Efficient excitatory transmission depends on a family of transporters that utilize the Na+-electrochemical gradient to maintain low synaptic concentrations of glutamate. These transporters consume substantial energy in the spatially restricted space of fine astrocytic processes. GLT-1 (EAAT2) mediates the bulk of this activity in forebrain. To date, relatively few proteins have been identified that associate with GLT-1. In the present study, GLT-1 immunoaffinity isolates were prepared from rat cortex using three strategies and analyzed by LC coupled tandem mass spectrometry. In addition to known interacting proteins, the analysis identified glycolytic enzymes and outer mitochondrial proteins. Using double label immunofluorescence, GLT-1 was shown to co-localize with the mitochondrial matrix protein, ubiquinol-cytochrome c reductase core protein II (UQCRC2) or the inner mitochondrial membrane protein, ADP/ATP translocase (ANT), in rat cortex. In biolistically transduced hippocampal slices, fluorescently tagged GLT-1 puncta overlapped with fluorescently tagged mitochondria along fine astrocytic processes. In a Monte Carlo-type computer simulation, this overlap was significantly more frequent than would occur by chance. Furthermore, fluorescently tagged hexokinase-1 overlapped with mitochondria or GLT-1, strongly suggesting that GLT-1, mitochondria, and the first step in glycolysis are co-compartmentalized in astrocytic processes. Acute inhibition of glycolysis or oxidative phosphorylation had no effect on glutamate uptake in hippocampal slices, but simultaneous inhibition of both processes significantly reduced transport. Together with previous results, these studies show that GLT-1 co-compartmentalizes with Na+/K+ ATPase, glycolytic enzymes, and mitochondria, providing a mechanism to spatially match energy and buffering capacity to the demands imposed by transport.
Many drug candidates fail in clinical trials due to a lack of efficacy from limited target engagement or an insufficient therapeutic index. Minimizing off-target effects while retaining the desired pharmacodynamic (PD) response can be achieved by reduced exposure for drugs that display kinetic selectivity in which the drug:target complex has a longer half-life than off-target:drug complexes. However, while slow-binding inhibition kinetics are a key feature of many marketed drugs1,2, prospective tools that integrate drug-target residence time into predictions of drug efficacy are lacking, hindering the integration of drug-target kinetics into the drug discovery cascade. Here we describe a mechanistic PD model that includes drug-target kinetic parameters including the on- and off-rates for the formation and breakdown of the drug-target complex. We demonstrate the utility of this model by using it to predict dose response curves for inhibitors of the LpxC enzyme from Pseudomonas aeruginosa in an animal model of infection.
Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na ϩ /K ϩ -ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate D-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na ϩ /Ca 2ϩ exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake.
The effectiveness and efficiency of outpatient geriatric evaluation and management (GEM) was compared with usual outpatient primary care (UPC). One hundred sixty frail elderly outpatients were assigned randomly to GEM or UPC and assessed at baseline and at 8 months on measures of (1) health and functional status, (2) psychosocial well-being, (3) quality of health and social care, (4) use of inpatient and outpatient services, and (5) cost of care. The results indicate that GEM was significantly more effective than UPC in reducing mortality, increasing patient satisfaction, and improving the quality of health and social care. However, it was not effective in reducing health care use or the cost of care.
Recently, mitochondria have been localized to astrocytic processes where they shape Ca 2ϩ signaling; this relationship has not been examined in models of ischemia/reperfusion. We biolistically transfected astrocytes in rat hippocampal slice cultures to facilitate fluorescent confocal microscopy, and subjected these slices to transient oxygen/glucose deprivation (OGD) that causes delayed excitotoxic death of CA1 pyramidal neurons. This insult caused a delayed loss of mitochondria from astrocytic processes and increased colocalization of mitochondria with the autophagosome marker LC3B. The losses of neurons in area CA1 and mitochondria in astrocytic processes were blocked by ionotropic glutamate receptor ( (1) in the cytoplasm surrounding mitochondria (mitochondrially centered) and (2) traversing the space between mitochondria (extramitochondrial). The spatial spread, kinetics, and frequency of these events were different. The amplitude of both types was doubled and the spread of both types changed by ϳ2-fold 24 h after OGD. Together, these data suggest that pathologic activation of glutamate transport and increased astrocytic Ca 2ϩ through reversed Na ϩ /Ca 2ϩ exchange triggers mitochondrial loss and dramatic increases in Ca 2ϩ signaling in astrocytic processes.
Outpatient GEM improves patient satisfaction and some aspects of the quality of care patients' receive but does not reduce the cost of outpatient or inpatient care. Longer-term follow-up studies are needed to determine whether reductions in emergency room use and inpatient admissions persist over time and result in reductions in the overall cost of care.
The effectiveness and efficiency of outpatient geriatric evaluation and management (GEM) was compared to usual outpatient primary care (UPC). Although GEM had no overall impact on health care utilization or cost of care for the entire study period, significant reductions were found during the sixteen- to twenty-four-month study period, suggesting a possible investment effect. In the first eight months of the study, GEM patients incurred 34.8% more in health care costs than UPC patients, but in the final eight months of the study the cost of care for UPC patients exceeded that for GEM patients by 37.8%.
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