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

Cholesterol-Rich Plasma Membrane Submicrodomains Can Be a Major Extramitochondrial Source of Reactive Oxygen Species in Partially Depolarized Mature Cerebellar Granule Neurons in Culture Sofia Fortalezas1, Joana Poejo1, Alejandro K Samhan-Arias2 and Carlos Gutierrez-Merino1* 1Department of Biochemistry and Molecular Biology, Faculty of Sciences, and Institute of Molecular Pathology Biomarkers, University of Extremadura, 06006Badajoz, Spain 2Department of Biochemistry, Autonomous University of Madrid (UAM), and ‘Alberto Sols’ Biomedical Research Institute (CSIC-UAM), c / Arturo Duperier 4, 28029-Madrid, Spain Research Open Access Journal of Neurophysiology and Neurological Disorders


Introduction
Oxidative stress induced by excessive production of reactive oxygen and nitrogen species can elicit neuronal cell death and brain neurodegeneration. This has been shown during last 20 years with neuronal cultures in vitro [1][2][3], with animal models of neurodegenerative diseases [4,5] and brain insults like transient ischemia [6,7], and also in human brains after brain stroke [8] or affected by major neurodegenerative diseases [2,[9][10][11][12]. At least in part, this is due to the fact that the cerebrospinal fluid is poorer in antioxidants than the blood, because of the low permeability and high selectivity of the blood-cerebrospinal fluid barrier.
Studies with primary cultures of cerebellar granule neurons (CGN) have shown that an overshot of reactive oxygen species (ROS) production plays a critical role for the entry in the irreversible stage of low potassium-induced apoptosis [13][14][15].
Noteworthy, the peak of this ROS overshot was at approximately 3 h after the change of CGN to low potassium medium, well before the drop of mitochondrial membrane potential and before a significant release of cytochrome c from mitochondria to the cytosol [16]. Furthermore, the addition of superoxide dismutase (SOD) to the extracellular medium of CGN cultures quenches more than 90% of this ROS overshot and also blocks the apop-totic process [13,14]. As superoxide anion has a low permeability across lipid bilayers [17], this ROS overshot must have a large component of superoxide anion released to the extracellular medium. Therefore, although mitochondria is widely accepted as the major source of intracellular superoxide anion in oxidative stress-induced neuronal death in cultures in vitro [2,3,18], extramitochondrial ROS production seems to play a major role in the superoxide anion overshot at the onset of CGN apoptosis induced by potassium deprivation of the extracellular medium.
On these grounds, the possibility that redox systems of the plasma membrane may be a major sub cellular source of superoxide anion deserves to be studied.
Plasma membrane NADPH oxidases of the NADPH oxidases family play a major role in superoxide anion production in glial, macrophages and endothelial cells [19]. However, we have reported previously that in brain synaptic membranes derived from rat synaptosomes and in CGN cultures the NA-DPH-dependent superoxide anion production is nearly ten-fold lower than the NADH-dependent superoxide anion production [14,20,21]. In previous works, we have shown that dysregulation of cytochrome b 5 reductase (Cb5R) associated with plasma membrane lipid rafts sub-microdomains is largely responsible for the overshot of superoxide anion observed at the onset of CGN apoptosis induced by 5 mM potassium concentration in the extracellular medium [16,22]. Briefly, our results showed that the mRNA levels of both Cb5R and cytochrome b 5 increases nearly three-fold and an enhanced translocation of Cb5R to the neuronal plasma membrane within 1-3 hours after switching CGN to a 5 mM K + medium [16]. The increase of the level of cytochrome b 5 can account for the stimulation of the NADH activity of the plasma membrane of CGN in this time window [14] because cellular cytochrome b 5 concentration is not saturating for Cb5R. It is to be noted that this correlated with the three to four-fold increase of superoxide anion production measured three hours after the change of CGN to a pro-apoptotic low potassium medium [16].
We have shown that the isoform 3 of cytochrome b 5 reductase (Cb5R3) and cytochrome b 5 are highly expressed in the cerebellar granule neurons of the adult rat brain, and also in cerebellar Purkinje cells and pyramidal neurons of the brain neocortex and neuronal motor nuclei of the brain stem [23]. Therefore, dysregulation of Cb5R3 is also likely to play a widespread role in brain neuronal degeneration in oxidative stress-mediated apoptosis and not only in CGN apoptosis. It is to be recalled here that over 40 mutations of Cb5R3 have been linked to recessive congenital type II methemoglobinemia [24,25]. In persons affected by this rare disease mild cyanosis is accompanied by generalized dystonia, movement disorders, failure to thrive, and cortical and subcortical atrophy [24][25][26][27], including cerebellar atrophy [28].
Methyl-β-cyclodextrin (MβCD) has been extensively used for cholesterol removal and lipid rafts disruption in different cell lines [36,37], and also in mature CGN in culture in a recent study of our laboratory [38]. Noteworthy, cyclodextrins are also used as antibrowning additives in foods and foodstuffs [39], as well as additives to improve the bioavailability of poorly soluble drugs [40,41].
The main goal of this work has been to evaluate in partially depolarized CGN cultures the contribution of ROS production and in particular of superoxide anion by redox systems associated with lipid rafts of the plasma membrane relative to mitochondrial production of these ROS. This study has been performed with mature CGN and also during maturation of CGN in culture, to seek for correlations between the increase of protein markers of lipid rafts, Cb5R and nNOS expression during CGN maturation and the overall ROS production and sensitivity to exposure to extracellular peroxynitrite. The results show that the redox systems associated with lipid rafts of the CGN plasma membrane can account for a production of ROS in partially depolarized CGN in culture much higher than usually recognized.

Preparation of rat cerebellar granule neurons (CGN)
CGN have been prepared from Wistar rats as described previously [14,22,30,31,38,[42][43][44] [31,38,44]. Only fields devoid of large aggregates of neurons forming granules or small grain-like structures were selected for image acquisition, to minimize distortion of images by the sum of the fluorescence contributions of juxtaposed layers of neurons. The mean ± s.e. intensity reading of fluorescence per pixel within CGN somas were obtained in experiments performed by triplicate (n>100 CGN somas in each case). 2' ,7'-dihydrodichloro fluorescein diacetate (H 2 D-CF-DA), a broad-range ROS indicator [45], has been used as an indicator of the overall oxidative stress. Dihydroethidium (DHE) has been used as an indicator of superoxide anion production [46,47]. 4,5-diaminofluorescein diacetate (DAF2-DA)has been used to monitor nitric oxide and ROS production upon the rapid reaction of nitric oxide with superoxide anion and ascorbate free radicals [48]. For image acquisition in this work, these dyes were added at the following concentrations: 10 µM (H 2 DCF-DA and DHE) and 5 µM (DAF2-DA). Images of CGN stained with

Fluorescence Resonance Energy Transfer (FRET) imaging
FRET imaging has been performed as in previous works [16,30,31,49], with CGN at the indicated days in vitro (DIV) in 24-well plates with 600,000 cells per well or in 35 mm diameter Petri plates with 2.5x10 6 cells per plate. The plates were washed twice with 1 mL MLocke's K25 buffer to wash the phenol red remaining in the plates. Then CGN were fixed with 2.5% paraformaldehyde, 3 mM MgCl These CGN plates will be named as (target protein-2)**Cy3. After finishing the acquisition of the images of these plates, CGN plates (target protein-1) **A488 were incubated during 1 h at 37 °C with the second primary antibody against target protein-2 in PBS supplemented with 0.2% Triton X-100, washed six times with PBS (washing step), and then incubated for 30 min with the appropriate Cy3-labelled secondary antibody in PBS supplemented with 0.2% Triton X-100, and finally washed again with PBS.
These CGN plates will be named as (target protein-1)**A488/ (target protein-2)**Cy3. The lack of significant fluorescence labelling of CGN treated with the Alexa488-and Cy3-secondary IgG antibodies used in this study in the absence of the primary antibodies was assessed before running FRET experiments, confirming that it yielded fluorescence intensity readings within the background range of control non-stained CGN. Control experiments were performed to quantify the effect of the treatment for labeling with the second primary antibody/IgG-Cy3 complex in the ratio between red and green fluorescence intensities (Ratio RF/GF) of CGN plates (target protein-1)**A488. To this end, CGN were subjected to treatments mimicking those followed for labeling with the second primary antibody (see above) but without the second primary antibody. These control experiments showed that on average the treatment done to label CGN plates  donor and acceptor dyes, plates (target protein-1)**A488/(target protein-2)**Cy3, were routinely started between 2 and 3 hours after finishing the acquisition of images of CGN stained only with the donor dye, plates (target protein-1)**A488. Finally, the exposure times were fixed at 0.3 s, i.e. as low as possible to ensure that the contribution of CGN autofluorescence was always lower than 10% of the fluorescence intensity readings of cells stained with the primary and secondary antibodies used in this work.
The average intensity of fluorescence per pixel within CGN somas were taken using the ROI tool of the Hamamatsu HC Image software to select somas as areas of interest in experiments performed by triplicate (n>200 CGN somas in each case).
Images of CGN stained only with anti-H-Ras/IgG-Cy3 (H-Ras*-Cy3) were acquired to substract the direct excitation component of the red fluorescence intensity per pixel. As discussed in previous works [30,31,49], significant FRET efficiency using this experimental approach implies that the selected protein pair is separated by ≤80 nm.

Cb5R3 silencing in CGN
For siRNA knockdown studies Accell™ siRNA delivery system was used (Dharmacon TM ). To knockdown Cb5R3 we used the E-089102-00-0005 Accell Rat Cyb5r3 (25035) siR-NA SMART pool and as control was used the D-001910-10-05 Accell Non-targeting pool. siRNAs were prepared following the instructions of the manufacturer. Briefly, 5x siRNA Buffer was diluted to 1x siRNA Buffer using sterile RNase-free water; a 100 μM siRNA solution was prepared in 1x siRNA buffer and up and down 3-5 times while avoiding introduction of bubbles; the solution was placed on an orbital mixer/shaker for 90 minutes at 37 °C and briefly centrifuged to collect solution from the bottom of the tube. In separate tubes the 100 μM siRNA was mixed with Accell Delivery Media 1:100 (delivery mix). CGN were prepared as previously described and at DIV 4 the growth medium was replaced by 500 μL of the appropriate delivery mix to each well (12 wells plate). Cells were incubated at 37 °C with 5% CO2 for 90 hours for Western blotting analysis, or by 72 hours for fluorescence microscopy measurements. Thereafter, cell lysates for Western Blotting or fluorescence microscopy measurements of ROS production with H 2 DCF-DA were performed as described above.

Statistical analysis
Results are expressed as mean ± standard error (s.e.).
Statistical analysis was carried out by Mann-Whitney non-parametric test. A significant difference was accepted at the p < 0.05 level. All the results were confirmed with at least triplicate measurements with different CGN preparations.  Figure 1. The intensity of fluorescence of these dyes is clearly higher in the neuronal somas. In addition, the long dendritic extensions connecting distant neuronal somas are more intensely stained with H 2 DCF-DA than with DHE. Indeed, these extensions are hardly seen with the red fluorescence of DHE staining. We have acquired images of DHE staining after preincubation for 1.5 hours with the superoxide anion scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron) [50,51], to evaluate the putative contribution to the measured DHE fluorescence of DNA-and RNA-oxidized ethidium complexes formation [45,46]. The results shown in Figure 1C

CGN maturation in vitro leads to an approximately 2 to 3-fold increase of the overall lipid rafts network of these neurons from 4 to 9 days in vitro, and a similar increase of the expression of nNOS.
CGN maturation has been experimentally assessed measuring the contribution of L-type calcium channels (LTCCs) activity to the cytosolic calcium homeostasis because of LTCCs activity increases during CGN maturation in the partially depolarized medium [52]. The decrease of the fluorescence ratio  Figure 4B). Moreover, quantitative fluorescence images acquired with the same exposure time and camera gain showed that they increase about two-fold during the in vitro CGN mat-10 uration process ( Figure 4B). This point was also confirmed with fluorescence intensity measurements of cell lysates using a fluorimeter ( Figure 4C). The increase is widespread within the neurons, but it is more readily seen at neuronal somas and interdendritic contact sites ( Figure 4B).  Figure   6). In this work, we have experimentally confirmed by FRET microscopy imaging that nNOS is also within FRET distance from H-Ras (Figure 7), i.e. both proteins co-localize within the same lipid rafts.

The production of ROS that is inhibited by MβCD decreased during CGN maturation and this is reverted by Cb5R3silencing
The production of ROS monitored by H 2 DCF-DA decreased between 3 and 4-fold from immature CGN at 5 DIV to mature CGN at 9-10 DIV, and this decrease largely accounts for the ROS production in CGN that is sensitive to MβCD ( Figure   8A). This result suggested that immature CGN should be more vulnerable to extracellular oxidative neurotoxic insults than mature CGN. We have experimentally assessed this point using 3-morpholinosydnonimine (SIN-1), a superoxide anion and nitric oxide donor that can trigger oxidative CGN death under controlled exposure conditions [43]. Loss of cell viability in- duced by CGN exposure to 0.5 mM SIN-1 in MLocke's K25 is reduced from 78±2% at 5 DIV to 49±2% at 9-10 DIV.
We have studied the effect of Cb5R3 silencing on ROS production by mature CGN following the protocol indicated in the Materials and Methods, Western blotting showed that we have achieved approximately 50% Cb5R3 silencing at the protein expression level with respect to control CGN, with a statistically non-significant 10-15% decrease of Cb5R3 induced by treatment with control-siRNA ( Figure 8B). This partial Cb5R3 silencing produced between 3.5 and 4.5-fold increase of ROS production relative to control and control-siRNA CGN, respectively, when monitored by H 2 DCF-DA ( Figure 8C). Furthermore, the flash-like increase of fluorescence intensity was also more than 90% attenuated by DPI ( Figure 9C)

Discussion
The results of this work show that a short-time treatment of CGN with the cholesterol-trapping agent MβCD strongly inhibits the rate of production of ROS by mature CGN in culture.
The total duration of the exposure of CGN to MβCD in our experiments has been 15-20 min because fluorescence microscopy images were acquired for 5-10 min after 5 min pre-incubation of CGN with MβCD before placing the Petri dishes in the fluorescence microscope and handlings to focus the selected field for images. As we have shown in a previous and recent publication [38],  [45,46], H 2 DCF-DA is a ROS probe that monitors a broad range of ROS [45] and DAF2-DA monitors not only nitric oxide but also the production of other ROS, like those generated upon the rapid reaction of nitric oxide with superoxide anion and ascorbate free radicals [48]. Noteworthy, the inhibition of ROS/RNS production monitored by H 2 DCF-DA and DAF2-DA is more sensitive to lower concentrations of MβCD than the inhibition of superoxide anion production monitored by DHE. H 2 D-CF-DA and DHE have been used to show the oxidative stress of CGN during the early stages of apoptosis [14][15][16]42], in L-glutamate-induced excitotoxic death [3,55,56] and in other oxidative stress-mediated neuronal death [45]. Furthermore, both dyes have been widely used to monitor oxidative-induced cell death in other neuronal cultures. On these grounds, we can conclude that this treatment elicits a generalized and strong impairment of the production of the most relevant ROS involved in oxidative stress-inducing conditions that are neurotoxic for these neurons in culture.
Therefore, these results suggested a major contribution of lipid rafts to overall ROS/RNS production in our mature CGN cultures. The rapid and nearly complete inhibition of the ROS production monitored by DHE and H 2 DCF-DA by extracellularly added SOD strongly support this hypothesis, because SOD does not permeate across the plasma membrane. Furthermore, this result indicates a major role of superoxide anion in the generation of another ROS produced in CGN and detected by H 2 D-CF-DA. Indeed, the lag phase in the production of ROS detected by H 2 DCF-DA, in contrast with the absence of lag phase in the production of superoxide anion monitored with DHE, also gives additional experimental support to this conclusion. A major role of extramitochondrial production of superoxide anion and of ROS detected by H 2 DCF-DA is supported also by the fact that the mitochondrial uncoupling protonophores FCCP and DNP produced less than 50% inhibition of the production of these ROS in mature CGN.
Previous works have shown that deregulation of several redox systems associated with neuronal lipid rafts can produce over shots of ROS and/or RNS, namely, superoxide anion by Cb5R3 [57,58], nitric oxide by nNOS [30,31,59,60], and hydrogen peroxide and superoxide anion by cytochromes P450 [61]. All these redox systems are flavoproteins, and DPI, a broad spectrum flavoprotein inhibitor, produced more than 90% inhibition of ROS/RNS production monitored by the dyes used in this work. In previous works, we have shown that both Cb5R3 and nNOS are present in caveolin-1/cholesterol-rich lipid rafts of mature CGN [16,30,31,49]. However, the expression of many cytochromes P450 is strongly downregulated in primary neuronal cultures maturated in vitro [62], as well as in other growth-stimulated mammalian cell cultures [63]. To experimentally assess this point in our CGN culture we selected the CYP46 isoform of cytochrome P450, which has 24S-cholesterol hydroxylase activity and is a cytochrome P450 isoform that should be expected to be associated with neuronal lipid rafts owing to its important role in brain cholesterol homeostasis [64]. As shown by Western blotting, during CGN maturation in the culture there is strong downregulation of the expression of CYP46 ( Supplementary Figure S3), and as a result, its expression level in mature CGN is negligible. Noteworthy, we have shown in previous works that there are large differences between the MβCD concentrations needed to inhibit two major calcium systems involved in the control of cytosolic calcium homeostasis of CGN, e.g. LTCCs and N-methyl D-aspartate receptors [31,38]. Therefore, the differences between Fluorescence microscopy images of CGN stained with fluorescent cholera toxin B and Western blotting of H-Ras content show more than two-fold increase of lipid rafts per neuron during the maturation of CGN cultures in vitro. Noteworthy, these increases are similar to the increase during maturation of CGN of redox systems associated with these lipid rafts, namely, ~2.5-fold increase of Cb5R3 [16,22] and ~2.5-fold increase of nNOS (this work). This latter result is in good agreement with the results reported by others during cerebellum maturation [65,66].
Moreover, FRET imaging results show that nNOS and the protein lipid rafts marker H-Ras are within the FRET distance range for the experimental approach used in this work. As discussed in previous works of our group [30,31,49],using this experimental FRET approach with fluorescent antibodies this means that they are at a distance ≤80 nm. Therefore, we can conclude that both nNOS and H-Ras are within the same lipid rafts. In previous works, we reached the same conclusion for Cb5R3 [31,49].
On the other hand, mature CGN at 9-10DIV are more resistant than immature CGN at 5DIV to the oxidative insult produced by extracellular exposure to SIN-1, a ROS/RNS releasing compound [67,68] that has been shown to mimic the extracellular oxidative stress associated with inflammation and ischemia-reperfusion in mammalian brain [32,68,69]. Consistently, we found that CGN maturation leads to a large decrease of the production of ROS detected by H 2 DCF-DA. This attenuation of ROS production in mature CGN can be reverted by silencing the expression of Cb5R3 to approximately 50%. Cb5R3 is a redox protein associated with lipid rafts, whose expression level increases approximately 2.5-fold from 5DIV to 9-10DIV [22], and whose deregulation leads to an overshot of superoxide anion production by CGN [16] and also of rat brain synaptic plasma membranes [14,57]. Therefore, our results show that Cb5R3 plays a major role in the attenuation of the overall ROS production in mature CGN, and reveal a major role of this protein as an antioxidant cellular defense against CGN exposure to extracellular neurotoxic oxidative insults.
DAF2 is a fluorescent dye that, besides being useful for nitric oxide detection under controlled experimental conditions [53,54], it has been shown to be largely photoactivated upon the irradiation with the 490nm light of a fluorescence microscope xenon lamp [54]. A remarkable photoactivation pattern is displayed by CGN stained with DAF2-DA upon irradiation with the fluorescence microscope Xenon lamp 470 nm blue light. The kinetics of ROS production by CGN detected with DAF2-DA is more than 90% inhibited by CGN pre-treatment with MβCD or DPI, as also found for the ROS production monitored with H 2 D-CF-DA. However, DAF2-DA revealed the occurrence of a clear space-temporal pattern of the increase of fluorescence within the CGN culture. Sequential images showed that a flash-like peak of fluorescence initiates at the center of the field irradiated with the light beam and rapidly spread away across neighbor neurons and through the dendritic extensions. This is a spreading fluorescent wave induced by ROS/RNS because it is approximately 70% attenuated by the peroxynitrite scavenger MnTBAP, and it is completely blocked by addition of SOD plus catalase to the extracellular medium. In addition, the latter result points out that it is largely due to ROS produced at or near the neuronal plasma membrane. Thus, ROS propagation in the culture is not isotropic, and the images sequences strongly suggest that ROS propagates along the neuronal plasma membrane. Taking into account that images were acquired every 5 s and the fluorescence propagation distance observed between sequential images in dendritic extensions, we can calculate a ROS propagation rate higher than 10 µm/s. The possibility that this was reflecting a depolarizing wave can be excluded because it is not affected by preincubation of the CGN with 1 µM tetrodotoxin (Supplementary Figure S4).
DAF2 complexation with calcium may also enhance the fluorescence of this dye [54]. However, the flash-like peak of DAF2-DA fluorescence was not affected by preincubation of CGN with 10 µM nifedipine nor by 10 µM MK-801, i.e. by inhibitors of LTCCs and N-methyl D-aspartate receptors which are the two major calcium entry systems present in the plasma membrane of these neurons (Supplementary Figure S4). Therefore, these results re- In summary, the results of this work unravel a major role of lipid rafts of the plasma membrane to the control of the overall ROS production by CGN in culture under partially depolarizing conditions that promote neuronal survival, with a major contribution of the lipid raft-associated Cb5R3 in this biological function. Moreover, lipid rafts of the plasma membrane also provide key structural elements to speed up ROS signaling propagation between neurons in mature CGN in culture.