Valentín Ceña

Sequential treatment of SH-SY5Y cells with retinoic acid and brain-derived neurotrophic factor gives rise to fully differentiated, neurotrophic factor-dependent, human neuron-like cells.

Abstract: A rapid and simple procedure is presented to obtain nearly pure populations of human neuron‐like cells from the SH‐SY5Y neuroblastoma cell line. Sequential exposure of SH‐SY5Y cells to retinoic acid and brain‐derived neurotrophic factor in serum‐free medium yields homogeneous populations of cells with neuronal morphology, avoiding the presence of other neural crest derivatives that would normally arise from those cells. Cells are withdrawn from the cell cycle, as shown by 5‐bromo‐2′‐deoxyuridine uptake and retinoblastoma hypophosphorylation. Cell survival is dependent on the continuous presence of brain‐derived neurotrophic factor, and removal of this neurotrophin causes apoptotic cell death accompanied by an attempt to reenter the cell cycle. Differentiated cells express neuronal markers, including neurofilaments, neuron‐specific enolase, and growth‐associated protein‐43 as well as neuronal polarity markers such as tau and microtubule‐associated protein 2. Moreover, differentiated cultures do not contain glial cells, as could be evidenced after the negative staining for glial fibrillary acidic protein. In conclusion, the protocol presented herein yields homogeneous populations of human neuronal differentiated cells that present many of the characteristics of primary cultures of neurons. This model may be useful to perform large‐scale biochemical and molecular studies due to its susceptibility to genetic manipulation and the availability of an unlimited amount of cells.

Adenosine released by astrocytes contributes to hypoxia-induced modulation of synaptic transmission

Astrocytes play a critical role in brain homeostasis controlling the local environment in normal as well as in pathological conditions, such as during hypoxic/ischemic insult. Since astrocytes have recently been identified as a source for a wide variety of gliotransmitters that modulate synaptic activity, we investigated whether the hypoxia‐induced excitatory synaptic depression might be mediated by adenosine release from astrocytes. We used electrophysiological and Ca2+ imaging techniques in hippocampal slices and transgenic mice, in which ATP released from astrocytes is specifically impaired, as well as chemiluminescent and fluorescence photometric Ca2+ techniques in purified cultured astrocytes. In hippocampal slices, hypoxia induced a transient depression of excitatory synaptic transmission mediated by activation of presynaptic A1 adenosine receptors. The glia‐specific metabolic inhibitor fluorocitrate (FC) was as effective as the A1 adenosine receptor antagonist CPT in preventing the hypoxia‐induced excitatory synaptic transmission reduction. Furthermore, FC abolished the extracellular adenosine concentration increase during hypoxia in astrocyte cultures. Several lines of evidence suggest that the increase of extracellular adenosine levels during hypoxia does not result from extracellular ATP or cAMP catabolism, and that astrocytes directly release adenosine in response to hypoxia. Adenosine release is negatively modulated by external or internal Ca2+ concentrations. Moreover, adenosine transport inhibitors did not modify the hypoxia‐induced effects, suggesting that adenosine was not released by facilitated transport. We conclude that during hypoxia, astrocytes contribute to regulate the excitatory synaptic transmission through the release of adenosine, which acting on A1 adenosine receptors reduces presynaptic transmitter release. Therefore, adenosine release from astrocytes serves as a protective mechanism by down regulating the synaptic activity level during demanding conditions such as transient hypoxia.

Barriers to Non-Viral Vector-Mediated Gene Delivery in the Nervous System

ABSTRACTEfficient methods for cell line transfection are well described, but, for primary neurons, a high-yield method different from those relying on viral vectors is lacking. Viral transfection has several drawbacks, such as the complexity of vector preparation, safety concerns, and the generation of immune and inflammatory responses when used in vivo. However, one of the main problems for the use of non-viral gene vectors for neuronal transfection is their low efficiency when compared with viral vectors. Transgene expression, or siRNA delivery mediated by non-viral vectors, is the result of multiple processes related to cellular membrane crossing, intracellular traffic, and/or nuclear delivery of the genetic material cargo. This review will deal with the barriers that different nanoparticles (cationic lipids, polyethyleneimine, dendrimers and carbon nanotubes) must overcome to efficiently deliver their cargo to central nervous system cells, including internalization into the neurons, interaction with intracellular organelles such as lysosomes, and transport across the nuclear membrane of the neuron in the case of DNA transfection. Furthermore, when used in vivo, the nanoparticles should efficiently cross the blood-brain barrier to reach the target cells in the brain.

Glitazones Differentially Regulate Primary Astrocyte and Glioma Cell Survival INVOLVEMENT OF REACTIVE OXYGEN SPECIES AND PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR-γ

The glitazones or thiazolidinediones are ligands of the peroxisome proliferator-activated receptor γ (PPARγ). The glitazones are used in the treatment of diabetes, regulate adipogenesis, inflammation, cell proliferation, and induce apoptosis in several cancer cell types. High grade astrocytomas are rapidly growing tumors derived from astrocytes, for which new treatments are needed. We determined the effects of two glitazones, ciglitazone and the therapeutic rosiglitazone, on the survival of serum-deprived primary rat astrocytes and glioma cell lines C6 and U251, which were assessed by the methylthiazolyl tetrazolium assay and lactate dehydrogenase release. Rosiglitazone (5–20 μm) decreased survival of glioma cells without affecting primary astrocytes, whereas ciglitazone at 20 μm was toxic for both cell types. Ciglitazone at 10 μm was cytoprotective for primary astrocytes but toxic to glioma cells. Cell death induced by ciglitazone, but not rosiglitazone, presented apoptotic features (Hoechst staining and externalization of phosphatidylserine). Two mechanisms to explain cytotoxicity were investigated: activation of PPARγ and production of reactive oxygen species (ROS). PPARγ does not seem to be the main mechanism involved, because the order of efficacy for cytotoxicity, ciglitazone > rosiglitazone, was inverse of their reported affinities for activating PPARγ. In addition, GW9662, an inhibitor of PPARγ, only slightly attenuated cytotoxicity. However, the rapid increase in ROS production and the marked reduction of cell death with the antioxidants ebselen and N-acetylcysteine, indicate that ROS have a key role in glitazone cytotoxicity. Ciglitazone caused a dose-dependent and rapid loss (in minutes) of mitochondrial membrane potential in glioma cells. Therefore, mitochondria are a likely source of ROS and early targets of glitazone cytotoxicity. Our results highlight the potential of rosiglitazone and related compounds for the treatment of astrogliomas.

Brain-derived neurotrophic factor modulates the severity of cognitive alterations induced by mutant huntingtin: Involvement of phospholipaseCγ activity and glutamate receptor expression

The involvement of brain-derived neurotrophic factor (BDNF) in cognitive processes and the decrease in its expression in Huntingtons disease suggest that this neurotrophin may play a role in learning impairment during the disease progression. We therefore analyzed the onset and severity of cognitive deficits in two different mouse models with the same mutant huntingtin but with different levels of BDNF (R6/1 and R6/1:BDNF+/- mice). We observed that BDNF modulates cognitive function in different learning tasks, even before the onset of motor symptoms. R6/1:BDNF+/- mice showed earlier and more accentuated cognitive impairment than R6/1 mice at 5 weeks of age in discrimination learning; at 5 weeks of age in procedural learning; and at 9 weeks of age in alternation learning. At the earliest age at which cognitive impairment was detected, electrophysiological analysis was performed in the hippocampus. All mutant genotypes showed reduced hippocampal long term potentiation (LTP) with respect to wild type but did not show differences between them. Thus, we evaluated the involvement of BDNF-trkB signaling and glutamate receptor expression in the hippocampus of these mice. We observed a decrease in phospholipaseCgamma activity, but not ERK, in R61, BDNF+/- and R6/1:BDNF+/- hippocampus at the age when LTP was altered. However, a specific decrease in the expression of glutamate receptors NR1, NR2A and GluR1 was detected only in R6/1:BDNF+/- hippocampus. Therefore, these results show that BDNF modulates the learning and memory alterations induced by mutant huntingtin. This interaction leads to intracellular changes, such as specific changes in glutamate receptors and in BDNF-trkB signaling through phospholipaseCgamma.

Glial Cell Line-derived Neurotrophic Factor Increases Intracellular Calcium Concentration ROLE OF CALCIUM/CALMODULIN IN THE ACTIVATION OF THE PHOSPHATIDYLINOSITOL 3-KINASE PATHWAY

Moderate increases of intracellular Ca2+ concentration ([Ca2+]i), induced by either the activation of tropomyosin receptor kinase (Trk) receptors for neurotrophins or by neuronal activity, regulate different intracellular pathways and neuronal survival. In the present report we demonstrate that glial cell line-derived neurotrophic factor (GDNF) treatment also induces [Ca2+]i elevation by mobilizing this cation from internal stores. The effects of [Ca2+]i increase after membrane depolarization are mainly mediated by calmodulin (CaM). However, the way in which CaM exerts its effects after tyrosine kinase receptor activation remains poorly characterized. It has been reported that phosphatidylinositol 3-kinase (PI 3-kinase) and its downstream target protein kinase B (PKB) play a central role in cell survival induced by neurotrophic factors; in fact, GDNF promotes neuronal survival through the activation of the PI 3-kinase/PKB pathway. We show that CaM antagonists inhibit PI 3-kinase and PKB activation as well as motoneuron survival induced by GDNF. We also demonstrate that endogenous Ca2+/CaM associates with the 85-kDa regulatory subunit of PI 3-kinase (p85). We conclude that changes of [Ca2+]i, induced by GDNF, promote neuronal survival through a mechanism that involves a direct regulation of PI 3-kinase activation by CaM thus suggesting a central role for Ca2+ and CaM in the signaling cascade for neuronal survival mediated by neurotrophic factors.

Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria

In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O2−) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. Addition of the reactive oxygen species (ROS) generator KO2 produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca2+ independent. Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O2− production detected with hydroethidine in mitochondrial preparations exposed to Ca2+ and the fact that ROS were required for Ca2+‐induced mitochondrial swelling. Superoxide anions, but not Ca2+, decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. Calcium, but not O2−, triggered a rapid loss of mitochondrial potential. Calcium‐induced Δψm dissipation was inhibited by Ruthenium Red, but not by CSA. Calcium‐ and superoxide‐induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A‐sensitive manner. In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.

Bcl-x L blocks mitochondrial multiple conductance channel activation and inhibits 6-OHDA-induced death in SH-SY5Y cells

Apoptosis is an active process that is regulated by different signalling pathways. One of the more important organelles involved in apoptosis regulation is the mitochondrion. Electron chain transport disruption increases free radical production leading to multiple conductance channel opening, release of cytochrome c and caspase activation. This death pathway can be blocked by anti‐apoptotic members of the Bcl‐2 protein family that might shift redox potential to a more reduced state, preventing free radical‐mediated damage. 6‐Hydroxydopamine (6‐OHDA) has been widely used to generate Parkinsons disease‐like models. It is able to generate free radicals and to induce catecholaminergic cell death. In this paper we have used the human neuroblastoma cell line SH‐SY5Y overexpressing Bcl‐xL as a model to gain insights into the mechanisms through which Bcl‐xL blocks 6‐OHDA‐induced cell death and to identify the molecular targets for this action. Herein, we present evidence supporting that the Bcl‐xL–anti‐apoptotic signal pathway seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase‐3 like activity following 6‐OHDA treatment in the human neuroblastoma cell line SH‐SY5Y.

Lifeguard/neuronal membrane protein 35 regulates Fas ligand-mediated apoptosis in neurons via microdomain recruitment

Fas ligand (FasL)‐receptor system plays an essential role in regulating cell death in the developing nervous system, and it has been implicated in neurodegenerative and inflammatory responses in the CNS. Lifeguard (LFG) is a protein highly expressed in the hippocampus and the cerebellum, and it shows a particularly interesting regulation by being up‐regulated during postnatal development and in the adult. We show that over‐expression of LFG protected cortical neurons from FasL‐induced apoptosis and decreased caspase‐activation. Reduction of endogenous LFG expression by small interfering RNA sensitized cerebellar granular neurons to FasL‐induced cell death and caspase‐8 activation, and also increased sensitivity of cortical neurons. In differentiated cerebellar granular neurons, protection from FasL‐induced cell death could be attributed exclusively to LFG and appears to be independent of FLICE inhibitor protein. Thus, LFG is an endogenous inhibitor of FasL‐mediated neuronal death and it mediates the FasL resistance of CNS differentiated neurons. Finally, we also demonstrate that LFG is detected in lipid rafts microdomains, where it may interact with Fas receptor and regulate FasL‐activated signaling pathways.

An activity-dependent switch from facilitation to inhibition in the control of excitotoxicity by group I metabotropic glutamate receptors.

Activation of group I metabotropic glutamate receptors (mGlu1 or ‐5 receptors) is known to either enhance or attenuate excitotoxic neuronal death depending on the experimental conditions. We have examined the possibility that these receptors may switch between two different functional modes in regulating excitotoxicity. In mixed cultures of cortical cells, the selective mGlu1/5 agonist, 3,5‐dihydroxyphenylglycine (DHPG), amplified neurodegeneration induced by a toxic pulse of NMDA. This effect was observed when DHPG was either combined with NMDA or transiently applied to the cultures prior to the NMDA pulse. However, two consecutive applications of DHPG consistently produced neuroprotection. Similar effects were observed with DHPG or quisqualate (a potent agonist of mGlu1/5 receptors) in pure cultures of cortical neurons virtually devoid of astrocytes. In cultures of hippocampal pyramidal neurons, however, only protective effects of DHPG were seen suggesting that, in these particular cultures, group I mGlu receptors were endogenously switched into a ‘neuroprotective mode’. The characteristics of the activity‐dependent switch from facilitation to inhibition were examined in mixed cultures of cortical cells. The switch in the response to DHPG was observed when the two applications of the drug were separated by an interval ranging from 1–45 min, but was lost when the interval was extended to 90 min. In addition, this phenomenon required the initial activation of mGlu5 receptors (as indicated by the use of subtype‐selective antagonists) and was mediated by the activation of protein kinase C. We conclude that group I mGlu receptors are subjected to an activity‐dependent switch in regulating excitotoxic neuronal death and, therefore, the recent ‘history’ of these receptors is critical for the response to agonists or antagonists.