Endosomal Accumulation of Lipids in U18666a- Induced NPC Leads to Hyperphosphorylation of GFAP and Vimentin in Culture of Astrocytes
Published Date: October 07, 2016
Endosomal Accumulation of Lipids in U18666a- Induced NPC Leads to Hyperphosphorylation of GFAP and Vimentin in Culture of Astrocytes
Daniela C. Santos1*, Carolina G. Fernandes2, Pierozan P2, Regina P. Pureur2 and Janice C. Coelho1
1Biochemistry Department, Lysosomal Storage Diseases Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
2Biochemistry Department, Instituto de Ciencias Basicas da Saude, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil
*Corresponding author: Daniela Copetti Santos, Biochemistry Department, Lysosomal Storage Diseases Laboratory, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil, E-mail: firstname.lastname@example.org
Citation: Santos DC, Fernandes CG, Pierozan P, Pureur RP, Coelho JC (2016) Endosomal Accumulation of Lipids in U18666a- Induced NPC Leads to Hyperphosphorylation of GFAP and Vimentin in Culture of Astrocytes. Enzym Metab J 1(1): 105.
U18666A is a drug that had been used in several models both in vivo and in vitro to mimic Niemann-Pick type C disease (NPC). MK-801 is an uncompetitive NMDA (N-Methyl-D-aspartate) receptor, a receptor of glutamate which is one of the brain’s primary neurotransmitters. Recent studies have reported the toxicity of glutamate on neuron cells (excitotoxicity). The goal of the present study is to observe the hyperphosphorylation of intermediate filaments (IF) GFAP (Glial fibrillary acidic protein) and vimentin in astrocyte cultures from Wistar rats, zero to three days old. These analyses were carried out using four different groups based on whether or not they used the U18666A drug or MK-801. Analyses in these groups show that U18666A, after causing an increase in cell cholesterol, leads to hyperphosphorylation of the intermediate filaments, both GFAP and vimentin. However, MK-801, being an NMDA antagonist and acting on the glutamate receptors, was shown to inhibit the excitotoxicity caused by U18666A. The results obtained may enable inferring that the substances to facilitate decrease in excitotoxicity caused by the higher cholesterol levels observed in NPC, such as the NMDA receptor antagonist MK-801, can be viable tools to aid in the treatment of neurodegenerative diseases related to glutamatergic excitotoxicity.
Keywords: U18666A; Niemann-Pick Disease; Cholesterol; Glutamate; Intermediate Filaments
Niemann-Pick type C disease (NPC) is characterized as a neurodegenerative disease caused by changes in lipid metabolism, particularly of cholesterol. Cholesterol levels are highly regulated among neurons and glia that changes in lipid metabolism, especially of cholesterol, lead to an imbalance causing NPC [1,2]. Lipoproteins and cholesterol in the central nervous system (CNS) do not come from peripheral blood, but are instead synthesized by glial cells, mainly astrocytes .
Glial fibrilary acidic protein (GFAP) is the main intermediate filament (IF) of mature astrocytes, thus it is considered a marker for these cells. Vimentin is the IF expressed in mesenchymal cells, besides being found in immature astrocytes . It is important to point out that cells with no vimentin IF are unable to carry LDL cholesterol derived from low-density lipoproteins from their lysosomes to the endoplasmic reticulum (ER)  resulting in a phenotype similar to that observed in NPC cells.
GFAP’s main structural role in astrocytes has been long known. However, in recent years, it has been shown to be important in several astrocyte functions such as regeneration after pathological processes, synaptic plasticity, reactive gliosis, shape maintenance, cell migration, and as the target of signal transduction pathways. Moreover, GFAP takes part in an elaborate communication system between astrocytes and neurons . Several diseases cause an increase in GFAP expression, such as Alzheimer’s disease, encephalomyelitis, multiple sclerosis , and Alexander disease . On the other hand, studies with GFAP knockout animals have shown that the lack of this protein seems to make astrocytes less efficient in dealing with acute states of CNS lesions .
IFs are the largest components of the cell cytoskeleton and are actively associated and disassociated via the phosphorylation-dephosphorylation cycle . A decrease in phosphorylation of soluble vimentin and increase of insoluble vimentin has been observed in human fibroblasts with NPC , which led to an imbalance in the vimentin association-disassociation cycle. This results in a state of dephosphorylated polymerized filaments (insoluble vimentin) and small depolymerized and phosphorylated units (soluble vimentin) [9,11,12]. This imbalance may occur due to the inhibition of several kinases, such as protein kinase C (PKC), which has been associated to vimentin phosphorylation .
Dizocilpine, also known as (+) 5-methyl-10,11-dihydro-5H-dibenzo[a,b]-cycloheptane-5-10-imine, or MK-801, is an uncompetitive antagonist of N-Methyl-D-aspartate (NMDA), a glutamate receptor. NMDA is one of the brain’s most important excitatory neurotransmitters. Studies have reported glutamate toxicity on neuron cells (excitotoxicity) and that the use of antagonists of the NMDA receptor (such as MK-801) might have a neuroprotective effect in several situations since they lower the activation of glutamatergic receptors [14,15].
NMDA receptor antagonists, including MK-801, have been extensively studied for use in the treatment of diseases with excitotoxicity components, such as stroke, traumatic brain injury, and neurodegenerative diseases such as Huntington’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. MK-801 has been effective in protecting neurons in cell cultures and in the excitotoxic neurodegeneration in animal models [16–18].
Phenotypes similar to NPC can be induced using several pathways and drugs, among which 3-β-[2-(diethylamino)ethoxy]androst-5-en-17-one (U18666A). This drug has been used to mimic the NPC phenotype, thus causing cholesterol accumulation in the intracellular spaces of treated cells, similar to human fibroblasts with NPC [19–23]. The U18666A inhibits the enzyme reductase desmosterol  responsible for reducing the desmosterol in cholesterol biosynthesis, and also blocks LDL-(low density lipoprotein) cholesterol transport from the lysosomes into the endoplasmic reticulum  thereby increasing the level of caveolina-1 located within the plasma membrane caveolae . Combinations of actin filaments, microtubules and intermediate filaments showed that there is a complex interaction between the cytoskeleton of cells and caveolae . It is therefore necessary to study the phosphorylation of GFAP and vimentin and the reversion of these settings after adding MK-801 to cultures of astrocytes with U18666A-induced NPC phenotype.
Pregnant Wistar rats (200-250 g) were obtained from the breeding stock of the Federal University of Rio Grande do Sul (UFRGS). Rats were maintained on a 12 h/12 h photoperiod in a colony room with constant temperature (22 C), with food and water at libitum. The animals were observed on gestational day 22. As soon as delivery finished, mothers and pups were sacrificed by decapitation without anesthesia. The experimental protocol followed the “Principles of Laboratory Animal Care” (NHI publication 85-23, revised 1996) and was approved by the Ethics Committee for Animal Research of the Federal University of Rio Grande do Sul (number 18266). All efforts were made to minimize the number of animals used and their suffering.
Cortical Astrocyte Culture
Astrocyte primary cultures were prepared from the cerebral cortex of newborn (0–3 day old) Wistar rats as described by Loureiro et al. . Briefly, rats were decapitated, brain structures were removed, and the meninges were carefully stripped off. Mechanically dissociated cells were plated in Dulbecco’s Modified Eagle’s Medium (DMEM)/10% FBS (pH 7.4) supplemented with glucose (33 mM), glutamine (2 mM), and sodium bicarbonate (3 mM) in a 15.6 and 34.8 mm diameter well (6 and 24 well plate) (Corning Inc., New York, New York) previously coated with polyornithine (1.5 μg/mL, Sigma, St. Louis, MO).
After astrocytes reached confluence, the culture medium was removed by suction and the cells were incubated for 1 h at 37°C in an atmosphere of 5% CO2 in DMEM without fetal bovine serum (FBS) in the absence (controls) or presence of U18666A at a dose of 0.25 µg/mL for 48 h as described below. To evaluate glutamatergic involvement on NPC astrocytes, cells were pre-incubated in the presence of the NMDA antagonist MK-801 (50 µM) for 30 min before U18666A treatment .
In order to investigate the effects of U18666A-induced cholesterol accumulation and of MK-801 on the hyperphosphorylation of IF, the astrocyte cultures were treated as follows: group 1 = cultures were without drug (controls); group 2 = cultures were with drug U18666A (0.25 µg/mL) (treated group); group 3 = cultures with MK-801 (50 µM) added to the medium; and group 4 = cultures with U18666A and MK-801 (50 µM) added to the medium. All culture plates were incubated for 48 h. Cell cultures containing over 95 % GFAP-positive cells were used in all trials.
In Vitro Phosphorylation
Astrocytes were incubated for 24 h in the presence or absence of U18666A, then the medium was changed and incubation for 1 h was carried out at 30°C with 1,000 mL of a medium containing 124 mM NaCl, 4 mM KCl, 1.2 mM MgSO4, 25 mM Na-HEPES (pH 7.4), 12 mM glucose, and 1 mM CaCl2 and the following protease inhibitors: 1 mM benzamidine, 0.1 mM leupeptin, 0.7 mM antipain, 0.7 mM pepstatin, 0.7 mM chymostatin, and 10 mCi [32P]-orthophosphate with or without addition of U18666A (0.25 µg/mL). In the experiments designed to study signaling mechanisms, cells were pre-incubated for 30 min in the presence or absence of 50 µM MK-801. The labeling reaction was normally allowed to proceed for 1 h at 30°C and stopped with 1 mL of cold stop buffer (150 mM NaF, 5 mM EDTA, 5 mM EGTA, Tris-HCl, 50 mM, pH 6.5, and the protease inhibitors as described above). Cells were then washed twice by decantation with stop buffer to remove excess radioactivity next, preparations of IF-enriched high-salt Triton-insoluble cytoskeletal fraction were obtained from striatal primary astrocytes, as described by Funchal C, et al. . Briefly, cells were homogenized in 200 µL of ice-cold high-salt buffer containing 5 mM KH2PO4 (pH 7.1), 600 mM KCl, 10 mM MgCl2, 2mM EGTA, 1% TritonX-100, and the protease inhibitors described above. The homogenate was centrifuged at 15,800 g for 10 min at 4°C in an Eppendorf centrifuge and the supernatant was discarded. The Triton-insoluble IF-enriched pellet, containing GFAP and vimentin, was dissolved in 1% SDS and protein concentration was determined using the method by Lowry .
Polyacrylamide Gel Electrophoresis (SDS-PAGE)
The cytoskeletal fraction was prepared as described above. Equal protein concentrations were loaded onto 10% polyacrylamide gels and analyzed by SDS-PAGE . After drying, the gels were exposed to T-MAT films at -70°C with intensifying screens and finally the autoradiograph was obtained. Cytoskeletal proteins were quantified by scanning the films with a Hewlett-Packard Scanjet 6100C scanner and optical densities were determined with an Optiquant version 02.00 software (Packard Instrument Company). Density values were obtained for the proteins studied.
Western Blot Analysis
The homogenates (50 µg) were analyzed by SDS-PAGE and transferred to nitrocellulose membranes (Trans- blot SD semi-dry transfer cell, Bio Rad) for 1 h at 15 V in transfer buffer (48 mM Trizma, 39 mM glycine, 20 % methanol and 0.25 % SDS). The nitrocellulose membranes were washed for 10min in Tris buffered saline (TBS, 0.5 M NaCl, 20 mM Trizma, pH 7.5), followed by 2 h incubation in blocking solution (TBS plus 5 % defatted dried milk or ambumine bovine). After incubation, the blot was washed twice or 5 min with TBS plus 0.05 % Tween-20 (T-TBS), and then incubated overnight at 4°C in blocking solution containing one of the following antibodies: anti-GFAP (clone G-A-5) diluted 1:500, anti-Vimentin (Vim 13-12) diluted 1:1400. The blot was then washed for 5 min with T-TBS and incubated for 2 h in TBS containing peroxidase conjugated anti-rabbit IgG diluted 1:2000 or peroxidase conjugated anti-mouse IgG diluted 1:2000. In addition, we used the following controls in the antibody experiments: primary antibody only, secondary antibody only and negative controls (lacking the sample containing the antigen of interest). These controls (not show) stated the specificity and sensibility of the antibodies. The blot was washed twice with T-TBS, incubated for 5 min each time and with TBS twice by incubating 5 min each time. The blot was then developed using a chemiluminescence ECL kit and quantified as described above.
Data was statistically analyzed by one-way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test when the ƒ-test was significant and necessary. All analyses were performed using the SPSS software program on an IBM-PC compatible computer.
Initially, the effects of U18666A on astrocyte cell cultures from Wistar rats zero to three days old on the endogenous phosphorylation system associated with enriched cytoskeletal IF fractions from the cerebral cortex regarding both vimentin and GFAP were studied. Astrocyte cultures in contact with U18666A showed an increase in IF protein hyperphosphorylation when compared with the control cells (drug free). These analyses indicate that phosphorylation increased both in GFAP and insoluble vimentin by nearly two fold, as shown in figure 1, with a significant difference (GFAP = p < 0.0001; vimentin = p < 0.0001). Western blot analyses on the IF subunits confirm that the cells in contact with U18666A do undergo hyperphosphorylation (figure 2). These results were observed due to the addition of 32P both in vimentin and in GFAP.
Figure 1: Effect of MK 801 and U18666A on the phosphorylation of IF of (A) GFAP and (B) vimentin in the cytoskeletal fraction from rats’ astrocytes cultures. Different statistically significant from control were determined by student t-test: *p < 0.0001, t = 6.86 (A), t = 6.66 (B). * Different from control, # different from control, MK-801 and MK-801 + U18666A. n = 6 per group for each experiment and the time in which the drug U18666A promoted the accumulation of cholesterol was 48 hours.
Figure 2: Sodium dodecyl sulfate polyacrylamide gel eletrophoresis (SDS-PAGE) and autoradiograph of the cytoskeletal fraction. The gel was stained with Coomasie blue molecular weight standards, representative stained gel of a control (C) and treated samples (T) (U18666A), corresponding autoradiograph, and SDS-Page for Vimentin and GFAP. Equal amounts of the cytoskeletal fraction (50µg) from control and treated samples were run in 7.5 % polyacrylamide gel electrophoresis. MW: molecular weight of standard proteins; n = 6 per group for each experiment.
Subsequently, drug free cultured cells were kept in contact with MK-801, an uncompetitive NMDA antagonist, and it was observed that this agent does not tends to alter the phosphorylation, remaining at normal levels, driving it to even lower levels than in the control cells, which shows a significant difference when the GFAP IFs are analyzed (p < 0.0001) (figure 1).
Concomitantly, cultures of astrocytes treated with U18666A were also put in contact with MK-801 and protein phosphorylation did not increase regarding both GFAP and insoluble vimentin. That shows, MK-801 inhibits the action of the drug U18666A, thus preventing hyperphosphorylation of the IF insoluble (figure 1).
U18666A (3-β-[2-(diethylamino) ethoxy]androst-5-en-17-one), a hydrophobic amine, has been used to mimic NPC in human and animal tissues, among which: fibroblasts of newborn rats , fibroblasts of NPC-free humans , NPC knockout mice (NPC-/-)  , and Wistar rat astrocyte cultures .
Considering that, from the development of the NPC phenotype with U18666A, changes have already been observed in the activity of lysosomal hydrolases which are also changed in human NPC  and that changes have already been observed in the cellular cytoskeleton in human NPC , the aim of this study was to observe that these changes are present in U18666A-induced NPC astrocyte cultures.
In the present study, first hyperphosphorylation was observed in cytoskeleton IF both in GFAP and insoluble vimentin in cultures of U18666A-induced NPC astrocytes. Kodam, et al.  also observed an insoluble hyperphosphorylation of vimentin in the hippocampus of mice NPC1-/-. The same was also observed by Walter et al.  in human fibroblasts with NPC1.
As to GFAP, our results also agree with the literature. Hovakimyan et al.  observed an increase of this protein insoluble phosphorylated in the olfactory bulb and Luan, et al.  and Bae, et al. , had the same result in astrocytes, both worked in an experimental model of NPC1-/- mice. The difference is that, in our work we did not use knockout rats and used only Wistar rats.
The intermediate filaments were also studied in other neurodegenerative diseases. An increase in GFAP phosphorylated was observed in Alzheimer's disease  and Parkinson's disease  and Huntington's disease  and induced neurodegeneration ditelluride . Vimentin insoluble phosphorylated also proved to be increased in Alzheimer disease , Huntington disease  and neurodegeneration induced ditelluride . Although there are works with other NMDA agonists, such as quinolinic acid  major emphasis of our research was the placement of U18666A drug in astrocytes of Wistar rats thus stimulating the accumulation of cholesterol, which has been described in previous studies  and the presence of MK-801.
These results were supported by the evidence that phosphorylation may regulate the dynamic properties of IF, thus leading to a reorganization of the IF network . In addition, the pathological hyperphosphorylation of the cytoskeletal protein is thought to be related to neuronal damage and formation of aggregates of cytoskeletal elements in different cell compartments, which can be considered a common characteristic of some neurodegenerative diseases .
Among the receptors involved in excitotoxicity, the NMDA receptor stands out as the main one taking part in this phenomenon. Astrocyte cultures in contact with MK-801, since it is an uncompetitive NMDA antagonist, enable full prevention by lowering hyperphosphorylation levels and leading to regular phosphorylation levels in the IFs both in vimentin and GFAP. This neuroprotective action has also been observed in cortical cells using MK-801, which inhibits the action of NMDA [46,47]. Hyperphosphorylation of IFs caused by the U18666A-induced cholesterol accumulation, was prevented by MK-801, thus showing the cytotoxic effects involved in cytoskeleton protein deregulation. As shown in this study, MK-801 fully prevented hyperphosphorylation both in vimentin and GFAP. Therefore, the mechanisms that led to IF hyperphosphorylation were the ones that dependent on the NMDA receptor, supporting U18666A as a glutamatergic agonist that acts on the cytoskeleton, besides being directly linked to increase in cholesterol.
The results obtained may enable inferring that substances that decrease the excitotoxicity caused by the higher cholesterol levels observed in NPC, such as the NMDA receptor antagonist MK-801, can be viable tools to aid in the treatment of neurodegenerative diseases related to glutamatergic excitotoxicity.
This work was supported by Coordenating Agency Training of Superior- Level Personnel (CAPES) and National Council for Scientific and Technological Development (CNPq).
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