RESUMEN
The transmembrane protein known as the mitochondrial calcium uniporter (MCU) mediates the influx of calcium ions (Ca2+) into the mitochondrial matrix. An overload of mitochondrial Ca2+ ( m Ca2+) is directly linked to damaging effects in pathological conditions. Therefore, inhibitors of the MCU are important chemical biology tools and therapeutic agents. Here, two new analogues of previously reported Ru- and Os-based MCU inhibitors Ru265 and Os245, of the general formula [(C10H15CO2)M(NH3)4(µ-N)M(NH3)4(O2CC10H15)](CF3SO3)3, where M = Ru (1) or Os (2), are reported. These analogues bear adamantane functional groups, which were installed to act as guests for the host molecule cucurbit-[7]-uril (CB[7]). These complexes were characterized and analyzed for their efficiency as guests for CB[7]. As shown through a variety of spectroscopic techniques, each adamantane ligand is encapsulated into one CB[7], affording a supramolecular complex of 1 : 2 stoichiometry. The biological effects of these compounds in the presence and absence of two equiv. CB[7] were assessed. Both complexes 1 and 2 exhibit enhanced cellular uptake compared to the parent compounds Ru265 and Os245, and their uptake is increased further in the presence of CB[7]. Compared to Ru265 and Os245, 1 and 2 are less potent as m Ca2+ uptake inhibitors in permeabilized cell models. However, in intact cell systems, 1 and 2 inhibit the MCU at concentrations as low as 1 µM, marking an advantage over Ru265 and Os245 which require an order of magnitude higher doses for similar biological effects. The presence of CB[7] did not affect the inhibitory properties of 1 and 2. Experiments in primary cortical neurons showed that 1 and 2 can elicit protective effects against oxygen-glucose deprivation at lower doses than those required for Ru265 or Os245. At low concentrations, the protective effects of 1 were modulated by CB[7], suggesting that supramolecular complex formation can play a role in these biological conditions. The in vivo biocompatibility of 1 was investigated in mice. The intraperitoneal administration of these compounds and their CB[7] complexes led to time-dependent induction of seizures with no protective effects elicited by CB[7]. This work demonstrates the potential for supramolecular interactions in the development of MCU inhibitors.
RESUMEN
BACKGROUND AND PURPOSE: Excitotoxicity due to mitochondrial calcium (Ca2+) overloading can trigger neuronal cell death in a variety of pathologies. Inhibiting the mitochondrial calcium uniporter (MCU) has been proposed as a therapeutic avenue to prevent calcium overloading. Ru265 (ClRu(NH3)4(µ-N)Ru(NH3)4Cl]Cl3) is a cell-permeable inhibitor of the mitochondrial calcium uniporter (MCU) with nanomolar affinity. Ru265 reduces sensorimotor deficits and neuronal death in models of ischemic stroke. However, the therapeutic use of Ru265 is limited by the induction of seizure-like behaviours. EXPERIMENTAL APPROACH: We examined the effect of Ru265 on synaptic and neuronal function in acute brain slices and hippocampal neuron cultures derived from mice, in control and where MCU expression was genetically abrogated. KEY RESULTS: Ru265 decreased evoked responses from calyx terminals and induced spontaneous action potential firing of both the terminal and postsynaptic principal cell. Recordings of presynaptic Ca2+ currents suggested that Ru265 blocks the P/Q type channel, confirmed by the inhibition of currents in cells exogenously expressing the P/Q type channel. Measurements of presynaptic K+ currents further revealed that Ru265 blocked a KCNQ current, leading to increased membrane excitability, underlying spontaneous spiking. Ca2+ imaging of hippocampal neurons showed that Ru265 increased synchronized, high-amplitude events, recapitulating seizure-like activity seen in vivo. Importantly, MCU ablation did not suppress Ru265-induced increases in neuronal activity and seizures. CONCLUSIONS AND IMPLICATIONS: Our findings provide a mechanistic explanation for the pro-convulsant effects of Ru265 and suggest counter screening assays based on the measurement of P/Q and KCNQ channel currents to identify safe MCU inhibitors.
Asunto(s)
Canales de Calcio , Neuronas , Compuestos de Rutenio , Transmisión Sináptica , Animales , Canales de Calcio/metabolismo , Canales de Calcio/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Compuestos de Rutenio/farmacología , Ratones , Transmisión Sináptica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Ratones Endogámicos C57BL , Masculino , Células Cultivadas , Calcio/metabolismoRESUMEN
The neurovascular unit (NVU) is composed of vascular cells, glia, and neurons that form the basic component of the blood brain barrier. This intricate structure rapidly adjusts cerebral blood flow to match the metabolic needs of brain activity. However, the NVU is exquisitely sensitive to damage and displays limited repair after a stroke. To effectively treat stroke, it is therefore considered crucial to both protect and repair the NVU. Mitochondrial calcium (Ca2+) uptake supports NVU function by buffering Ca2+ and stimulating energy production. However, excessive mitochondrial Ca2+ uptake causes toxic mitochondrial Ca2+ overloading that triggers numerous cell death pathways which destroy the NVU. Mitochondrial damage is one of the earliest pathological events in stroke. Drugs that preserve mitochondrial integrity and function should therefore confer profound NVU protection by blocking the initiation of numerous injury events. We have shown that mitochondrial Ca2+ uptake and efflux in the brain are mediated by the mitochondrial Ca2+ uniporter complex (MCUcx) and sodium/Ca2+/lithium exchanger (NCLX), respectively. Moreover, our recent pharmacological studies have demonstrated that MCUcx inhibition and NCLX activation suppress ischemic and excitotoxic neuronal cell death by blocking mitochondrial Ca2+ overloading. These findings suggest that combining MCUcx inhibition with NCLX activation should markedly protect the NVU. In terms of promoting NVU repair, nuclear hormone receptor activation is a promising approach. Retinoid X receptor (RXR) and thyroid hormone receptor (TR) agonists activate complementary transcriptional programs that stimulate mitochondrial biogenesis, suppress inflammation, and enhance the production of new vascular cells, glia, and neurons. RXR and TR agonism should thus further improve the clinical benefits of MCUcx inhibition and NCLX activation by increasing NVU repair. However, drugs that either inhibit the MCUcx, or stimulate the NCLX, or activate the RXR or TR, suffer from adverse effects caused by undesired actions on healthy tissues. To overcome this problem, we describe the use of nanoparticle drug formulations that preferentially target metabolically compromised and damaged NVUs after an ischemic or hemorrhagic stroke. These nanoparticle-based approaches have the potential to improve clinical safety and efficacy by maximizing drug delivery to diseased NVUs and minimizing drug exposure in healthy brain and peripheral tissues.
RESUMEN
Dysregulation of mitochondrial calcium uptake mediated by the mitochondrial calcium uniporter (MCU) is implicated in several pathophysiological conditions. Dinuclear ruthenium complexes are effective inhibitors of the MCU and have been leveraged as both tools to study mitochondrial calcium dynamics and potential therapeutic agents. In this study, we report the synthesis and characterization of Os245 ([Os2(µ-N)(NH3)8Cl2]3+) which is the osmium-containing analogue of our previously reported ruthenium-based inhibitor Ru265. This complex and its aqua-capped analogue Os245' ([Os2(µ-N)(NH3)8(OH2)2]5+) are both effective inhibitors of the MCU in permeabilized and intact cells. In comparison to the ruthenium-based inhibitor Ru265 (k obs = 4.92 × 10-3 s-1), the axial ligand exchange kinetics of Os245 are two orders of magnitude slower (k obs = 1.63 × 10-5 s-1) at 37 °C. The MCU-inhibitory properties of Os245 and Os245' are different (Os245 IC50 for MCU inhibition = 103 nM; Os245' IC50 for MCU inhibition = 2.3 nM), indicating that the axial ligands play an important role in their interactions with this channel. We further show that inhibition of the MCU by these complexes protects primary cortical neurons against lethal oxygen-glucose deprivation. When administered in vivo to mice (10 mg kg-1), Os245 and Os245' induce seizure-like behaviors in a manner similar to the ruthenium-based inhibitors. However, the onset of these seizures is delayed, a possible consequence of the slower ligand substitution kinetics for these osmium complexes. These findings support previous studies that demonstrate inhibition of the MCU is a promising therapeutic strategy for the treatment of ischemic stroke, but also highlight the need for improved drug delivery strategies to mitigate the pro-convulsant effects of this class of complexes before they can be implemented as therapeutic agents. Furthermore, the slower ligand substitution kinetics of the osmium analogues may afford new strategies for the development and modification of this class of MCU inhibitors.
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Fingolimod, used to treat relapsing-remitting multiple sclerosis (RRMS), reduces motor deficits in mice with established experimental autoimmune encephalomyelitis (EAE). To better characterize the therapeutic effects of fingolimod, kinematic gait analysis was employed to precisely measure movements of a hindleg while EAE mice walked on a treadmill. Relative to the vehicle group, oral dosing with fingolimod, beginning after disease onset (1 mg/kg/day), increased hip heights and knee joint movements, and reduced spinal cord demyelination. These findings suggest that fingolimod preserves gait in RRMS patients by protecting motor circuits in the spinal cord.
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Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple Recurrente-Remitente , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Clorhidrato de Fingolimod/farmacología , Clorhidrato de Fingolimod/uso terapéutico , Marcha , Ratones , Ratones Endogámicos C57BL , Médula EspinalRESUMEN
CHKB encodes one of two mammalian choline kinase enzymes that catalyze the first step in the synthesis of the membrane phospholipid phosphatidylcholine. In humans and mice, inactivation of the CHKB gene (Chkb in mice) causes a recessive rostral-to-caudal muscular dystrophy. Using Chkb knockout mice, we reveal that at no stage of the disease is phosphatidylcholine level significantly altered. We observe that in affected muscle a temporal change in lipid metabolism occurs with an initial inability to utilize fatty acids for energy via mitochondrial ß-oxidation resulting in shunting of fatty acids into triacyglycerol as the disease progresses. There is a decrease in peroxisome proliferator-activated receptors and target gene expression specific to Chkb-/- affected muscle. Treatment of Chkb-/- myocytes with peroxisome proliferator-activated receptor agonists enables fatty acids to be used for ß-oxidation and prevents triacyglyerol accumulation, while simultaneously increasing expression of the compensatory choline kinase alpha (Chka) isoform, preventing muscle cell injury.
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Enfermedades Musculares , Distrofias Musculares , Animales , Colina Quinasa/genética , Colina Quinasa/metabolismo , Ácidos Grasos , Metabolismo de los Lípidos/genética , Mamíferos/metabolismo , Ratones , Distrofias Musculares/genética , Distrofias Musculares/terapia , Fosfatidilcolinas/metabolismoRESUMEN
Blooms of the diatom genus Pseudo-nitzschia occur annually in the Southern California Bight (SCB), and domoic acid (DA) associated with these events can contaminate fisheries, presenting both human and wildlife health risks. Recent studies have suggested that marine sediments may act as a reservoir for DA, extending the risk of food web contamination long after water column blooms have ended. In this study, we conducted a regional assessment of the extent and magnitude of DA in the benthic environment, and monthly observations of sediments and benthic infauna at multiple stations over a 16-month period. DA was widespread in continental shelf sediments of the SCB. The toxin was detected in 54% of all shelf habitats sampled. Detectable concentrations ranged from 0.11 ng/g to 1.36 ng/g. DA was consistently detected in benthic infauna tissues over the monthly timeseries, while the DA concentrations in sediments during the same period were commonly below detection or at low concentrations. The presence of DA in the benthic environment did not always have an apparent water column source, raising the possibility of lateral transport, retention/preservation in sediments or undetected blooms in subsurface waters. In most cases, DA was detected in tissues but not in the co-located surface sediments. Coarse taxonomic sorting of the infauna revealed that the accumulation of DA varied among taxa. We observed that DA was widespread among lower trophic level organisms in this study, potentially acting as a persistent source of DA to higher trophic levels in the benthos.
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Diatomeas , Ácido Kaínico , Cadena Alimentaria , Sedimentos Geológicos , Ácido Kaínico/análogos & derivadosRESUMEN
Claudin (Cldn)-10 tight junction (TJ) proteins are hypothesized to form the paracellular Na+ secretion pathway of hyposmoregulating mummichog (Fundulus heteroclitus) branchial epithelia. Organ-specific expression profiles showed that only branchial organs [the gill and opercular epithelium (OE)] exhibited abundant cldn-10 paralog transcripts, which typically increased following seawater (SW) to hypersaline (2SW) challenge. Post-translational properties, protein abundance, and ionocyte localization of Cldn-10c, were then examined in gill and OE. Western blot analysis revealed two Cldn-10c immunoreactive bands in the mummichog gill and OE at â¼29â kDa and â¼40â kDa. The heavier protein could be eliminated by glycosidase treatment, demonstrating the novel presence of a glycosylated Cldn-10c. Protein abundance of Cldn-10c increased in gill and OE of 2SW-exposed fish. Cldn-10c localized to the sides of gill and OE ionocyte apical crypts and partially colocalized with cystic fibrosis transmembrane conductance regulator and F-actin, consistent with TJ complex localization. Cldn-10c immunofluorescent intensity increased but localization was unaltered by 2SW conditions. In support of our hypothesis, cldn-10/Cldn-10 TJ protein dynamics in gill and OE of mummichogs and TJ localization are functionally consistent with the creation and maintenance of salinity-responsive, cation-selective pores that facilitate Na+ secretion in hyperosmotic environments.
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Aclimatación/fisiología , Claudinas/metabolismo , Epitelio/metabolismo , Iones/metabolismo , Lóbulo Temporal/metabolismo , Animales , Fundulidae , SalinidadRESUMEN
Sleep and circadian rhythm disruptions commonly occur in individuals with schizophrenia. Stable tubule only polypeptide (STOP) knockout (KO) mice show behavioral impairments resembling symptoms of schizophrenia. We previously reported that STOP KO mice slept less and had more fragmented sleep and waking than wild-type littermates under a light/dark (LD) cycle. Here, we assessed the circadian phenotype of male STOP KO mice by examining wheel-running activity rhythms and EEG/EMG-defined sleep/wake states under both LD and constant darkness (DD) conditions. Wheel-running activity rhythms in KO and wild-type mice were similarly entrained in LD, and had similar free-running periods in DD. The phase delay shift in response to a light pulse given early in the active phase under DD was preserved in KO mice. KO mice had markedly lower activity levels, lower amplitude activity rhythms, less stable activity onsets, and more fragmented activity than wild-type mice in both lighting conditions. KO mice also spent more time awake and less time in rapid eye movement sleep (REMS) and non-REMS (NREMS) in both LD and DD conditions, with the decrease in NREMS concentrated in the active phase. KO mice also showed altered EEG features and higher amplitude rhythms in wake and NREMS (but not REMS) amounts in both lighting conditions, with a longer free-running period in DD, compared to wild-type mice. These results indicate that the STOP null mutation in mice altered the regulation of sleep/wake physiology and activity rhythm expression, but did not grossly disrupt circadian mechanisms.
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Proteínas Asociadas a Microtúbulos/genética , Esquizofrenia , Animales , Ritmo Circadiano/genética , Oscuridad , Masculino , Ratones , Actividad Motora , Péptidos , Esquizofrenia/genética , SueñoRESUMEN
High-capacity mitochondrial calcium (Ca2+) uptake by the mitochondrial Ca2+ uniporter (MCU) is strategically positioned to support the survival and remyelination of axons in multiple sclerosis (MS) by undocking mitochondria, buffering Ca2+ and elevating adenosine triphosphate (ATP) synthesis at metabolically stressed sites. Respiratory chain deficits in MS are proposed to metabolically compromise axon survival and remyelination by suppressing MCU activity. In support of this hypothesis, clinical scores, mitochondrial dysfunction, myelin loss, axon damage and inflammation were elevated while remyelination was blocked in neuronal MCU deficient (Thy1-MCU Def) mice relative to Thy1 controls subjected to experimental autoimmune encephalomyelitis (EAE). At the first sign of walking deficits, mitochondria in EAE/Thy1 axons showed signs of activation. By contrast, cytoskeletal damage, fragmented mitochondria and large autophagosomes were seen in EAE/Thy1-MCU Def axons. As EAE severity increased, EAE/Thy1 axons were filled with massively swollen mitochondria with damaged cristae while EAE/Thy1-MCU Def axons were riddled with late autophagosomes. ATP concentrations and mitochondrial gene expression were suppressed while calpain activity, autophagy-related gene mRNA levels and autophagosome marker (LC3) co-localization in Thy1-expressing neurons were elevated in the spinal cords of EAE/Thy1-MCU Def compared to EAE/Thy1 mice. These findings suggest that MCU inhibition contributes to axonal damage that drives MS progression.
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Canales de Calcio/deficiencia , Encefalomielitis Autoinmune Experimental/patología , Mitocondrias/metabolismo , Proteínas Mitocondriales/deficiencia , Vaina de Mielina/patología , Neuronas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Autofagia/genética , Axones/patología , Canales de Calcio/genética , Trastornos Neurológicos de la Marcha/genética , Trastornos Neurológicos de la Marcha/patología , Expresión Génica/genética , Masculino , Ratones , Ratones Noqueados , Proteínas Mitocondriales/genética , Dilatación Mitocondrial , Fagosomas/patología , Médula Espinal/patologíaRESUMEN
Chronic sleep restriction (CSR) negatively impacts brain functions. Whether microglia, the brain's resident immune cells, play any role is unknown. We studied microglia responses to CSR using a rat model featuring slowly rotating wheels (3 h on/1 h off), which was previously shown to induce both homeostatic and adaptive responses in sleep and attention. Adult male rats were sleep restricted for 27 or 99 h. Control rats were housed in locked wheels. After 27 and/or 99 h of CSR, the number of cells immunoreactive for the microglia marker ionized calcium-binding adaptor molecule-1 (Iba1) and the density of Iba1 immunoreactivity were increased in 4/10 brain regions involved in sleep/wake regulation and cognition, including the prelimbic cortex, central amygdala, perifornical lateral hypothalamic area, and dorsal raphe nucleus. CSR neither induced mitosis in microglia (assessed with bromodeoxyuridine) nor impaired blood-brain barrier permeability (assessed with Evans Blue). Microglia appeared ramified in all treatment groups and, when examined quantitatively in the prelimbic cortex, their morphology was not affected by CSR. After 27 h, but not 99 h, of CSR, mRNA levels of the anti-inflammatory cytokine interleukin-10 were increased in the frontal cortex. Pro-inflammatory cytokine mRNA levels (tumor necrosis factor-α, interleukin-1ß, and interleukin-6) were unchanged. Furthermore, cortical microglia were not immunoreactive for several pro- and anti-inflammatory markers tested, but were immunoreactive for the purinergic P2Y12 receptor. These results suggest that microglia respond to CSR while remaining in a physiological state and may contribute to the previously reported homeostatic and adaptive responses to CSR.
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Microglía , Privación de Sueño , Animales , Encéfalo , Homeostasis , Masculino , Ratas , SueñoRESUMEN
The mitochondrial calcium (Ca2+) uniporter (MCU) mediates high-capacity mitochondrial Ca2+ uptake implicated in ischemic/reperfusion cell death. We have recently shown that inducible MCU ablation in Thy1-expressing neurons renders mice resistant to sensorimotor deficits and forebrain neuron loss in a model of hypoxic/ischemic (HI) brain injury. These findings encouraged us to compare the neuroprotective effects of Ru360 and the recently identified cell permeable MCU inhibitor Ru265. Unlike Ru360, Ru265 (2-10 µM) reached intracellular concentrations in cultured cortical neurons that preserved cell viability, blocked the protease activity of Ca2+-dependent calpains and maintained mitochondrial respiration and glycolysis after a lethal period of oxygen-glucose deprivation (OGD). Intraperitoneal (i.p.) injection of adult male C57Bl/6 mice with Ru265 (3 mg/kg) also suppressed HI-induced sensorimotor deficits and brain injury. However, higher doses of Ru265 (10 and 30 mg/kg, i.p.) produced dose-dependent increases in the frequency and duration of seizure-like behaviours. Ru265 is proposed to promote convulsions by reducing Ca2+ buffering and energy production in highly energetic interneurons that suppress brain seizure activity. These findings support the therapeutic potential of MCU inhibition in the treatment of ischemic stroke but also indicate that such clinical translation will require drug delivery strategies which mitigate the pro-convulsant effects of Ru265.
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Canales de Calcio/efectos de los fármacos , Hipoxia-Isquemia Encefálica/metabolismo , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Compuestos de Rutenio/farmacología , Animales , Canales de Calcio/metabolismo , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Glucosa/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Oxígeno/metabolismo , Convulsiones/inducido químicamenteRESUMEN
In a companion paper, the structural integrity, conformational stability, and degradation mechanisms of 3 recombinant fusion-protein antigens comprising a non-replicating rotavirus (NRRV) vaccine candidate (currently being evaluated in early-stage clinical trials) are described. In this work, we focus on the aggregation propensity of the 3 NRRV antigens coupled to formulation development studies to identify common frozen bulk candidate formulations. The P2-VP8-P[8] antigen was most susceptible to shaking and freeze-thaw-induced aggregation and particle formation. Each NRRV antigen formed aggregates with structurally altered protein (with exposed apolar regions and intermolecular ß-sheet) and dimers containing a non-native disulfide bond. From excipient screening studies with P2-VP8-P[8], sugars or polyols (e.g., sucrose, trehalose, mannitol, sorbitol) and various detergents (e.g., Pluronic F-68, polysorbate 20 and 80, PEG-3350) were identified as stabilizers against aggregation. By combining promising additives, candidate bulk formulations were optimized to not only minimize agitation-induced aggregation, but also particle formation due to freeze-thaw stress of P2-VP8-P[8] antigen. Owing to limited material availability, stabilization of the P2-VP8-P[4] and P2-VP8-P[6] was confirmed with the lead candidate P2-VP8-P[8] formulations. The optimization of these bulk NRRV candidate formulations is discussed in the context of subsequent drug product formulations in the presence of aluminum adjuvants.
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Antígenos Virales/química , Excipientes/química , Agregado de Proteínas , Proteínas Recombinantes de Fusión/química , Vacunas contra Rotavirus/química , Composición de Medicamentos , Estabilidad de Medicamentos , Almacenaje de Medicamentos , Drogas en Investigación/química , Congelación , Tamaño de la Partícula , Estabilidad Proteica , Vacunas de Subunidad/químicaRESUMEN
Although live attenuated Rotavirus (RV) vaccines are available globally to provide protection against enteric RV disease, efficacy is substantially lower in low- to middle-income settings leading to interest in alternative vaccines. One promising candidate is a trivalent nonreplicating RV vaccine, comprising 3 truncated RV VP8 subunit proteins fused to the P2 CD4+ epitope from tetanus toxin (P2-VP8-P[4/6/8]). A wide variety of analytical techniques were used to compare the physicochemical properties of these 3 recombinant fusion proteins. Various environmental stresses were used to evaluate antigen stability and elucidate degradation pathways. P2-VP8-P[4] and P2-VP8-P[6] displayed similar physical stability profiles as function of pH and temperature while P2-VP8-P[8] was relatively more stable. Forced degradation studies revealed similar chemical stability profiles with Met1 most susceptible to oxidation, the single Cys residue (at position 173/172) forming intermolecular disulfide bonds (P2-VP8-P[6] was most susceptible), and Asn7 undergoing the highest levels of deamidation. These results are visualized in a structural model of the nonreplicating RV antigens. The establishment of key structural attributes of each antigen, along with corresponding stability-indicating methods, have been applied to vaccine formulation development efforts (see companion paper), and will be utilized in future analytical comparability assessments.
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Antígenos Virales/genética , Infecciones por Rotavirus/prevención & control , Vacunas contra Rotavirus , Rotavirus/inmunología , Composición de Medicamentos , Estabilidad de Medicamentos , Escherichia coli/genética , Proteínas Recombinantes de Fusión/genética , Vacunas contra Rotavirus/química , Vacunas contra Rotavirus/genética , Vacunas contra Rotavirus/inmunología , Vacunas de Subunidad/química , Vacunas de Subunidad/genética , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/química , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Proteínas no Estructurales Virales/genéticaRESUMEN
Algal toxins (domoic acid, saxitoxin, okadaic acid) were monitored at seven locations off southern California using Solid Phase Adsorption Toxin Tracking. At least two types of toxins were found at all locations, with co-occurrence of two and three toxins in 12% and 10% of samples, respectively. This study expands our limited understanding of the simultaneous presence of multiple algal toxins along the coast and raises questions regarding the potential health ramifications of such co-occurrences.
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Ácido Kaínico/análogos & derivados , Ácido Ocadaico/análisis , Saxitoxina/análisis , Adsorción , California , Monitoreo del Ambiente , Floraciones de Algas Nocivas , Ácido Kaínico/análisis , Toxinas Marinas/análisis , Agua de Mar/químicaRESUMEN
Experimental autoimmune encephalomyelitis (EAE) and lysophosphatidylcholine (LPC)-induced demyelination were combined to study remyelination in a pro-inflammatory context. Two groups of female C57BL/6 mice were subjected either to EAE (EAE mice) or injected with just complete Freund's adjuvant (CFA) and pertussis toxin (PTX) followed by bilateral LPC and phosphate buffered saline injections in the corpus callosum on day 7 (CFA controls). Relative to CFA controls, EAE accelerated remyelination and increased innate immune cell activation, lymphocyte infiltration and cytokine gene expression in the LPC lesions. However, compared to CFA mice, remyelination was reduced (day 14) suggesting this aggressive immune response also compromised myelin repair in EAE mice.
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Cuerpo Calloso/inmunología , Enfermedades Desmielinizantes/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Inmunidad Innata/inmunología , Lisofosfatidilcolinas/toxicidad , Remielinización/inmunología , Animales , Cuerpo Calloso/efectos de los fármacos , Cuerpo Calloso/patología , Enfermedades Desmielinizantes/inducido químicamente , Enfermedades Desmielinizantes/patología , Encefalomielitis Autoinmune Experimental/inducido químicamente , Encefalomielitis Autoinmune Experimental/patología , Femenino , Adyuvante de Freund/toxicidad , Inmunidad Innata/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Remielinización/efectos de los fármacosRESUMEN
BACKGROUND: Paired exercise and cognitive training have the potential to enhance cognition by "priming" the brain and upregulating neurotrophins. METHODS: Two-site randomized controlled trial. Fifty-two patients >6 months poststroke with concerns about cognitive impairment trained 50 to 70 minutes, 3× week for 10 weeks with 12-week follow-up. Participants were randomized to 1 of 2 physical interventions: Aerobic (>60% VO2peak using <10% body weight-supported treadmill) or Activity (range of movement and functional tasks). Exercise was paired with 1 of 2 cognitive interventions (computerized dual working memory training [COG] or control computer games [Games]). The primary outcome for the 4 groups (Aerobic + COG, Aerobic + Games, Activity + COG, and Activity + Games) was fluid intelligence measured using Raven's Progressive Matrices Test administered at baseline, posttraining, and 3-month follow-up. Serum neurotrophins collected at one site (N = 30) included brain-derived neurotrophic factor (BDNF) at rest (BDNFresting) and after a graded exercise test (BDNFresponse) and insulin-like growth factor-1 at the same timepoints (IGF-1rest, IGF-1response). RESULTS: At follow-up, fluid intelligence scores significantly improved compared to baseline in the Aerobic + COG and Activity + COG groups; however, only the Aerobic + COG group was significantly different (+47.8%) from control (Activity + Games -8.5%). Greater IGF-1response at baseline predicted 40% of the variance in cognitive improvement. There was no effect of the interventions on BDNFresting or BDNFresponse; nor was BDNF predictive of the outcome. CONCLUSIONS: Aerobic exercise combined with cognitive training improved fluid intelligence by almost 50% in patients >6 months poststroke. Participants with more robust improvements in cognition were able to upregulate higher levels of serum IGF-1 suggesting that this neurotrophin may be involved in behaviorally induced plasticity.
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Terapia Cognitivo-Conductual/métodos , Terapia por Ejercicio/métodos , Factor I del Crecimiento Similar a la Insulina/análisis , Inteligencia , Rehabilitación de Accidente Cerebrovascular/métodos , Accidente Cerebrovascular/psicología , Anciano , Enfermedad Crónica/psicología , Enfermedad Crónica/rehabilitación , Terapia Combinada/métodos , Femenino , Humanos , Masculino , Persona de Mediana Edad , Accidente Cerebrovascular/sangre , Resultado del TratamientoRESUMEN
Recent evidence suggests that clozapine and quetiapine (atypical antipsychotics), tamoxifen (selective-estrogen receptor modulator) and pioglitazone (PPARγ agonist) may improve functional recovery in multiple sclerosis (MS). We have compared the effectiveness of oral administration of these drugs, beginning at peak disease, at reducing ascending paralysis, motor deficits and demyelination in mice subjected to experimental autoimmune encephalomyelitis (EAE). Mice were immunized with an immunogenic peptide corresponding to amino acids 35-55 of the myelin oligodendrocyte glycoprotein (MOG35-55) in complete Freund's adjuvant and injected with pertussis toxin to induce EAE. Unlike clozapine, quetiapine and tamoxifen, administration of pioglitazone beginning at peak disease decreased both clinical scores and lumbar white matter loss in EAE mice. Using kinematic gait analysis, we found that pioglitazone also maintained normal movement of the hip, knee and ankle joints for at least 44â¯days after MOG35-55 immunization. This long-lasting preservation of hindleg joint movements was accompanied by reduced white matter loss, microglial and macrophage activation and the expression of pro-inflammatory genes in the lumbar spinal cords of EAE mice. These results support clinical findings that suggest pioglitazone may reduce the progressive loss of motor function in MS by decreasing inflammation and myelin damage.
Asunto(s)
Clozapina/administración & dosificación , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Pioglitazona/administración & dosificación , Fumarato de Quetiapina/administración & dosificación , Tamoxifeno/administración & dosificación , Administración Oral , Animales , Antidepresivos/administración & dosificación , Encefalomielitis Autoinmune Experimental/metabolismo , Antagonistas de Estrógenos/administración & dosificación , Femenino , Hipoglucemiantes/administración & dosificación , Mediadores de Inflamación/antagonistas & inhibidores , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Antagonistas de la Serotonina/administración & dosificación , Resultado del TratamientoRESUMEN
The mitochondrial calcium uniporter (MCU) mediates high-capacity mitochondrial calcium uptake that stimulates energy production. However, excessive MCU activity can cause ischemic heart injury. To examine if the MCU is also involved in hypoxic/ischemic (HI) brain injury, we have generated conditional MCU knockout mice by tamoxifen (TMX) administration to adult MCU-floxed (MCUfl/fl) mice expressing a construct encoding Thy1-cre/ERT2-eYFP. Relative to TMX/Thy1-cre/ERT2-eYFP controls, HI-induced sensorimotor deficits, forebrain neuron loss and mitochondrial damage were decreased for conditional MCU knockout mice. MCU knockdown by siRNA-induced silencing in cortical neuron cultures also reduced cell death and mitochondrial respiratory deficits following oxygen-glucose deprivation. Furthermore, MCU silencing did not produce metabolic abnormalities in cortical neurons observed previously for global MCU nulls that increased reliance on glycolysis for energy production. Based on these findings, we propose that brain-penetrant MCU inhibitors have strong potential to be well-tolerated and highly-efficacious neuroprotectants for the acute management of ischemic stroke.
Asunto(s)
Canales de Calcio/metabolismo , Técnicas de Silenciamiento del Gen , Hipoxia-Isquemia Encefálica/metabolismo , Hipoxia-Isquemia Encefálica/prevención & control , Neuronas/metabolismo , Tamoxifeno/farmacología , Antígenos Thy-1/metabolismo , Animales , Canales de Calcio/deficiencia , Respiración de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Glucosa/deficiencia , Glucólisis/efectos de los fármacos , Hipoxia-Isquemia Encefálica/fisiopatología , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/patología , Oxígeno , Fosforilación/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from freshwater (FW) to seawater (SW, 35) and from SW to hypersaline SW (2SW, 60) in a time course with transfer control groups (FW to FW, and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and cldn-10e twofold, whilst cldn-10c and cldn-10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d, and transiently altered cldn-10e abundance, but increased cldn-10c and cldn-10f fourfold. This was coincident with an increased number of single-stranded junctions (observed by transmission electron microscopy). For both salinity transfers, (1) cldn-10e mRNA was acutely responsive (i.e. after 24â h), (2) other responsive cldn-10 isoforms increased later (3-7â days), and (3) cystic fibrosis transmembrane conductance regulator (cftr) mRNA was elevated in accordance with established changes in transcellular Cl- movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and cldn-10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na+>K+>Rb+â¼Cs+>Li+ Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes.