Milagros Ramos

Developmental changes in the Ca2+-regulated mitochondrial aspartate–glutamate carrier aralar1 in brain and prominent expression in the spinal cord

Aralar1 and citrin are two isoforms of the mitochondrial carrier of aspartate-glutamate (AGC), a calcium regulated carrier, which is important in the malate-aspartate NADH shuttle. The expression and cell distribution of aralar1 and citrin in brain cells has been studied during development in vitro and in vivo. Aralar1 is the only isoform expressed in neurons and its levels undergo a marked increase during in vitro maturation, which is higher than the increase in mitochondrial DNA in the same time window. The enrichment in aralar1 per mitochondria during neuronal maturation is associated with a prominent rise in the function of the malate-aspartate NADH shuttle. Paradoxically, during in vivo development of rat or mouse brain there is very little postnatal increase in total aralar1 levels per mitochondria. This is explained by the fact that astrocytes develop postnatally, have aralar1 levels much lower than neurons, and their increase masks that of aralar1. Aralar1 mRNA and protein are widely expressed throughout neuron-rich areas in adult mouse CNS with clear enrichments in sets of neuronal nuclei in the brainstem and, particularly, in the ventral horn of the spinal cord. These aralar1-rich neurons represent a subset of the cytochrome oxidase-rich neurons in the same areas. The presence of aralar1 could reflect a tonic activity of these neurons, which is met by the combination of high malate-aspartate NADH shuttle and respiratory chain activities.

Essential role of aralar in the transduction of small Ca2+ signals to neuronal mitochondria.

Aralar, the neuronal Ca2+-binding mitochondrial aspartate-glutamate carrier, has Ca2+ binding domains facing the extramitochondrial space and functions in the malate-aspartate NADH shuttle (MAS). Here we showed that MAS activity in brain mitochondria is stimulated by extramitochondrial Ca2+ with an S0.5 of 324 nm. By employing primary neuronal cultures from control and aralar-deficient mice and NAD(P)H imaging with two-photon excitation microscopy, we showed that lactate utilization involves a substantial transfer of NAD(P)H to mitochondria in control but not aralar-deficient neurons, in agreement with the lack of MAS activity associated with aralar deficiency. The increase in mitochondrial NAD(P)H was greatly potentiated by large [Ca2+]i signals both in control and aralar-deficient neurons, showing that these large signals activate the Ca2+ uniporter and mitochondrial dehydrogenases but not MAS activity. On the other hand, small [Ca2+]i signals potentiate the increase in mitochondrial NAD(P)H only in control but not in aralar-deficient neurons. We concluded that neuronal MAS activity is selectively activated by small Ca2+ signals that fall below the activation range of the Ca2+ uniporter and plays an essential role in mitochondrial Ca2+ signaling.

Cyclosporin A targets involved in protection against glutamate excitotoxicity

The toxicity of glutamate in neuronal cultures has been attributed in part to a mitochondrial dysfunction involving the permeability transition pore. The participation of the permeability transition pore in this process has been pharmacologically demonstrated by the use of cyclosporin A, which inhibits pore opening by interaction with mitochondrial cyclophilin and, thus, prevents cell death and upstream events. Since cyclosporin A also acts on calcineurin, we have investigated which of the targets of cyclosporin A was responsible for the inhibition of glutamate-excitotoxicity in cerebrocortical primary neuronal cultures. Reactive oxygen species production and early (30 min to 2 h) drop in ATP levels are initial events in glutamate excitotoxicity taking place before neuronal death. Cyclosporin A did not inhibit reactive oxygen species production, but reduced the drop in ATP levels and subsequent neuronal death. However, cyclosporin derivatives that do not bind to calcineurin had smaller effect on survival than cyclosporin A, (regardless of whether they were able to bind cyclophilin), indicating that cyclosporin A protects against glutamate toxicity also through calcineurin-related mechanisms. Consistent with this view, ATP loss appears to result from nitric oxide synthase (NOS) activation (including calcineurin-dependent dephosphorylation) and nitric oxide (NO)/peroxinitrite-dependent increase in poly (ADP-ribose) polymerase activity, since it was reduced by inhibitors of these activities. Collectively, these results suggest that cyclosporin A exerts its protective effects through calcineurin-dependent and independent mechanisms.

Pyruvate protection against β‐amyloid‐induced neuronal death: Role of mitochondrial redox state

The mechanism by which β‐amyloid protein (Aβ) causes degeneration in cultured neurons is not completely understood, but several lines of evidence suggest that Aβ‐mediated neuronal death is associated with an enhanced production of reactive oxygen species (ROS) and oxidative damage. In the present study, we address whether supplementation of glucose‐containing culture media with energy substrates, pyruvate plus malate (P/M), protects rat primary neurons from Aβ‐induced degeneration and death. We found that P/M addition attenuated cell death evoked by β‐amyloid peptides (Aβ25–35 and Aβ1–40) after 24 hr treatment and that this effect was blocked by α‐ciano‐3‐hydroxycinnamate (CIN), suggesting that it requires mitochondrial pyruvate uptake. P/M supply to control and Aβ‐treated neuronal cultures increases cellular reducing power, as indicated by the ability to reduce the dye 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT). The early increases in ROS levels, measured by dichlorofluorescein (DCF) fluorescence, and caspase‐3 activity that follow exposure to Aβ were notably reduced in the presence of P/M. These results place activation of caspase‐3 most likely downstream of oxidative damage to the mitochondria and indicate that mitochondrial NAD(P) redox status plays a central role in the neuroprotective effect of pyruvate. Inhibition of respiratory chain complexes and mitochondrial uncoupling did not block the early increase in ROS levels, suggesting that Aβ could initiate oxidative stress by activating a source of ROS that is not accesible to the antioxidant defenses fueled by mitochondrial substrates.

Differential effects of guanine nucleotides on kainic acid binding and on adenylate cyclase activity in chick optic tectum

In G protein‐coupled receptors, neurotransmitter‐induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor—G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP‐PNP, a non‐hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP‐binding sites are pre‐saturated with GMP‐PNP. Experiments carried out with these membranes show that GMP‐PNP, GDP‐S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP‐PNP activates adenylate cyclase activity by binding to G proteins.

Role of aralar, the mitochondrial transporter of aspartate‐glutamate, in brain N‐acetylaspartate formation and Ca2+ signaling in neuronal mitochondria

Aralar, the Ca2+‐dependent mitochondrial aspartate‐glutamate carrier expressed in brain and skeletal muscle, is a member of the malate–aspartate NADH shuttle. Disrupting the gene for aralar, SLC25a12, in mice has enabled the discovery of two new roles of this carrier. On the one hand, it is required for synthesis of brain aspartate and N‐acetylaspartate, a neuron‐born metabolite that supplies acetate for myelin lipid synthesis; and on the other, it is essential for the transmission of small Ca2+ signals to mitochondria via an increase in mitochondrial NADH.

In Vitro and in Vivo Enhanced Generation of Human A9 Dopamine Neurons from Neural Stem Cells by Bcl-XL

Human neural stem cells derived from the ventral mesencephalon (VM) are powerful research tools and candidates for cell therapies in Parkinson disease. Previous studies with VM dopaminergic neuron (DAn) precursors indicated poor growth potential and unstable phenotypical properties. Using the model cell line hVM1 (human ventral mesencephalic neural stem cell line 1; a new human fetal VM stem cell line), we have found that Bcl-XL enhances the generation of DAn from VM human neural stem cells. Mechanistically, Bcl-XL not only exerts the expected antiapoptotic effect but also induces proneural (NGN2 and NEUROD1) and dopamine-related transcription factors, resulting in a high yield of DAn with the correct phenotype of substantia nigra pars compacta (SNpc). The expression of key genes directly involved in VM/SNpc dopaminergic patterning, differentiation, and maturation (EN1, LMX1B, PITX3, NURR1, VMAT2, GIRK2, and dopamine transporter) is thus enhanced by Bcl-XL. These effects on neurogenesis occur in parallel to a decrease in glia generation. These in vitro Bcl-XL effects are paralleled in vivo, after transplantation in hemiparkinsonian rats, where hVM1-Bcl-XL cells survive, integrate, and differentiate into DAn, alleviating behavioral motor asymmetry. Bcl-XL then allows for human fetal VM stem cells to stably generate mature SNpc DAn both in vitro and in vivo and is thus proposed as a helpful factor for the development of cell therapies for neurodegenerative conditions, Parkinson disease in particular.

Specific binding of [3H]GppNHp to extracellular membrane receptors in chick cerebellum: possible involvement of kainic acid receptors

Guanine nucleotides (GNs), including GMP, displace [3H]kainic acid binding to chick cerebellar lysed and vesiculated membranes. Saturation studies of [3H]GppNHp binding, under conditions that prevent the occupation of the nucleotide binding sites in G‐proteins, demonstrate the existence of extracellular membrane receptors specific for guanine nucleotides. Affinity‐labeling of a vesicle preparation with [α‐32P]GTP gives one single labeled band, upon electrophoresis, with an apparent molecular mass of 50 kDa. Additional experiments with partially purified kainate receptors suggest that the GN extracellular sites may overlap, at least partially, the kainic acid binding sites, being then responsible for the displacement of [3H]kainic acid by GNs. The physiological significance of these findings remains unclear.

Functional properties of the human ventral mesencephalic neural stem cell line hVM1.

The human fetal ventral mesencephalon-derived stem cell line, hVM1, yields high number of tyrosine hydroxylase-expressing presumed dopaminergic neurons upon in vitro differentiation. Here we report that cells generated from this line differentiate into a neuronal phenotype, express electrophysiological properties of functional neurons and respond to neurotransmitters in vitro. However, the electrophysiological properties are immature and the cells require longer maturation time than possible under in vitro conditions.

A Streptomyces fradiae protease dissociates structurally preserved neurons and glial cells from the embryonic and adult central nervous system of vertebrates

Nerve cell dissociation has become a key procedural tool in the implementation of a number of techniques in cellular and molecular neurobiology. We report that a protease preparation from Streptomyces fradiae (henceforth SF-protease) dissociates viable and morphologically identifiable embryonic and mature neurons and glial cells from the central nervous system of chick and rat, when used under strictly controlled conditions. Typical dendritic and axonal growth cones, with their lamellipodia and filopodia, are seen in many neuroblast types - growth cones in the case of embryonic glial cells and even the thinnest processes of some cells, such as the microvilli of adult chick retinal Müller (glial) cells, or the cilia of photoreceptors appear intact. Our results suggest that the SF-protease releases cells from tissue in a way that ensures the continuity of the plasma membrane and cuts through the transmembrane attachment systems (either cell-cell or cell-extracellular matrix) without compromising the cytoskeletal integrity underlying native cell shape.