Linda L. Bambrick

Astrocyte mitochondrial mechanisms of ischemic brain injury and neuroprotection.

Research on ischemic brain injury has established a central role of mitochondria in neuron death (1–3). Astrocytes are also damaged by ischemia (4), although the participation of mitochondria in their injury is ill defined. As astrocytes are responsible for neuronal metabolic and trophic support, astrocyte dysfunction (5) will compromise postischemic neuronal survival. Ischemic alterations to astrocyte energy metabolism and the uptake and metabolism of the excitatory amino acid transmitter glutamate may be particularly important. Despite the significance of ischemic astrocyte injury, little is known of the mechanisms responsible for astrocyte death and dysfunction. This review focuses on differences between astrocyte and neuronal metabolism and mitochondrial function, and on neuronal–glial interactions. The potential for astrocyte mitochondria to serve as targets of neuroprotective interventions is also discussed.

In Vivo Restoration of Physiological Levels of Truncated TrkB.T1 Receptor Rescues Neuronal Cell Death in a Trisomic Mouse Model

Imbalances in neurotrophins or their high-affinity Trk receptors have long been reported in neurodegenerative diseases. However, a molecular link between these gene products and neuronal cell death has not been established. In the trisomy 16 (Ts16) mouse there is increased apoptosis in the cortex, and hippocampal neurons undergo accelerated cell death that cannot be rescued by administration of brain-derived neurotrophic factor (BDNF). Ts16 neurons have normal levels of the TrkB tyrosine kinase receptor but an upregulation of the TrkB.T1 truncated receptor isoform. Here we show that restoration of the physiological level of the TrkB.T1 receptor by gene targeting rescues Ts16 cortical cell and hippocampal neuronal death. Moreover, it corrects resting Ca2+ levels and restores BDNF-induced intracellular signaling mediated by full-length TrkB in Ts16 hippocampal neurons. These data provide a direct link between neuronal cell death and abnormalities in Trk neurotrophin receptor levels.

Modulation of two functionally distinct Ca2+ stores in astrocytes: Role of the plasmalemmal Na/Ca exchanger

Mechanisms that regulate the amount of releasable Ca2+ in intracellular stores of cultured mouse astrocytes were investigated using digital imaging of fura‐2 loaded cells. At rest, the cytoplasmic Ca2+ concentration, [Ca2+]cyt, was about 110 nM. In the absence of extracellular Ca2+, cyclopiazonic acid (CPA), an inhibitor of the endoplasmic reticulum (ER) Ca2+‐ATPase, induced a transient, four‐fold increase in [Ca2+]cyt due to the release of Ca2+ from inositol triphosphate (IP3) sensitive stores. Caffeine (CAF), which releases Ca2+ from Ca2+‐sensitive stores, induced a two‐fold increase in [Ca2+]cyt. The CPA‐ and CAF‐sensitive stores could be released independently. Changes in the amplitudes of the Ca2+ transients were taken as a measure of changes in store content. Removal of extracellular Na+ or addition of ouabain, which inhibit Ca2+ extrusion and promote Ca2+ entry across the plasmalemma via the Na/Ca exchanger, caused minimal increases in resting [Ca2+]cyt but greatly potentiated both CPA‐ and CAF‐induced Ca2+ transients. The amount of Ca2+ releasable from the IP3 (CPA) sensitive store was directly proportional to cytosolic Na+ concentration (i.e., inversely proportional to the transmembrane Na+ electrochemical gradient). Under these reduced Na+ gradient conditions, little, if any, Ca2+ destined for the ER stores enters the cells through voltage‐dependent Ca2+ channels. These results demonstrate that mouse astrocytes contain two distinct ER Ca2+ stores, the larger, IP3‐ (CPA‐) sensitive, and the smaller, Ca2+‐ (CAF‐) sensitive. The Ca2+ content of both ER stores can be regulated by the Na/Ca exchanger. Thus, the magnitude of cellular responses to signals that are mediated by Ca2+ release induced by the two second messengers, IP3 and Ca2+, can be modulated by factors that affect the net transport of Ca2+ across the plasmalemma.

Glutamate-mediated excitotoxicity in neonatal hippocampal neurons is mediated by mGluR-induced release of Ca++ from intracellular stores and is prevented by estradiol

Hypoxic/ischemic (HI) brain injury in newborn full‐term and premature infants is a common and pervasive source of life time disabilities in cognitive and locomotor function. In the adult, HI induces glutamate release and excitotoxic cell death dependent on NMDA receptor activation. In animal models of the premature human infant, glutamate is also released following HI, but neurons are largely insensitive to NMDA or AMPA/kainic acid (KA) receptor‐mediated damage. Using primary cultured hippocampal neurons we have determined that glutamate increases intracellular calcium much more than kainic acid. Moreover, glutamate induces cell death by activating Type I metabotropic glutamate receptors (mGluRs). Pretreatment of neurons with the gonadal steroid estradiol reduces the level of the Type I metabotropic glutamate receptors and completely prevents cell death, suggesting a novel therapeutic approach to excitotoxic brain damage in the neonate.

Organotypic slice cultures for analysis of proliferation, cell death, and migration in the embryonic neocortex

Dynamic cellular interactions during neocortical neurogenesis are critical for proper cortical development, providing both trophic and tropic support. Although cell proliferation and programmed cell death have been characterized in dissociated primary cell cultures, many in vivo processes during cortical neurogenesis depend on cell-cell interactions and therefore on the three-dimensional environment of the proliferating neuroblasts and their progeny. Here we describe a murine organotypic neocortical slice preparation that retains major morphological and functional in vivo characteristics of the developing neocortex and is viable (exhibits very low levels of cell death) for up to three days. Moreover, this slice preparation is amenable to direct experimental manipulation of potential diffusible regulators of neurogenesis. Using a variety of biochemical and physiological methods including time-lapse and quantitative confocal microscopy, we demonstrate that this system can be used effectively to investigate cellular mechanisms important for brain growth and maturation, including neurogenesis, apoptosis, and neuronal migration.

Sulforaphane protects immature hippocampal neurons against death caused by exposure to hemin or to oxygen and glucose deprivation.

Oxidative stress is a mediator of cell death following cerebral ischemia/reperfusion and heme toxicity, which can be an important pathogenic factor in acute brain injury. Induced expression of phase II detoxification enzymes through activation of the antioxidant response element (ARE)/Nrf2 pathway has emerged as a promising approach for neuroprotection. Little is known, however, about the neuroprotective potential of this strategy against injury in immature brain cells. In this study, we tested the hypothesis that sulforaphane (SFP), a naturally occurring isothiocyanate that is also a known activator of the ARE/Nrf2 antioxidant pathway, can protect immature neurons from oxidative stress‐induced death. The hypothesis was tested with primary mouse hippocampal neurons exposed to either O2 and glucose deprivation (OGD) or hemin. Treatment of immature neurons with SFP immediately after the OGD during reoxygenation was effective in protecting immature neurons from delayed cell death. Exposure of immature hippocampal neurons to hemin induced significant cell death, and both pre‐ and cotreatment with SFP were remarkably effective in blocking cytotoxicity. RT‐PCR analysis indicated that several Nrf2‐dependent cytoprotective genes, including NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO1), and glutamate‐cysteine ligase modifier subunit (GCLM), which is involved in glutathione biosynthesis, were up‐regulated following SFP treatment both in control neurons and following exposure to OGD and hemin. These results indicate that SFP activates the ARE/Nrf2 pathway of antioxidant defense and protects immature neurons from death caused by stress paradigms relevant to those associated with ischemic and traumatic injury to the immature brain.

Ca2+-dependent Regulation of TrkB Expression in Neurons

The neurotrophin brain-derived neurotrophic factor (BDNF), via activation of its receptor, tyrosine receptor kinase B (trkB), regulates a wide variety of cellular processes in the nervous system, including neuron survival and synaptic plasticity. Although the expression of BDNF is known to be Ca2+-dependent, the regulation of trkB expression has not been extensively studied. Here we report that depolarization of cultured mouse cortical neurons increased the expression of the full-length, catalytically active isoform of trkB without affecting expression of the truncated isoform. This increase in protein expression was accompanied by increased levels of transcripts encoding full-length, but not truncated, trkB. Depolarization also regulated transcription of the gene, TRKB, via entry of Ca2+ through voltage-gated Ca2+ channels and subsequent activation of Ca2+-responsive elements in the two TRKB promoters. Using transient transfection of neurons with TRKB promoter-luciferase constructs, we found that Ca2+ inhibited the upstream promoter P1 but activated the downstream promoter P2. Ca2+-dependent stimulation of TRKB expression requires two adjacent, non-identical CRE sites located within P2. The coordinated regulation of BDNF and trkB by Ca2+ may play a role in activity-dependent survival and synaptic plasticity by enhancing BDNF signaling in electrically active neurons.

Prolongation and enhancement of γ‐aminobutyric acidA receptor mediated excitation by chronic treatment with estradiol in developing rat hippocampal neurons

GABAA receptor activation during brain development is a critical source of excitation. This is due to the positive equilibrium potential for chloride relative to resting membrane potential, resulting in membrane depolarization sufficient to open voltage sensitive calcium channels. The gonadal steroid estradiol has pronounced trophic effects on the developing hippocampus, promoting cell survival and synaptogenesis. In the current study, we investigated the effect of estradiol on GABAA receptor‐mediated calcium transients in cultured neonatal hippocampal neurons, from Sprague–Dawley rats, using the calcium sensitive dye, Fura‐2‐AM. Treatment of hippocampal neurons with physiological levels of estradiol significantly increased the peak amplitude of calcium transients, increased the number of cells responding to the GABAA agonist muscimol with membrane depolarization, and delayed the rate of clearance of free intracellular calcium. These effects were significantly attenuated by pretreatment with the oestrogen receptor antagonist ICI‐182,780. This suggests that estradiol, via its action on the oestrogen receptor, prolongs the developmental duration of depolarizing GABA. Estradiol likely maintains GABA‐mediated excitation by promoting increased protein levels of the active/phosphorylated form of the chloride cotransporter Na+K+2CL– and L‐type voltage sensitive calcium channels containing the α1C subunit. We propose that a component of the trophic effects of estradiol on hippocampal development results from enhanced calcium influx subsequent to GABAA receptor activation.

Failure of Brain-Derived Neurotrophic Factor-Dependent Neuron Survival in Mouse Trisomy 16

The neurotrophin, brain derived neurotrophic factor (BDNF), exerts multiple effects on the development and maintenance of the nervous system, including regulating synaptic plasticity and promoting neuron survival. Here we report the selective failure of BDNF-dependent survival in cultured hippocampal neurons from the trisomy 16 (Ts16) mouse, an animal model of Down syndrome. This failure is accompanied by overexpression of a truncated, kinase-deficient isoform (T1) of the BDNF receptor tyrosine receptor kinase B (trkB). Adenovirus-mediated introduction of exogenous full-length trkB into Ts16 neurons fully restored BDNF-dependent survival, whereas exogenous truncated trkB expression in normal, euploid neurons reproduced the Ts16 BDNF signaling failure. Thus, the failure of Ts16 neurons to respond to BDNF is caused by dysregulation of trkB isoform expression. Such a neurotrophin signaling defect could contribute to developmental and degenerative disorders of the nervous system.

Calcineurin activity is required for depolarization‐induced, CREB‐dependent gene transcription in cortical neurons

Cyclic AMP response element binding protein (CREB) functions as an activity‐dependent transcription factor in the nervous system. Increases in intracellular Ca2+ due to neuronal activity lead to the phosphorylation and subsequent activation of CREB. Although phosphorylation of CREB at Ser‐133 is necessary for the stimulation of transcriptional activity, it is not sufficient. Here we demonstrate that in mouse cortical neurons, inhibition of the Ca2+‐dependent protein phosphatase calcineurin by FK506 or cyclosporine A blocks CREB‐dependent gene expression induced by depolarization without inhibiting depolarization‐induced Ca2+ influx or CREB Ser‐133 phosphorylation. Over‐expression of a constitutively‐active allele of the transducer of regulated CREB activity could not bypass the requirement for calcineurin activity. Stimulation of a CRE‐luciferase reporter gene by depolarization was sensitive to FK506 throughout the entire time course of the transcriptional response, revealing that calcineurin activity is required to maintain CREB‐dependent transcription. Stimulation of CRE‐luciferase expression by forskolin and 8‐Br‐cAMP also required calcineurin activity. These results suggest that calcineurin functions as a critical determinant in shaping genome responses to CREB activation in cortical neurons.