Posttraumatic GABA(A)-mediated [Ca2+]i increase is essential for the induction of brain-derived neurotrophic factor-dependent survival of mature central neurons.

A shift of GABA(A)-mediated responses from hyperpolarizing to depolarizing after neuronal injury leads to GABA(A)-mediated increase in [Ca2+](i). In addition, central neurons become dependent on BDNF for survival. Whether these two mechanisms are causally interrelated is an open question. Here, we show in lesioned CA3 hippocampal neurons in vitro and in axotomized corticospinal neurons in vivo that posttraumatic downregulation of the neuron-specific K-Cl cotransporter KCC2 leads to intracellular chloride accumulation by the Na-K-2Cl cotransporter NKCC1, resulting in GABA-induced [Ca2+](i) transients. This mechanism is required by a population of neurons to survive in a BDNF-dependent manner after injury, because blocking GABA(A)-depolarization with the NKCC1 inhibitor bumetanide prevents the loss of neurons on BDNF withdrawal. The resurgence of KCC2 expression during recovery coincides with loss of BDNF dependency for survival. This is likely mediated through BDNF itself, because injured neurons reverse their response to this neurotrophin by switching the BDNF-induced downregulation of KCC2 to upregulation.

ApoE receptor 2 controls neuronal survival in the adult brain.

A central pathogenic feature of neurodegenerative diseases and neurotrauma is the death of neurons. A mechanistic understanding of the factors and conditions that induce the dysfunction and death of neurons is essential for devising effective treatment strategies against neuronal loss after trauma or during aging. Because Apolipoprotein E (ApoE) is a major risk factor for several neurodegenerative diseases, including Alzheimer's disease , a direct or indirect role of ApoE receptors in the disease process is likely. Here we have used gene targeting in mice to investigate possible roles of ApoE receptors in the regulation of neuronal survival. We demonstrate that a differentially spliced isoform of an ApoE receptor, ApoE receptor 2 (Apoer2), is essential for protection against neuronal cell loss during normal aging. Furthermore, the same splice form selectively promotes neuronal cell death after injury through mechanisms that may involve serine/threonine kinases of the Jun N-terminal kinase (JNK) family. These findings raise the possibility that ApoE and its receptors cooperatively regulate common mechanisms that are essential to neuronal survival in the adult brain.

Functional dissection of Reelin signaling by site-directed disruption of Disabled-1 adaptor binding to apolipoprotein E receptor 2: distinct roles in development and synaptic plasticity.

The Reelin signaling pathway controls neuronal positioning in human and mouse brain during development as well as modulation of long-term potentiation (LTP) and behavior in the adult. Reelin signals by binding to two transmembrane receptors, apolipoprotein E receptor 2 (Apoer2) and very-low-density lipoprotein receptor. After Reelin binds to the receptors, Disabled-1 (Dab1), an intracellular adaptor protein that binds to the cytoplasmic tails of the receptors, becomes phosphorylated on tyrosine residues, initiating a signaling cascade that includes activation of Src-family kinases and Akt. Here, we have created a line of mutant mice (Apoer2 EIG) in which the Apoer2 NFDNPVY motif has been altered to EIGNPVY to disrupt the Apoer2-Dab1 interaction to further study Reelin signaling in development and adult brain. Using primary neuronal cultures stimulated with recombinant Reelin, we find that normal Reelin signaling requires the wild-type NFDNPVY sequence and likely the interaction of Apoer2 with Dab1. Furthermore, examination of hippocampal, cortical, and cerebellar layering reveals that the NFDNPVY sequence of Apoer2 is indispensable for normal neuronal positioning during development of the brain. Adult Apoer2 EIG mice display severe abnormalities in LTP and behavior that are distinct from those observed for mice lacking Apoer2. In Apoer2 EIG slices, LTP degraded to baseline within 30 min, and this was prevented in the presence of Reelin. Together, these findings emphasize the complexity of Reelin signaling in the adult brain, which likely requires multiple adaptor protein interactions with the intracellular domain of Apoer2.

Lack of correlation between NF-kappaB activation and induction of programmed cell death in PC12 pheochromocytoma cells treated with 6-hydroxydopamine or the cannabinoid receptor 1-agonist CP55,940.

NF-kappaB is a transcriptional regulator that plays a key role in immunity, inflammation and programmed cell death. We generated a PC12 cell line termed PC12kappaBluc that contains an integrated NF-kappaB-responsive reporter gene to directly measure NF-kappaB activity. The "classical" activators of NF-kappaB, phorbol 12-O-tetradecanoate-13-acetate and tumor necrosis factor alpha, strongly induced NF-kappaB activity in PC12kappaBluc cells. Activation of NF-kappaB could be attenuated by preincubating the cells with the cAMP analogue dbcAMP or via expression of the superrepressor IkappaBalphaS32A/S36A. PC12kappaBluc cells were subjected to several apoptotic paradigms, including treatment with 6-hydroxydopamine, H2O2, K2Cr2O7, MnCl2, C2-ceramide or the cannabinoid receptor-1 agonist CP55,940. A simultaneous measurement of the NF-kappaB activity revealed that only administration of 6-hydroxydopamine or CP55,940 increased NF-kappaB activity. Using pharmacological and genetic strategies to attenuate NF-kappaB transcriptional activity, we demonstrate that the elevation of NF-kappaB activity by 6-hydroxydopamine and CP55,940 is not an integral part of the apoptotic signaling cascade in PC12 cells.

Neuronal cell death induced by antidepressants: lack of correlation with Egr-1, NF-kappa B and extracellular signal-regulated protein kinase activation.

The tricyclic antidepressants (TCA) amitriptyline and desipramine and the serotonin reuptake inhibitor fluoxetine induce, at microM concentrations, cell death in HT22 immortalized hippocampal neurons and PC12 pheochromocytoma cells. Here, we show that these neurotoxic effects are accompanied by a selective activation of extracellular signal-regulated protein kinase (ERK), the biosynthesis of the transcription factor Egr-1 and an increase in the transcriptional activity of NF-kappa B. However, an impairment of both ERK activation and Egr-1 biosynthesis by the MAP kinase kinase-1 (MEK-1) inhibitor PD98059 did not block cell death. Moreover, stimulation of ERK phosphorylation and Egr-1 biosynthesis by sphingosine-1-phosphate did not induce cell death, indicating that stimulation of the ERK signaling pathway and Egr-1 biosynthesis are not required for neuronal cell death induced by antidepressants. Likewise, attenuation of antidepressant-induced NF-kappa B activity by elevation of the intracellular cAMP concentration or by retroviral driven expression of the non-degradable superrepressor I kappa B alpha S32A/S36A demonstrated that the elevation of NF-kappa B activity by amitriptyline, desipramine and fluoxetine is not an integral part of the apoptotic signaling cascade triggered by these compounds.

Neuronal development.

Biological tools that are unleashed in malignancies are employed in a controlled manner during neuronal development. By default, early embryonic cells would become neuronal stem cells, a path that is blocked by specific signaling pathways. The future nervous system only develops where this blockade is inhibited by inductive signals from the 'organizer'. Once the future brain and spinal cord regions are determined, the mitotic potential in this region must be maintained long enough to produce all cells required, but also be controlled to avoid excessive over-production of cells. Newly generated cells must then migrate to their future destination, they must know where to settle down, and they must differentiate. To shape the developing nervous system and to adapt its functionality to the postnatal environment, cell survival must be regulated, i.e. survival of some cells is supported while death of others is induced. Thus, inductive events, proliferation, cell migration, differentiation, cell survival and cell death are highly regulated during neuronal development, while these functions are de-regulated in malignancies. The molecular pathways for neuronal development mutually modulate each other and are still present in the adult nervous system. Because many of these pathways are implicated in tumors, neurons may affect these conditions.

Alternative splicing switches the divalent cation selectivity of TRPM3 channels.

TRPM3 is a poorly understood member of the large family of transient receptor potential (TRP) ion channels. Here we describe five novel splice variants of TRPM3, TRPM3alpha1-5. These variants are characterized by a previously unknown amino terminus of 61 residues. The differences between the five variants arise through splice events at three different sites. One of these splice sites might be located in the pore region of the channel as indicated by sequence alignment with other, better-characterized TRP channels. We selected two splice variants, TRPM3alpha1 and TRPM3alpha2, that differ only in this presumed pore region and analyzed their biophysical characteristics after heterologous expression in human embryonic kidney 293 cells. TRPM3alpha1 as well as TRPM3alpha2 induced a novel, outwardly rectifying cationic conductance that was tightly regulated by intracellular Mg(2+). However, these two variants are highly different in their ionic selectivity. Whereas TRPM3alpha1-encoded channels are poorly permeable for divalent cations, TRPM3alpha2-encoded channels are well permeated by Ca(2+) and Mg(2+). Additionally, we found that currents through TRPM3alpha2 are blocked by extracellular monovalent cations, whereas currents through TRPM3alpha1 are not. These differences unambiguously show that TRPM3 proteins constitute a pore-forming channel subunit and localize the position of the ion-conducting pore within the TRPM3 protein. Although the ionic selectivity of ion channels has traditionally been regarded as rather constant for a given channel-encoding gene, our results show that alternative splicing can be a mechanism to produce channels with very different selectivity profiles.

Neuroprotection of immortalized hippocampal neurones by brain-derived neurotrophic factor and Raf-1 protein kinase: role of extracellular signal-regulated protein kinase and phosphatidylinositol 3-kinase.

We have investigated the molecular mechanisms of neurotrophin-mediated cell survival in HT22 cells, a murine cell line of hippocampal origin, expressing the brain-derived neurotrophic factor (BDNF) receptor TrkB as well as the TrkB.T1 splice variant. Stimulation with BDNF protected HT22-TrkB cells, but not HT22-TrkB.T1 cells, against programmed cell death induced by serum deprivation. BDNF did not, however, provide protection against oxidative glutamate toxicity, indicating that serum deprivation-induced cell death differs substantially from glutamate-induced cell death. Using a pharmacological strategy to block either the extracellular signal-regulated protein kinase (ERK) or the phosphatidylinositol 3-kinase (PI3) pathway, we show that activation of PI3 kinase is required for the neuroprotective activity of BDNF in HT22 cells. To further analyse the role of ERK in neuroprotection we expressed an inducible deltaRaf-1:ER fusion protein in HT22 cells. Activation of this conditionally active form of Raf-1 induced a sustained phosphorylation of ERK, and protected the cells from serum withdrawal-induced cell death. Inhibition of ERK activation at different time points revealed that a prolonged activation of ERK is essential to protect HT22 cells from cell death triggered by the withdrawal of serum, indicating that the duration of ERK activation is of major importance for its neuroprotective biological function.

Role of c-Jun concentration in neuronal cell death.

A dimer of the basic region leucine zipper proteins c-Jun and c-Fos constitutes the classical activator protein-1 (AP-1) transcription factor. c-Jun is thought to play essential roles in many important cellular pathways, including the control of proliferation and cell death. To investigate the roles of c-Jun and c-Fos concentrations in the regulation of neuronal cell death, we generated conditional alleles by fusing c-Jun and c-Fos to the ligand binding domain of the murine estrogen receptor (ER), with the aim of controlling the biological activities of c-Jun and c-Fos by the synthetic ligand 4-hydroxytamoxifen (4OHT). Transient transfection experiments revealed an increase in AP-1 activity following transfection of an expression vector encoding a c-Jun/estrogen receptor fusion protein (c-JunER) and stimulation with 4OHT. In contrast, a c-Fos/estrogen receptor fusion protein (c-FosER) was only weakly active in HT22 immortalized hippocampal cells and in PC12 pheochromocytoma cells. Highest levels of AP-1 activity were obtained by cotransfection of c-FosER and c-JunER and stimulation with 4OHT. Using retroviral gene transfer, we generated HT22 and PC12 cells expressing either c-JunER or c-FosER. The AP-1 activity was moderately increased in 4OHT-treated HT22 and PC12 cells expressing c-JunER, whereas no biological activity was observed in cells expressing c-FosER. We tested the influence of 4OHT-activated c-JunER or c-FosER upon cell survival and cell death by quantification of mitochondrial reduction capacity of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan dye crystals. We did not observe any 4OHT-dependent decrease in cell survival in cells expressing c-JunER or c-FosER. Likewise, the number of pycnic nuclei did not increase in HT22 or PC12 cells expressing c-JunER or c-FosER. We conclude that an increase in the c-Jun concentration is not sufficient to trigger neuronal cell death. We propose that it is not the concentration of c-Jun that is critical for cell survival but rather the concentration of active, i.e., phosphorylated c-Jun.