Biomedical subjects

R Sattler

Publications and source records attributed to R Sattler.

At least 19 recordsLinked to original sources

PubMed2006

Regulation and dysregulation of glutamate transporters.

Glutamate is the primary excitatory neurotransmitter in the central nervous system. During synaptic activity, glutamate is released into the synaptic cleft and binds to glutamate receptors on the pre- and postsynaptic membrane as well as on neighboring astrocytes in order to start a number of intracellular signaling cascades. To allow for an efficient signaling to occur, glutamate levels in the synaptic cleft have to be maintained at very low levels. This process is regulated by glutamate transporters, which remove excess extracellular glutamate via a sodium-potassium coupled uptake mechanism. When extracellular glutamate levels rise to about normal, glutamate overactivates glutamate receptors, triggering a multitude of intracellular events in the postsynaptic neuron, which ultimately results in neuronal cell death. This phenomenon is known as excitotoxicity and is the underlying mechanisms of a number of neurodegenerative diseases. A dysfunction of the glutamate transporter is thought to contribute to cell death during excitotoxicity. Therefore, efforts have been made to understand the regulation of glutamate transporter function. Transporter activity can be regulated in different ways, including through gene expression, transporter protein targeting and trafficking and through posttranslational modifications of the transporter protein. The identification of these mechanisms has helped to understand the role of glutamate transporters during pathology and will aid in the development of therapeutic strategies with the transporter as a desirable target.

Animals↗

PubMed2001

The influence of glutamate receptor 2 expression on excitotoxicity in Glur2 null mutant mice.

AMPA receptor (AMPAR)-mediated ionic currents that govern gene expression, synaptic strength, and plasticity also can trigger excitotoxicity. However, native AMPARs exhibit heterogeneous pharmacological, biochemical, and ionic permeability characteristics, which are governed partly by receptor subunit composition. Consequently, the mechanisms governing AMPAR-mediated excitotoxicity have been difficult to elucidate. The GluR2 subunit is of particular interest because it influences AMPAR pharmacology, Ca(2+) permeability, and AMPAR interactions with intracellular proteins. In this paper we used mutant mice lacking the AMPAR subunit GluR2 to study AMPAR-mediated excitotoxicity in cultured cortical neurons and in hippocampal neurons in vivo. We examined the hypothesis that in these mice the level of GluR2 expression governs the vulnerability of neurons to excitotoxicity and further examined the ionic mechanisms that are involved. In cortical neuronal cultures AMPAR-mediated neurotoxicity paralleled the magnitude of kainate-evoked AMPAR-mediated currents, which were increased in neurons lacking GluR2. Ca(2+) permeability, although elevated in GluR2-deficient neurons, did not correlate with excitotoxicity. However, toxicity was reduced by removal of extracellular Na(+), the main charge carrier of AMPAR-mediated currents. In vivo, the vulnerability of CA1 hippocampal neurons to stereotactic kainate injections and of CA3 neurons to intraperitoneal kainate administration was independent of GluR2 level. Neurons lacking the GluR2 subunit did not demonstrate compensatory changes in the distribution, expression, or function of AMPARs or of Ca(2+)-buffering proteins. Thus GluR2 level may influence excitotoxicity by effects additional to those on Ca(2+) permeability, such as effects on agonist potency, ionic currents, and synaptic reorganization.

Adenosine Monophosphate↗

PubMed2001

The neuronal Rho-GEF Kalirin-7 interacts with PDZ domain-containing proteins and regulates dendritic morphogenesis.

Spine function requires precise control of the actin cytoskeleton. Kalirin-7, a GDP/GTP exchange factor for Rac1, interacts with PDZ proteins such as PSD-95, colocalizing with PSD-95 at synapses of cultured hippocampal neurons. PSD-95 and Kalirin-7 interact in vivo and in heterologous expression systems. In primary cortical neurons, transfected Kalirin-7 is targeted to spines and increases the number and size of spine-like structures. A Kalirin-7 mutant unable to interact with PDZ proteins remains in the cell soma, inducing local formation of aberrant filopodial neurites. Kalirin-7 with an inactivated GEF domain reduces the number of spines below control levels. These results provide evidence that PDZ proteins target Kalirin-7 to the PSD, where it regulates dendritic morphogenesis through Rac1 signaling to the actin cytoskeleton.

Actins↗

PubMed2000

Distinct roles of synaptic and extrasynaptic NMDA receptors in excitotoxicity.

Excitatory synaptic activity governs excitotoxicity and modulates the distribution of NMDA receptors (NMDARs) among synaptic and extrasynaptic sites of central neurons. We investigated whether NMDAR localization was functionally linked to excitotoxicity by perturbing F-actin, a cytoskeletal protein that participates in targeting synaptic NMDARs in dendritic spines. Depolymerizing F-actin did not affect NMDA-evoked whole-cell currents. However, the number of dendritic NMDAR clusters and the NMDAR-mediated component of miniature spontaneous EPSCs were reduced, whereas the number of AMPA receptor clusters and AMPA receptor-mediated component of EPSCs was unchanged. This selective perturbation of synaptically activated NMDARs had no effect on neuronal death or the accumulation of (45)Ca(2+) evoked by applying exogenous NMDA or L-glutamate, which reach both synaptic and extrasynaptic receptors. However, it increased survival and decreased (45)Ca(2+) accumulation in neurons exposed to oxygen glucose deprivation, which causes excitotoxicity by glutamate release at synapses. Thus, synaptically and extrasynaptically activated NMDARs are equally capable of excitotoxicity. However, their relative contributions vary with the location of extracellular excitotoxin accumulation, a factor governed by the mechanism of extracellular neurotransmitter accumulation, not the synaptic activation of NMDARs.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed2000

Molecular mechanisms of calcium-dependent excitotoxicity.

Excitotoxicity is thought to be a major mechanism contributing to neurodegeneration during central nervous system ischemia, trauma, and other neurological disorders. Briefly, synaptic overactivity leads to the excessive release of glutamate, the major excitatory neurotransmitter in the mammalian central nervous system. Glutamate activates a number of postsynaptic cell membrane receptors, which upon activation open their associated ion channel pore to produce ion influx or efflux. This leads to a disturbance of the intracellular ionic environment, the best characterized feature of which is the influx of sodium, chloride, and Ca2+. An excess of Ca2+ ions then activates intracellular Ca2+-dependent signaling cascades that eventually lead to neuronal cell death. Despite intensive research in the field of Ca2+-dependent neurotoxicity the precise molecular mechanisms leading to cell death remain poorly understood. In particular, the question of the precise relationship between Ca2+ loading and neurotoxicity has been controversial. Many glutamate receptors are clustered and localized at the postsynaptic density. Recently, increasing knowledge of the molecular composition of the postsynaptic density has allowed us to extend our understanding of the molecular mechanisms of Ca2+-dependent excitotoxicity and to propose that distinct, membrane receptor-specific, neurotoxic signaling pathways transduce Ca2+-dependent excitotoxicity. These findings may have significant implications in the search for precisely targeted therapeutic drugs for a range of neurological disorders.

Animals↗

PubMed1999

Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein.

The efficiency with which N-methyl-D-aspartate receptors (NMDARs) trigger intracellular signaling pathways governs neuronal plasticity, development, senescence, and disease. In cultured cortical neurons, suppressing the expression of the NMDAR scaffolding protein PSD-95 (postsynaptic density-95) selectively attenuated excitotoxicity triggered via NMDARs, but not by other glutamate or calcium ion (Ca2+) channels. NMDAR function was unaffected, because receptor expression, NMDA currents, and 45Ca2+ loading were unchanged. Suppressing PSD-95 blocked Ca2+-activated nitric oxide production by NMDARs selectively, without affecting neuronal nitric oxide synthase expression or function. Thus, PSD-95 is required for efficient coupling of NMDAR activity to nitric oxide toxicity, and imparts specificity to excitotoxic Ca2+ signaling.

Animals↗

PubMed1998

Distinct influx pathways, not calcium load, determine neuronal vulnerability to calcium neurotoxicity.

Many forms of neurodegeneration are ascribed to excessive cellular Ca2+ loading (Ca2+ hypothesis). We examined quantitatively whether factors other than Ca2+ loading were determinants of excitotoxic neurodegeneration. Cell survival, morphology, free intracellular Ca2+ concentration ([Ca2+]i), and 45Ca2+ accumulation were measured in cultured cortical neurons loaded with known quantities of Ca2+ through distinct transmembrane pathways triggered by excitatory amino acids, cell membrane depolarization, or Ca2+ ionophores. Contrary to the Ca2+ hypothesis, the relationships between Ca2+ load and cell survival, free [Ca2+]i, and Ca2+-induced morphological alterations depended primarily on the route of Ca2+ influx, not the Ca2+ load. Notably, Ca2+ loading via NMDA receptor channels was toxic, whereas identical Ca2+ loads incurred through voltage-sensitive Ca2+ channels were completely innocuous. Furthermore, accounting quantitatively for Ca2+ loading via NMDA receptors uncovered a previously unreported component of L-glutamate neurotoxicity apparently not mediated by ionotropic or metabotropic glutamate receptors. It was synergistic with toxicity attributable to glutamate-evoked Ca2+ loading, and correlated with enhanced cellular ATP depletion. This previously unrecognized toxic action of glutamate constituted a chief excitotoxic mechanism under conditions producing submaximal Ca2+ loading. We conclude that (a) Ca2+ neurotoxicity is a function of the Ca2+ influx pathway, not Ca2+ load, and (b) glutamate toxicity may not be restricted to its actions on glutamate receptors.

Animals↗

PubMed1998

Characterization of neuroprotection from excitotoxicity by moderate and profound hypothermia in cultured cortical neurons unmasks a temperature-insensitive component of glutamate neurotoxicity.

Although profound hypothermia has been used for decades to protect the human brain from hypoxic or ischemic insults, little is known about the underlying mechanism. We therefore report the first characterization of the effects of moderate (30 degrees C) and profound hypothermia (12 degrees to 20 degrees C) on excitotoxicity in cultured cortical neurons exposed to excitatory amino acids (EAA; glutamate, N-methyl-D-aspartate [NMDA], AMPA, or kainate) at different temperatures (12 degrees to 37 degrees C). Cooling neurons to 30 degrees C and 20 degrees C was neuroprotective, but cooling to 12 degrees C was toxic. The extent of protection depended on the temperature, the EAA receptor agonist employed, and the duration of the EAA challenge. Neurons challenged briefly (5 minutes) with all EAA were protected, as were neurons challenged for 60 minutes with NMDA, AMPA, or kainate. The protective effects of hypothermia (20 degrees and 30 degrees C) persisted after rewarming to 37 degrees C, but rewarming from 12 degrees C was deleterious. Surprisingly, however, prolonged (60 minutes) exposures to glutamate unmasked a temperature-insensitive component of glutamate neurotoxicity that was not seen with the other, synthetic EAA; this component was still mediated via NMDA receptors, not by ionotropic or metabotropic non-NMDA receptors. The temperature-insensitivity of glutamate toxicity was not explained by effects of hypothermia on EAA-evoked [Ca2+]i increases measured using high- and low-affinity Ca2+ indicators, nor by effects on mitochondrial production of reactive oxygen species. This first characterization of excitotoxicity at profoundly hypothermic temperatures reveals a previously unnoticed feature of glutamate neurotoxicity unseen with the other EAA, and also suggests that hypothermia protects the brain at the level of neurons by blocking, rather than slowing, excitotoxicity.

Animals↗

PubMed1997

A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.

The inability to determine the precise intracellular location of non-fluorescent organic calcium chelators such as BAPTA is a persistent problem which has precluded much detailed analysis of the chelators' spatial or temporal dynamics in live cells. Similarly, following physiological experiments with fluorescent indicators like Fura-2, it has often been desirable to maintain the dye within the cell for later analysis by additional histological techniques. Based on chemical considerations, and its prior use in tissue fixation, we examined the water soluble reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) as a potential fixative for diverse calcium chelators. The utility of EDC, but not other common fixatives, was confirmed through electrophysiological means, through a novel ELISA, which exploits anti-BAPTA antibodies to assess the extent and kinetics of fixation; by autoradiography of neurons loaded with [14C]-BAPTA, and by immunocytochemistry and imaging of intracellular BAPTA or Calcium Green in neurons. At concentrations > 0.1 mg/ml, EDC caused virtually instantaneous, irreversible, fixation of > 95% of BAPTA free acid. Fixation of intracellular BAPTA was confirmed in hippocampal brain slices loaded with BAPTA/AM ester, and showed biphasic kinetics consistent with rapid loading and subsequent extrusion of the chelator. Immunocytochemistry on neurons microinjected with BAPTA free acid and the dye Lucifer Yellow showed BAPTA-specific staining which was distributed in the cell similarly to that of the accompanying marker dye. Application of EDC also efficiently fixed in situ analogs of BAPTA such as Calcium Green (a fluorescent Ca2+ indicator) as shown by confocal imaging of EDC-fixed brain slices loaded with this indicator. Taken together, these data show that EDC is an effective, inexpensive and versatile fixative for calcium chelators in diverse cells. The availability of a suitable fixative now makes it possible to determine the distributions of such chelators at both the light and, possibly, the electron microscope level. Two important features of EDC, arise from its specificity for free carboxyl groups. First, the ability to fix, selectively, the chelators but not their AM esters; and, second, its enormous potential as a fixative for the numerous other carboxyl-containing chelators, dyes and pH indicators currently available.

Animals↗

PubMed1997

Preparation, characterization and utility of a novel antibody for resolving the spatial and temporal dynamics of the calcium chelator BAPTA.

In spite of its importance as a tool to manipulate cell calcium, the versatility of the octadentate chelator BAPTA in cell physiological and diverse other applications is limited by the difficulty with which it can be quantified and its cell and tissue distributions determined. Conventional approaches, such as HPLC analysis or autoradiography, are of limited sensitivity and resolution and have attendant biohazard risks. We now describe a versatile, facile and inexpensive means for quantifying and determining the distribution of BAPTA which exploits an immunological approach based on our generation of novel antibodies to BAPTA. Antibodies to BAPTA were prepared by immunizing rabbits with BAPTA conjugated to keyhole limpet hemocyanin via a zero-order cross-linking reagent-EDC. The ability of anti-BAPTA IgGs to recognize free or conjugated BAPTA was confirmed using enzyme-linked and immunoblotting assays made possible by our introduction of a BAPTA-BSA adduct. Using such assays, we show that the anti-BAPTA antibodies possess marked selectivity for BAPTA compared to several structurally-related BAPTA analogs. The utility of the anti-BAPTA antibodies in cell calcium research has been confirmed in two ways. First, by determining the spatial distribution of BAPTA through immunocytochemistry and confocal microscopy of cortical neurons loaded with BAPTA/AM and, second, by determination of the kinetics of loading and efflux of BAPTA through enzyme-linked cell immunoassays (ELISA) and immunocytochemistry. Together, these data demonstrate that anti-BAPTA antibodies are a powerful new tool with which to quantify BAPTA and to define the spatial and temporal distribution of this important calcium chelator in live cells. Such information should greatly aid the design of cell physiological experiments, the development of new chelators and the identification of sources of chelator selectivity in emerging therapeutic applications.

Animals↗

PubMed1997

Determination of the time course and extent of neurotoxicity at defined temperatures in cultured neurons using a modified multiwell plate fluorescence scanner.

The cellular and molecular mechanisms of hypoxic/ischemic neurodegeneration are sensitive to numerous factors that modulate the time course and degree of neuronal death. Among such factors is hypothermia, which can dramatically protect neurons from injury. To examine and control for temperature-dependent effects, we developed a technique that provides for a high-throughput, accurate, and reproducible determination of the time course and degree of neurotoxicity in cultured cortical neurons at precisely defined temperatures. We used a fluorescence multiwell plate scanner, modified by us to permit the control of temperature, to perform serial quantitative measurements of propidium iodide (PI) fluorescence in cortical neuronal cultures exposed to excitotoxic insults. In validating this approach, we show that these time course measurements correlate highly with manual counts of PI-stained cells in the same cultures (r = 0.958, p < 0.0001) and with lactate dehydrogenase release (r = 0.964, p < 0.0001). This method represents an efficient approach to mechanistic and quantitative studies of cell death as well as a high-throughput technique for screening new neuroprotective therapies in vitro.

Animals↗

PubMed1996

Voltage-sensitive calcium channels mediate calcium entry into cultured mammalian sympathetic neurons following neurite transection.

Calcium ion entry following mechanical neurite transection was examined in cultured sympathetic neurons loaded with the Ca2+ indicator fluo-3. Neurite transection produced a rapid [Ca2+]i rise in the cell soma which preceded any [Ca2+]i rise in the neurite (n = 30). Blocking sodium channels with tetrodotoxin had no effect on the Ca2+ rise, but inactivating voltage-sensitive Ca2+ channels by bath-applying 140 mM potassium prior to the transection, and the simultaneous application of nimodipine and omega-conotoxin GVIA, blockers of L-type and N-type Ca2+ channels, respectively, considerably attenuated the Ca2+ rise in the soma and neurites. These data contradict the intuitive hypothesis that Ca2+ entry following mechanical neurite transection occurs via non-specific influx pathways produced by cell-membrane disruption and provide direct evidence in mammalian neurons that immediate, traumatically-induced, increases in neuronal [Ca2+]i are amenable to pharmacological manipulation.

Animals↗

PubMed1978

[Myocardial potassium and H-ion loss during normothermic, ischemic heart arrest].

Arterial and venous [K+] and [H+] concentrations were determined during normothermic ischemic arrest in cat hearts intermittently perfused without resuscitation. The slight loss in [K+] was taken as evidence of a stable membrane potential. The decrease in [H+] activity appearing in the perfusate correlated well with the previously described increase in coronary resistance. This was interpreted as a sign of restricted coronary perfusion and not as evidence for reduced anaerobic metabolic activity. Thus the functional impairment of the coronary system was confirmed to be a limiting factor when resuscitating ischemically arrested hearts.

Animals↗

PubMed1975