Sprague-Dawley , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/metabolismo , Sinaptossomos/metabolismo , Sinaptossomos/ultraestrutura , Fatores de Transcrição/deficiênciaRESUMO
GABA transporter subtype 1 (GAT-1) and GABA transporter subtype 3 (GAT-3) are the main transporters that regulate inhibitory GABAergic transmission in the mammalian brain through GABA reuptake. In this study, we characterized the ultrastructural localizations and determined the respective roles of these transporters in regulating evoked inhibitory postsynaptic currents (eIPSCs) in globus pallidus (GP) neurons after striatal stimulation. In the young and adult rat GP, GAT-1 was preferentially expressed in unmyelinated axons, whereas GAT-3 was almost exclusively found in glial processes. Except for rare instances of GAT-1 localization, neither of the two transporters was significantly expressed in GABAergic terminals in the rat GP. 1-(4,4-Diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) (10 µm), a GAT-1 inhibitor, significantly prolonged the decay time, but did not affect the amplitude, of eIPSCs induced by striatal stimulation (15-20 V). On the other hand, the semi-selective GAT-3 inhibitor 1-(2-[tris(4-methoxyphenyl)methoxy]ethyl)-(S)-3-piperidinecarboxylic acid (SNAP 5114) (10 µm) increased the amplitude and prolonged the decay time of eIPSCs. The effects of transporter blockade on the decay time and amplitude of eIPSCs were further increased when both inhibitors were applied together. Furthermore, SKF 89976A or SNAP 5114 blockade also increased the amplitude and frequency of spontaneous IPSCs, but did not affect miniature IPSCs. Significant GABA(A) receptor-mediated tonic currents were induced in the presence of high concentrations of both SKF 89976A (30 µm) and SNAP 5114 (30 µm). In conclusion, these data indicate that GAT-1 and GAT-3 represent different target sites through which GABA reuptake may subserve complementary regulation of GABAergic transmission in the rat GP.
Assuntos
Proteínas da Membrana Plasmática de Transporte de GABA/metabolismo , Globo Pálido/metabolismo , Animais , Anisóis/farmacologia , Bicuculina/farmacologia , Estimulação Elétrica , Feminino , GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A/farmacologia , Globo Pálido/citologia , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Masculino , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Ácidos Nipecóticos/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-DawleyRESUMO
Previous efforts to improve the efficiency of cellular reprogramming for the generation of induced pluripotent stem cells (iPSCs) have focused mainly on transcription factors and small molecule combinations. Here, we report the results of our focus instead on the phenotype of the cells targeted for reprogramming. We find that adult mouse pancreatic tissue stem cells derived by the method of suppression of asymmetric cell kinetics (SACK) acquire increased potency simply by culture under conditions for the production and maintenance of pluripotent stem cells. Moreover, supplementation with the SACK agent xanthine, which promotes symmetric self-renewal, significantly increases the efficiency and degree of acquisition of pluripotency properties. In transplantation analyses, clonal reprogrammed pancreatic stem cells produce slow-growing tumors with tissue derivative of all three embryonic germ layers. This acquisition of pluripotency, without transduction with exogenous transcription factors, supports the concept that tissue stem cells are predisposed to cellular reprogramming, particularly when symmetrically self-renewing.
Assuntos
Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco/fisiologia , Fosfatase Alcalina/metabolismo , Animais , Linhagem Celular , Proliferação de Células , Reprogramação Celular , Feminino , Histocitoquímica , Proteínas de Homeodomínio/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos SCID , Neoplasias Experimentais/patologia , Pâncreas/citologia , Fenótipo , Células-Tronco/citologia , Células-Tronco/metabolismo , Transativadores/metabolismoRESUMO
INTRODUCTION: While studies suggest that innate immune memory acquired by circulating monocytes may mediate the benefit of bacillus Calmette-Guérin (BCG) in the treatment of patients with high-risk non-muscle-invasive bladder cancer (NMIBC), prospective studies are lacking. Innate immune memory is defined by enhanced release of pro-inflammatory cytokines by innate immune cells following a secondary challenge with pattern recognition receptor (PRR) ligands. METHODS: Peripheral blood monocytes isolated from 33 patients with intermediate- or high-risk NMIBC before and after two or five induction BCG instillations were stimulated with the PRR ligand lipopolysaccharide (LPS). Inflammatory cytokine levels in the culture medium were measured. Extent of innate immune memory acquisition was determined by dividing the levels of cytokines released after BCG instillation by the levels released prior to BCG therapy. RESULTS: Monocytes secreted variable levels of TNFα, IL-1ß, IL-6, IFNγ, IL-12, and IL-10. Compared with patients with recurrences, the post-BCG:pre-BCG ratio of IL-12 in monocyte cultures from patients without recurrences after five BCG instillations was significantly increased. Patients with no innate immune memory (based on IL-12 ratios) had significantly shorter time to recurrence than patients with innate immune memory (p<0.001). Eighty-four percent (16/19) of patients with innate immune memory vs. only 22% (2/9) of patients without memory had disease-free survival of over 500 days. CONCLUSIONS: Results demonstrate a potential link between BCG-induced innate immune memory peripherally and local anti-tumor responses. Further validation will increase our understanding of the mode of action of BCG and, therefore, will be used to enhance its effectiveness.