Cholesterol-Rich Plasma Membrane Submicrodomains Can Be a Major Extram- itochondrial Source of Reactive Oxygen Species in Partially Depolarized Mature Cerebellar Granule Neurons in Culture

Abstract

Excessive production of reactive oxygen and nitrogen species can elicit neuronal cell death and brain neurodegeneration. In previous studies, we have shown that lipid rafts nanodomains of mature cerebellar granule neurons (CGN) contain most of the neuronal nitric oxide synthase and also of cytochrome b 5 reductase isoform 3 (Cb5R3), which is the component of neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c. In this work, we show that CGN maturation in vitro elicits between two-and three-fold increase of the overall lipid rafts, and a similar increase of the expression of neuronal nitric oxide synthase and Cb5R3. Only 5-15 min incubation of CGN with millimolar concentrations of methyl-β-cyclodextrin led to more than 90% attenuation of the kinetics of reactive oxygen species (ROS) production monitored by dihydro dichlorofluorescein diacetate (H 2 DCF-DA), dihydroethidium (DHE) and 4,5-diaminofluorescein diacetate (DAF2-DA). In contrast, the results obtained using H 2 DCF-DA and DHE pointed out that preincubation of CGN with 2,4-dinitrophenol or carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP) only produced a decrease of 38±5% and 42±5% of the rate of ROS production, respectively. In addition, superoxide dismutase added to the extracellular medium of CGN also elicited more than 90% attenuation of the increase of DHE fluorescence that monitors superoxide anion production. CGN maturation leads to a large decrease of the production of ROS detected by H 2 DCF-DA, and this attenuation of ROS production in mature CGN can be reverted by silencing the expression of Cb5R3 to approximately 50%. The kinetics of ROS/reactive nitrogen species (RNS) production by CGN detected with DAF2-DA revealed the occurrence of a spreading ROS/RNS wave rapidly propagating along the neuronal plasma membrane. In conclusion, our results point out that redox systems associated with lipid rafts make a large contribution to total ROS/RNS production in mature CGN cultures under partially depolarizing conditions.

Internalized Amyloid-β (1-42) Peptide Inhibits the Store-Operated Calcium Entry in HT-22 Cells

“…Increasing evidence suggests that Aβ peptides are internalized in neuronal cells through different pathways, which also depend on the cell type [ 22 , 58 , 59 ]. It has been reported that neuronal lipid rafts microdomains play a major role in Aβ formation/oligomerization and Aβ uptake by neurons, reviewed in [ 60 ], and we showed in a previous study that mature cerebellar granule neurons express high levels of lipid rafts markers like cholera toxin B binding sites, caveolin-1, flotillin, and HRas [ 61 , 62 ]. In contrast, we found in this work that HT-22 cells were not stained with cholera toxin B-Alexa488, demonstrating the lack of lipid rafts in this mouse hippocampal cell line HT-22 [ 63 ].…”

Store-Operated Calcium Entry Inhibition and Plasma Membrane Calcium Pump Upregulation Contribute to the Maintenance of Resting Cytosolic Calcium Concentration in A1-like Astrocytes

“…Green fluorescence (GF) images of Control and A1 cells were acquired with an excitation filter of 470 nm and a 510 nm dichroic mirror/520 nm emission filter, using the exposure times indicated for each case in the legends of the figures. Quantitative analysis of the average fluorescence intensity per pixel of selected neuronal soma was performed via HCImage software using the ROI tool, as in previous works [ 30 , 60 , 61 , 62 ].…”