Inflammatory Milieu Induces Mitochondrial Alterations and Neuronal Activations in Hypothalamic POMC Neurons in a Time-Dependent Manner.

Inflammation has been associated with numerous neurological disorders. Inflammatory environments trigger a series of cellular and physiological alterations in the brain. However, how inflammatory milieu affects neuronal physiology and how neuronal alterations progress in the inflammatory environments are not fully understood. In this study, we examined the effects of pro-inflammatory milieu on mitochondrial functions and neuronal activities in the hypothalamic POMC neurons. Treating mHypoA-POMC/GFP1 with the conditioned medium collected from LPS activated macrophage were employed to mimic the inflammatory milieu during hypothalamic inflammation. After a 24-h treatment, intracellular ROS/RNS levels were elevated, and the antioxidant enzymes were reduced. Mitochondrial respiration and mitochondrial functions, including basal respiratory rate, spared respiration capacity, and maximal respiration, were all significantly compromised by inflammatory milieu. Moreover, pro-inflammatory cytokines altered mitochondrial dynamics in a time-dependent manner, resulting in the elongation of mitochondria in POMC neurons after a 24-h treatment. Additionally, the increase of C-Fos and Pomc genes expression indicated that the neurons were activated upon the stimulation of inflammatory environment. This neuronal activation of were confirmed on the LPS-challenged mice. Collectively, a short-term to midterm exposure to inflammatory milieu stimulated metabolic switch and neuronal activation, whereas chronic exposure triggered the elevation of oxidative stress, the decrease of the mitochondrial respiration, and the alterations of mitochondrial dynamics.

AMP-activated protein kinase mediates lipopolysaccharide-induced proinflammatory responses and elevated bone resorption in differentiated osteoclasts.

Systemic and intracellular metabolic states are critical factors affecting immune cell functions. The metabolic regulator AMP-activated protein kinase (AMPK) senses AMP levels and mediates cellular responses to energy-restrained conditions. The ubiquitously expressed AMPK participates in various biological functions in numerous cell types, including innate immune cell macrophages and osteoclasts, which are their specialized derivatives in bone tissues. Previous studies have demonstrated that the activation of AMPK promotes macrophage polarization toward anti-inflammatory M2 status. Additionally, AMPK acts as a negative regulator of osteoclastogenesis, and upregulation of AMPK disrupts the differentiation of osteoclasts. However, the regulation and roles of AMPK in differentiated osteoclasts have not been characterized. Here, we report that inflammatory stimuli-regulated-AMPK activation of differentiated and undifferentiated osteoclasts in opposite ways. Lipopolysaccharide (LPS) inhibited the phosphorylation of AMPK in macrophages and undifferentiated osteoclasts, but it activated AMPK in differentiated osteoclasts. Inactivating AMPK decreased cellular responses against the activation of toll-like receptor signaling, including the transcriptional activation of proinflammatory cytokines and the bone resorption genes TRAP, and MMP9. The elevation of bone resorption by LPS stimulation was disrupted by AMPK inhibitor, indicating the pivotal roles of AMPK in inflammation-induced activities in differentiated osteoclasts. The AMPK activator metformin did not increase proinflammatory responses, possibly because other factors are also required for this regulation. Notably, changing the activation status of AMPK did not alter the expression levels of bone resorption genes in unstimulated osteoclasts, indicating the essential roles of AMPK in cellular responses to inflammatory stimuli but not in the maintenance of basal levels. Unlike its M2-polarizing roles in macrophages, AMPK was not responsive to the M2 stimulus of interleukin-4. Our observations revealed differences in the cellular properties of macrophages and osteoclasts as well as the complexity of regulatory mechanisms for osteoclast functions.

Accumulation of cholesterol suppresses oxidative phosphorylation and altered responses to inflammatory stimuli of macrophages.

Hypercholesterolemia induces intracellular accumulation of cholesterol in macrophages and other immune cells, causing immunological dysfunctions. On cellular levels, cholesterol enrichment might lead to mitochondrial metabolic reprogramming and change macrophage functions. Additionally, as cholesterol is permeable to the plasma membrane and might integrate into the membranous organelles, such as endoplasmic reticulum or mitochondria, cholesterol enrichment might change the functions or properties of these organelles, and ultimately alters the cellular functions. In this study, we investigate the mitochondrial alterations and intracellular oxidative stress induced by accumulation of cholesterol in the macrophages, and the possible immunological impacts caused by these alterations. Macrophage cells RAW264.7 were treated with cholesterol to induce intracellular accumulation of cholesterol, which further triggered the reduced production of reactive oxygen/nitrogen species, as well as decrease of oxidative phosphorylation. Basal respiration rate, ATP production and non-mitochondrial oxygen consumption are all suppressed. In contrast, glycolysis remained unaltered in this cholesterol-enriched condition. Previous studies demonstrated that metabolic profiles are associated with macrophage polarization. We further verified whether this metabolic reprogramming influences the macrophage responses to pro-inflammatory or anti-inflammatory stimuli. Our results showed the changes of transcriptional regulations in both pro-inflammatory and anti-inflammatory genes, but not specific toward M1 or M2 polarization. Collectively, the accumulation of cholesterol induced mitochondrial metabolic reprogramming and suppressed the production of oxidative stress, and induced the alterations of macrophage functions.

4-Methylmethcathinone (mephedrone): neuropharmacological effects of a designer stimulant of abuse.

The designer stimulant 4-methylmethcathinone (mephedrone) is among the most popular of the derivatives of the naturally occurring psychostimulant cathinone. Mephedrone has been readily available for legal purchase both online and in some stores and has been promoted by aggressive Web-based marketing. Its abuse in many countries, including the United States, is a serious public health concern. Owing largely to its recent emergence, there are no formal pharmacodynamic or pharmacokinetic studies of mephedrone. Accordingly, the purpose of this study was to evaluate effects of this agent in a rat model. Results revealed that, similar to methylenedioxymethamphetamine, methamphetamine, and methcathinone, repeated mephedrone injections (4× 10 or 25 mg/kg s.c. per injection, 2-h intervals, administered in a pattern used frequently to mimic psychostimulant "binge" treatment) cause a rapid decrease in striatal dopamine (DA) and hippocampal serotonin (5-hydroxytryptamine; 5HT) transporter function. Mephedrone also inhibited both synaptosomal DA and 5HT uptake. Like methylenedioxymethamphetamine, but unlike methamphetamine or methcathinone, repeated mephedrone administrations also caused persistent serotonergic, but not dopaminergic, deficits. However, mephedrone caused DA release from a striatal suspension approaching that of methamphetamine and was self-administered by rodents. A method was developed to assess mephedrone concentrations in rat brain and plasma, and mephedrone levels were determined 1 h after a binge treatment. These data demonstrate that mephedrone has a unique pharmacological profile with both abuse liability and neurotoxic potential.

Methamphetamine-induced dopamine transporter complex formation and dopaminergic deficits: the role of D2 receptor activation.

Methamphetamine (METH) abuse is a serious public health issue. Of particular concern are findings that repeated high-dose administrations of METH cause persistent dopaminergic deficits in rodents, nonhuman primates, and humans. Previous studies have also revealed that METH treatment causes alterations in the dopamine transporter (DAT), including the formation of higher molecular mass DAT-associated complexes. The current study extends these findings by examining mechanisms underlying DAT complex formation. The association among DAT complex formation and other METH-induced phenomena, including alterations in vesicular monoamine transporter 2 (VMAT2) immunoreactivity, astrocytic activation [as assessed by increased glial fibrillary acidic protein (GFAP) immunoreactivity], and persistent dopaminergic deficits was also explored. Results revealed that METH-induced DAT complex formation and reductions in VMAT2 immunoreactivity precede increases in GFAP immunoreactivity. Furthermore, and as reported previously for DAT complexes, pretreatment with the D2 receptor antagonist eticlopride [S-(-)-3-chloro-5-ethyl-N-[(1-ethyl-2-pyrrolidinyl)methyl]-6-hydroxy-2-methoxybenzamide hydrochloride] attenuated the decrease in VMAT2 immunoreactivity as assessed 24 h after METH treatment. DAT complexes distinct from those present 24 h after METH treatment, decreases in VMAT2 immunoreactivity, and increased GFAP immunoreactivity were present 48 to 72 h after METH treatment. Pretreatment with eticlopride attenuated each of these phenomena. Finally, DAT complexes were present 7 days after METH treatment, a time point at which VMAT2 and DAT monomer immunoreactivity were also reduced. Eticlopride pretreatment attenuated each of these phenomena. These findings provide novel insight into not only receptor-mediated mechanisms underlying the effects of METH but also the interaction among factors that probably are associated with the persistent dopaminergic deficits caused by the stimulant.

Methamphetamine alters vesicular monoamine transporter-2 function and potassium-stimulated dopamine release.

This report demonstrates that a repeated 'challenge' high-dose methamphetamine (METH) injection regimen rapidly decreases striatal K(+)-stimulated dopamine (DA) release concurrent with decreases in both synaptosomal membrane-associated (referred to herein as membrane-associated) and previously reported decreases in non-synaptosomal membrane-associated (presumably cytoplasmic) vesicular DA uptake and content. Resembling previously reported effects involving cytoplasmic vesicles wherein uptake was decreased 48 h after treatment, the decrease in membrane-associated uptake persisted 72 h. Cytoplasmic and membrane-associated vesicular DA uptakes were decreased 7 days after the challenge regimen. A single METH injection also rapidly decreased K(+)-stimulated DA release, membrane-associated DA content, and membrane-associated DA uptake; however, unlike after the challenge regimen, the decrease in uptake recovered by 24 h. Pre-treatment with the D(2) receptor antagonist, eticlopride, did not attenuate the decrease in membrane-associated uptake as assessed 1 h after either a single or challenge treatment. However, eticlopride attenuated the decrease in membrane-associated uptake caused by the challenge regimen as assessed 24 h later. These data reveal complex effects of METH on vesicular function that vary according to the vesicle population under study, dosing regimen, and time after treatment. These may contribute to both the decrease in K(+)-stimulated DA release and the persistent dopaminergic deficits caused by METH.

Differential regional effects of methamphetamine on dopamine transport.

Multiple high-dose methamphetamine administrations cause long-lasting (>1 week) deficits in striatal dopaminergic neuronal function. This stimulant likewise causes rapid (within 1 h) and persistent (at least 48 h) decreases in activities of striatal: 1) dopamine transporters, as assessed in synaptosomes; and 2) vesicular monoamine transporter -2 (VMAT-2), as assessed in a non-membrane-associated (referred to herein as cytoplasmic) vesicular subcellular fraction. Importantly, not all brain areas are vulnerable to methamphetamine-induced long-lasting deficits. Similarly, the present study indicates that methamphetamine exerts differential acute effects on monoaminergic transporters according to brain region. In particular, results revealed that in the nucleus accumbens, methamphetamine rapidly, but reversibly (within 24 h), decreased plasmalemmal dopamine transporter function, without effect on plasmalemmal dopamine transporter immunoreactivity. Methamphetamine also rapidly and reversibly (within 48 h) decreased cytoplasmic VMAT-2 function in this region, with relatively little effect on cytoplasmic VMAT-2 immunoreactivity. In contrast, methamphetamine did not alter either dopamine transporter or VMAT-2 activity in the hypothalamus. Noteworthy, the nucleus accumbens and hypothalamus did not display the persistent long-lasting striatal dopamine depletions caused by the stimulant. Taken together, these data suggest that deficits in plasmalemmal and vesicular monoamine transporter activity lasting greater than 24-48 h may be linked to the long-lasting dopaminergic deficits caused by methamphetamine and appear to be region specific.