6-cyano-7-nitroquinoxaline-2-3-dione and Neuroblastoma

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H(2)O(2), 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Animals; Apoptosis; Autophagy; Caspase 3; Cell Line, Tumor; Cell Survival; Cerebral Cortex; Dizocilpine Maleate; Dose-Response Relationship, Drug; Drug Interactions; Electric Stimulation; Enzyme Inhibitors; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Humans; In Vitro Techniques; Insecticides; Kaempferols; Membrane Potential, Mitochondrial; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Neuroblastoma; Neuroprotective Agents; Oxygen Consumption; Protein Carbonylation; Rats; Rats, Wistar; Reactive Oxygen Species; Rotenone; Time Factors; Transfection

Parkinson's disease (PD) is the most common representative of a group of disorders known as synucleinopathies, in which misfolding and aggregation of α-synuclein (a-syn) in various brain regions is the major pathological hallmark. Indeed, the motor symptoms in PD are caused by a heterogeneous degeneration of brain neurons not only in substantia nigra pars compacta but also in other extrastriatal areas of the brain. In addition to the well known motor dysfunction in PD patients, cognitive deficits and memory impairment are also an important part of the disorder, probably due to disruption of synaptic transmission and plasticity in extrastriatal areas, including the hippocampus. Here, we investigated the impact of a-syn aggregation on AMPA and NMDA receptor-mediated rat hippocampal (CA3-CA1) synaptic transmission and long-term potentiation (LTP), the neurophysiological basis for learning and memory. Our data show that prolonged exposure to a-syn oligomers, but not monomers or fibrils, increases basal synaptic transmission through NMDA receptor activation, triggering enhanced contribution of calcium-permeable AMPA receptors. Slices treated with a-syn oligomers were unable to respond with further potentiation to theta-burst stimulation, leading to impaired LTP. Prior delivery of a low-frequency train reinstated the ability to express LTP, implying that exposure to a-syn oligomers drives the increase of glutamatergic synaptic transmission, preventing further potentiation by physiological stimuli. Our novel findings provide mechanistic insight on how a-syn oligomers may trigger neuronal dysfunction and toxicity in PD and other synucleinopathies.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Synuclein; Animals; Biophysics; Biotinylation; Cell Line, Tumor; Excitatory Amino Acid Antagonists; Excitatory Postsynaptic Potentials; Extracellular Fluid; Hippocampus; Humans; Insulin; L-Lactate Dehydrogenase; Long-Term Potentiation; Male; Neuroblastoma; Organ Culture Techniques; Patch-Clamp Techniques; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Synapses; Synaptic Transmission; Valine

Impaired zinc homeostasis is implicated in many cases of brain injury and pathogenesis. While several routes of zinc influx have been identified in neurons under depolarizing conditions, zinc uptake mechanisms during resting conditions are unknown. We have previously detected Zip6 at the plasma membrane of rat neurons, suggesting a role for Zip6 in neuronal zinc uptake. Zinc uptake under resting and depolarizing membrane potentials was measured in SH-SY5Y neuroblastoma cells using 65Zn. Zinc uptake was higher under depolarizing conditions, compared with resting conditions, and could be reduced by high extracellular calcium, gadolinium, or nimodipine, which suggests that L-type calcium channels are significant routes of zinc uptake under depolarizing membrane potential. In contrast, zinc uptake under resting conditions was not affected by calcium or calcium channel antagonists. Zip6 was localized to the plasma membrane in SH-SY5Y cells, and siRNA-mediated down-regulation of Zip6 expression reduced zinc uptake during resting, but not depolarizing conditions. Zinc treatment (100 microM Zn) reduced zinc uptake under resting, but not depolarizing conditions, which was associated with lower plasma membrane-associated and total Zip6 protein abundance. These results demonstrate that Zip6 functions as a zinc import protein in neuroblastoma cells, that zinc influx during resting and depolarizing conditions occurs via distinctly different processes in these cells, and suggest that neuronal zinc uptake may be down-regulated by excess zinc levels, but only under resting conditions.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; Analysis of Variance; Calcium; Calcium Channel Blockers; Cation Transport Proteins; Cell Line, Tumor; Dizocilpine Maleate; Drug Combinations; Excitatory Amino Acid Antagonists; Gadolinium; Gene Expression Regulation, Neoplastic; Humans; Membrane Potentials; Neoplasm Proteins; Neuroblastoma; Protein Transport; RNA, Small Interfering; Time Factors; Zinc

Positive allosteric modulation of AMPA receptor function has therapeutic potential in a number of psychiatric disorders and neurodegenerative diseases. AMPA receptor potentiators can induce neurite sprouting in vivo. Using a strategy of combined morphological and biochemical analyses, we investigated the effect of the AMPA receptor potentiator LY404187 on neurite growth in the SH-SY5Y human neuroblastoma cell line. LY404187 (0.1-10 microM) increased average neurite length and neurofilament expression when co-administered with s-AMPA. Co-incubation with s-AMPA and LY404187 also increased Trk receptor expression. All actions of LY404187 were sensitive to AMPA receptor blockade by the selective antagonist CNQX (10 microM). Antibody sequestration of BDNF attenuated neurite growth following AMPA receptor potentiator administration, suggesting that LY404187 increases neurite length in vitro by a BDNF mediated mechanism. AMPA receptor potentiation activates multiple intracellular neurochemical cascades and the present report identifies BDNF as one key mediator of the neurotrophic effects of AMPA receptor potentiation.

Topics: 6-Cyano-7-nitroquinoxaline-2,3-dione; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Antibodies; Brain-Derived Neurotrophic Factor; Cell Line, Tumor; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Gene Expression Regulation; Humans; Neurites; Neuroblastoma; Neurofilament Proteins; Receptor, trkA; Receptors, AMPA; Sulfonamides