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Cyp46-mediated cholesterol loss promotes survival in stressed hippocampal neurons
Neurobiology of Aging 32 (2011) 933–943
Cyp46-mediated cholesterol loss promotes survival in stressed hippocampal neurons Mauricio G. Martin a,∗,1 , Laura Trovò a,1 , Simona Perga b , Agniezska Sadowska a , Andrea Rasola c , Federica Chiara c,d , Carlos G. Dotti a,∗ a
VIB Department of Developmental Molecular Genetics and Katholieke Universiteit Leuven Department of Human Genetics, Heerestraat 49, 3000 Leuven, Belgium b Cavalieri Ottolenghi Scientific Institute, Università degli Studi di Torino, A.O. San Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano, TO, Italy c Department of Biomedical Sciences, Università degli Studi di Padova, 35122 Padova, Italy d Department of Environmental Medicine and Public Health, Università degli Studi di Padova, 35122, Padova, Italy Received 26 February 2009; received in revised form 23 April 2009; accepted 30 April 2009 Available online 3 June 2009
Abstract Aged neurons constitute an outstanding example of survival robustness, outliving the accumulation of reactive oxygen species (ROS) derived from various physiological activities. Since during aging hippocampal neurons experience a progressive loss of membrane cholesterol and, by virtue of this, a gradual and sustained increase in the activity of the survival receptor tyrosine kinase TrkB, we have tested in this study if cholesterol loss is functionally associated to survival robustness during aging. We show that old neurons that did not undergo the cholesterol drop, upon knockdown of the cholesterol hydroxylating enzyme Cyp46, presented low TrkB activity and increased apoptotic levels. In further agreement, inducing cholesterol loss in young neurons led to the early appearance of TrkB activity. In vivo, Cyp46 knockdown led to the appearance of damaged hippocampal neurons in old mice exposed to exogenous stressful stimuli. Cholesterol loss seems therefore to contribute to neuronal survival in conditions of prominent stress, either acute or chronic. The relevance of this pathway in health and disease is discussed. © 2009 Elsevier Inc. All rights reserved. Keywords: Cholesterol; TrkB; Akt; Survival; Stress; CYP46
1. Introduction Ageing is characterized by a decline in cognitive functions such as reduced learning ability and memory. In early reports these deficits were linked to neuronal loss in the hippocampus, one of the brain regions where task memory centres are located (Burke and Barnes, 2006; Driscoll et al., 2006; Morrison and Hof, 1997). These observations were consistent with subsequent data suggesting that neuronal death during senescence could be due to the decrease in the availability ∗ Corresponding authors at: VIB/KUL, Heerestraat 49, 3000 Leuven, Belgium. Tel.: +32 163 30 519; fax: +32 163 46 522. E-mail addresses: [email protected] (M.G. Martin), [email protected] (C.G. Dotti). 1 Equal contributing authors.
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.04.022
of growth factors (Hattiangady et al., 2005; Mattson and Magnus, 2006; Shetty et al., 2004; Silhol et al., 2005). Conversely, a plethora of recent evidences show that neuronal density in the hippocampus is unaffected during ageing in both, animal models and humans, despite the decrease in neurotrophin concentration (Burke and Barnes, 2006). This last scenario indicates that post-differentiated neurons must possess a robust and long-term survival strategy to outlive the noxious effects of trophic factor deficiency under the prolonged pressure of multiple stress insults, which act as major determinants of ageing (Andersen, 2004; Balaban et al., 2005; Benn and Woolf, 2004; Schmitt, 2003). The mechanisms behind survival under such adverse conditions remain largely undefined. The Trk family of receptor tyrosine kinases is one of the better characterized survival-promoter pathways in
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hippocampal neurons, especially during development, through activation of the PI3K/Akt signal transduction pathway (Airaksinen and Saarma, 2002; Huang and Reichardt, 2003; Zheng and Quirion, 2004). Receptor tyrosine kinases are activated either following cognate growth factor stimulation, and/or after receptor clustering within cholesterol-rich plasma membrane micro-domains, also known as DRMs (detergent resistant membrane domains) or lipid rafts. In previous work we have demonstrated that under conditions of ligand paucity, the age-associated increase in TrkB activity is correlated with a mild yet progressive loss of cholesterol and this, in turn, is correlated with the increased expression of the cholesterol catabolic enzyme cholesterol 24-hydroxylase. Direct cause–effect, cholesterol loss-TrkB activity, was demonstrated by pharmacological means and by manipulating the levels of cholesterol 24-hydroxylase (Martin et al., 2008). It remains undetermined however, if cholesterol loss is a mechanism employed by cells to promote survival. This was tackled in the current work.
2. Materials and methods 2.1. Cell culture and reagents Primary cultures of rat embryo hippocampal neurons were prepared as previously described (Goslin et al., 1991). For biochemical analysis, 105 cells were plated into 3 cm plastic dishes coated with poly-l-lysine (0.1 mg/ml) and containing minimal essential medium with N2 supplements (MEM-N2). Neurons were kept under 5% CO2 at 37 ◦ C. Where indicated, TrkB kinase inhibitor K-252a (Alexis Biochemicals, San Diego, CA) was added to medium to a final concentration of 50 nM (Lee and Chao, 2001). Cells were incubated with the inhibitor for 2 h to assess target inhibition or for 36 h to measure cell death. Monoclonal anti-p53 was from SantaCruz Biotechnology (Santa Cruz, CA); polyclonal rabbit anti-Akt, polyclonal rabbit anti-phospho-Akt (Ser473) polyclonal anti-phospho-SAPK/JNK and anti-SAPK/JNK kinase, mouse monoclonal anti-Tubulin were all from Cell Signaling (Beverly, MA).
ferred to nitrocellulose membranes and probed with primary antibodies for 16 h. Species-specific peroxidase-conjugated secondary antibodies were subsequently used to perform enhanced chemiluminescence (Amersham, Little Chalfont, UK). Images were recorded by use of a FujiFilm LAS 3000 CCD camera and quantification of the images was performed using the NIH-image software. 2.3. Membrane cholesterol quantification, reduction and replenishment Cholesterol was measured in samples containing equal amount of protein using Ecoline 25 (Merck, Darmstadt, Germany). The Ecoline assay relies on the production of H2 O2 by cholesterol oxidase. H2 O2 is converted into a colored quinonimine in a reaction with 4-aminoantipyrine and salicylic alcohol catalyzed by peroxidase. The optical density was measured at 500 nm and we used pure cholesterol (Sigma) solutions as standards. To reduce membrane cholesterol levels, 0.4 M mevilonin and 0.5 mM M-cyclodextrin (MCD) were added daily to 5-day-old neurons during 4 days (96 h). At the end cholesterol was measured (∼25%), to confirm that the treatment did not result in more then 25% of reduction. Cells were scraped with MBS Buffer (25 mM Hepes pH 7.0; 5 mM DTT; 2 mM EDTA) supplemented with CLAP at 4 ◦ C, and centrifuged at 1000 × g. Post-nuclear supernatants were further centrifuged at 100,000 × g, 1 h at 4 ◦ C to get the membrane pellet. Protein and cholesterol concentrations were measured after resuspension in MBS-CLAP with 0.1% Triton-X 100. Cholesterol–MCD inclusion complexes were prepared as described (Klein et al., 1995). These complexes, containing 0.3 mM cholesterol, were added to the medium at a final concentration of 0.03 mM together with 2 mg/ml of free cholesterol. The treatment was performed for 15 min and then cells were washed and scraped with MBS-CLAP 0.1% Triton-X 100 at 4 ◦ C. 2.4. Cell death determination by cytofluorimetric analysis
2.2. Western blotting Mouse hippocampal tissues were homogenized in PBS containing 9% sucrose, protease inhibitors (CLAP: pepstatin, antipain, chymostatin, leupeptin) each at a final concentration of 25 mM/ml) and 1 mM sodium orthovanadate using a dounce homogenizer and 10 passages through a 22-gauge syringe. Samples were centrifuged for 10 min at 2500 × g and supernatants were considered as total extracts. A further centrifugation was performed at 100,000 × g for 1 h at 4 ◦ C to pellet the membrane fraction. Extracts were clarified by centrifugation and protein concentration was quantified by the BCA method (Bio-Rad Laboratories, Hercules, CA). Proteins were then trans-
Hippocampal neuron cell death was measured 18 h after treatment by assessing the number of hypodiploid nuclei with the DNAcon3 kit (ConsulTS, Rivalta, Italy). Briefly, cells were gently lysed with 0.1% TritonX100 in the presence of RNAse and of a chromatin stabilizer, and DNA was stained with 50 mg/ml propidium iodide. Samples were kept for 1 h in the dark at room temperature and the DNA index was then measured by cytofluorimetric analysis on a FACSCalibur flow cytometer (Becton Dickinson, San Josè, CA) using CellQuest software. Hypodiploid, subG0/G1 nuclei were defined as those displaying a PI staining value lower than that of cells in the G0/G1 cell cycle phase (diploid DNA peak, Fraker et al., 1995).
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Fig. 1. Age-associated increase of stress by-products in the mouse hippocampus. (A) ROS levels detected by DHR staining. Fields with comparable cell density were chosen as indicated by DAPI staining in (B). Note the dramatic increase of ROS as mice age from 1 to 20 months, both in the number of cells revealing ROS and in the amount of ROS within cells. All images were taken with identical exposure and light intensity parameters. (C) Lipofuscin auto-fluorescent granules (yellow) in the mouse hippocampus. Same cells are labeled with the DAPI dye, to reveal the cells’ nuclei. Note that lipofuscin accumulation with age parallels the increase in ROS. (D) Western blot on whole hippocampus extracts from 1, 10 and 20 months old mice. The presence of stress in aging brain in situ is evidenced by the up-regulation phospho-Jnk, P53 and P21. The relative amount of pJnk/Sap, p53 and p21 corrected for tubulin is shown on the right.
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Fig. 2. Age-associated increase of stress by-products in hippocampal neurons in vitro. (A) Cells of 10, 15 and 26 DIV were stained with dihydrorhodamine-123 (DHR-123), converted into fluorescent RH-123 by hydroxyl radicals. The fluorescence microscopy images (lower row) make evident the increase in ROS with age in vitro. This is however shown in quantitative terms in the graph bar at the right of the images (mean ± s.d., n = 3). The phase-contrast images in the upper row (phase) demonstrate that ROS accumulation is not accompanied by cell death. (B) Lipofuscin granules in the lysosomes of aged neurons. Auto-fluorescent lipofuscin granules (taken in the red channel) are only found in old neurons, 26 DIV (arrow). (C) Western blot of whole cell extracts from hippocampal neurons maintained in vitro for 10, 15 and 26 days. The time-accumulation of stress is indicated by the increased levels of phospho-Jnk, p53 and p21. The relative amount of pJnk/Sap, p53 and p21 corrected for tubulin is shown on the right.
2.5. SiRNAs and plasmids The cyp46A1 targeting sequence was designed by using the RNAi design algorithm at www.dharmacon.com/ DesignCenter/DesignCenterPage.aspx and 46-sense and 46antisense oligos encoding cyp46A1-shRNA were designed as reported by Rubinson et al. (2007). pSi46 plasmid was constructed by cloning the 46-sense/antisense duplexes in pLentiLox 3.7, as previously described (Martin et al., 2008). 2.6. Lentiviral preparation and rat injections Lentiviral particles were produced using the pSi46 plasmid and the packaging system described by Rubinson et
al. (2007). Cells were transfected using FuGene 6 reagent (Roche) in 3.5 ml medium and viral particles were collected from filtered medium by centrifugation during 2 h at 25,000 RPM after transfection. Viral particles were resuspended in 50 l of 1× PBS, quickly frozen in liquid nitrogen and stored at −80 ◦ C. Rats were injected into C1 region of the hippocampus with 2 l of the viral suspension, following the coordinated described in the stereotaxic Rat Brain Atlas (Paxinos and Watson) and animals were sacrificed 10 days after injection. Kainic acid (15 mg/kg) was injected intraperitoneally 9 days after viral injection and animal were sacrificed 24 h later. Rats were perfused via the left ventricle with 4% neutral buffered PFA.
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Animals were anaesthetized by intraperitoneal administration of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg). 2.7. Brain sections After perfusion in 4% PFA, brains were removed and equilibrated in successively in 10, 20 and 30% sucrose. Finally brains were frozen in OCT medium and cut in 10 or 20 m thick slices. 2.8. Neuronal transfection Primary dissociated hippocampal cells isolated from rat embryos (on embryonic day 18) were transfected using the Rat Neuron Nucleofector Kit from Amaxa biosystems. SiRNA duplexes were delivered to neurons by use of Lipofectamine LTX (Invitrogen). 2.9. Immunofluorescence microscopy
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levels notoriously increased in 10-month-old mice, reaching a maximum at 20 months (Fig. 1A). Fields with comparable cell density were chosen, as can be assessed by DAPI staining in Fig. 1B. Accordingly, lipofuscin deposits were not identified in the hippocampus of young mice but they were evident in slices obtained from 10-month-old mice. Again, further increase was observed between 10 and 20 months (Fig. 1C). The accumulation of free radicals and oxidized cellular residues during aging in vivo is paralleled by the age-associated increase in the activity of death and stressrepairing pathways mediated by phospho-Jnk, P53 and P21 (Fig. 1D). Embryonic hippocampal neurons in primary culture undergo a most stereotyped process of differentiation: axonal and dendritic differentiation takes place in the first 7 days in vitro (DIV), synapse formation by about 10–12 DIV and death around 25–30 DIV (Goslin et al., 1991). To establish if this system could be used as experimental model to investigate the mechanisms involved in survival during aging, the levels of the above, in situ, stress/anti-stress parameters were analyzed in hippocampal neurons maintained in vitro for dif-
Neurons on glass coverslips were washed in PBS (0.01 M), fixed with 4% paraformaldehyde, and processed for immunofluorescence. Samples were analyzed on an Olympus IX81 fluorescence microscope. Quantification was performed using the NIHImageJ software and signal intensities were normalized by area. Apoptotic nuclei were identified by use of In Situ Cell Death Detection Kit, TMR red (Roche). 2.10. Measurement of hydrogen peroxide and peroxynitrite levels in mitochondria Cells were treated with 10 M DHR (Molecular Probes, Eugene, OR) for 20 min and then washed. The fluorescent product, rhodamine-123, was measured by fluorescence detection with excitation and emission wavelengths of 500 and 536 nm, respectively. Images were captured under Olympus IX81 fluorescence microscope.
3. Results 3.1. Signs of aging in hippocampal neurons in vitro and in situ Aging cells present numerous changes, morphological, biochemical and functional. Simple parameters to identify aging cells are the presence of cumuli of reactive oxygen species (ROS) in mitochondria (Chen et al., 1998), lipofuscin granules (Gray and Woulfe, 2005; Szweda et al., 2003), high activity of the Jnk MAP kinase (Pham et al., 2005) and up-regulation of p53 and p21 (Wu et al., 2004). Fig. 1 shows that similar changes occur in the aging hippocampus: ROS are undetectable in the brains of 1-month-old mice but the
Fig. 3. TrkB/Akt pathway promotes survival in aged neurons in vitro. (A) Effect of Trk inhibitor K252a on Akt activity in 26 DIV cells, detected by immunoblot analysis with anti-pAkt antibodies. Protein loading was assessed with anti-Akt antibody. (B) Hippocampal neuron cell death rate, assessed by cytofluorimetric analysis of subG0/G1, hypodiploid nuclei, in 15 DIV neurons untreated or treated with K252a for 36 h. K252a significantly increases death rate of treated cells (C). Bars represent mean ± s.d. of the percentages of hypodiploid nuclei from four different experiments (T-test, p < 0.05).
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ferent lengths of time. As shown in Fig. 2A, the levels of ROS increased with aging in vitro, from 10 to 26 DIV and the formation of lipofuscin granules was exclusive of old neurons (Fig. 2B). Again, increased levels of phospho-Jnk, P53 and P21 were observed as neurons age from 10 to 26 DIV (Fig. 2C). Since stress/death signaling begins at around 15 DIV (Fig. 2C) and apoptosis only reaches significant levels after 25 DIV, this was the time window utilized to dissect the possible mechanisms of survival under stress. 3.2. TrkB/Akt activity is required for survival of aging hippocampal neurons in vitro In a previous work we have shown that one characteristic of neuronal aging is the progressive increase in the levels of phosphorylated TrkB and its downstream kinase Akt (Martin et al., 2008). Therefore, we assessed the relevance of Trk/Akt pathway on the survival of hippocampal neurons of more than 15 DIV, time at which stress products start accumulating (see above). We started by inhibiting TkrB phosphorylation by adding to cells the K252a kinase inhibitor (Lee and Chao, 2001) and then determined cell death rate by
cytofluorimetric analysis of subG0/G1, hypo-diploid nuclei (see Section 2). Fig. 3A shows that incubation of 15 DIV cells with this inhibitor markedly reduced Akt phosphorylation, directly proving inhibitor efficacy (see below for specificity test). Furthermore, the treatment significantly increased cell death rate compared to untreated neurons in which the Trk activation is left unaffected (Fig. 3B and C). Because K252a has more targets than TrkB, we next assessed the contribution of TrkB/Akt to survival of aged neurons by knocking down TrkB by siRNA methodology. Fig. 4 shows that TrkB knockdown resulted in a 100% increase of apoptosis compared to cells treated with a negative control siRNA. These results leave little doubt of the relevance of TrkB/Akt activity for survival of hippocampal neurons under the stressful environment of the tissue culture dish. 3.3. Cholesterol loss is required for TrkB survival activity of aging hippocampal neurons in vitro Since TrkB activity in aging neurons is triggered by cholesterol loss, independently of BDNF (Martin et al., 2008), we next investigated whether or not cholesterol loss at
Fig. 4. TrkB is required for survival of aged hippocampal neurons in vitro. siRNA duplexes designed to knock down TrkB (siTrkB) were delivered to neurons and apoptosis rate was measured in 17 DIV neurons by the TUNEL assay. In the control, scrambled siRNA duplexes (siNeg) were used. (A) Phase contrast and fluorescence microscopy overlay images showing apoptotic (red) nuclei in siTrkB transfected neurons (green). Lower images is the enlarged view of the cells in the area boxed in the upper phase/fluorescence images, to render more evident the apoptosis in TrkB siRNA neurons. (B) Neuronal death rate, assessed by TUNEL assay, in control cells and in cells where TrkB was knocked down. The plot shows a significant (100%) increase in death rate in siTrkB-treated cells compared to control neurons. Bars represent mean ± s.d. from three different experiments (T-test, p < 0.05).
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this stage of life participated actively in the survival of neurons with accumulated stress, either in old neurons in vivo (Fig. 1) or in post-differentiation neurons in vitro (Fig. 2). The first (in vivo stress accumulation) reflect the normal metabolic stress situation whereas the second (in vitro stress accumulation) may reflect a supra-physiological/pathological, exogenous plus endogenous, stress condition. To analyze the role of cholesterol loss we directly reduced membrane cholesterol in 10 DIV neurons, time at which membrane cholesterol content is highest (Martin et al., 2008). For this, the cholesterol synthesis inhibitor mevilonin and a cholesterol removal drug (-methyl-cyclodextrin, MCD) were added 10 DIV neurons (see Section 2). Fig. 5A (↓chol), shows that cholesterol reduction resulted in the clear activation of Akt. To prove that this effect is a true consequence of the loss of cholesterol and not secondary to any other possible effect of the drugs, cholesterol was added back to the drugtreated 10 DIV cells (see Section 2). This treatment restored the low basal levels of p-AKT to levels of control 10 DIV cells (Fig. 5A; ↑chol). From here we conclude that cholesterol loss is a sufficient stimulus to activate the survival branch of TrkB. Next, we analyzed if cholesterol loss is required for survival of older neurons, which present abundant intracellular stress (see Fig. 2). For this, membrane cholesterol levels of 26 DIV hippocampal neurons were raised to levels of younger cells by adding cholesterol–MCD mix (see Section 2). Survival was then analyzed by cytofluorimetric analysis. As shown in Fig. 5B, the addition of cholesterol (↑chol) to old neurons led to reduced Akt activity, to almost undetectable levels. Consistent with a true functional link, cholesterol addition to these cells increased the rate of neuronal apoptosis in comparison to their untreated counterparts (Fig. 5C and D). 3.4. Cyp46 activity is required for the survival of aging hippocampal neurons, in vitro and in vivo To further test the importance of cholesterol loss in neuronal survival, the cholesterol-hydroxylating enzyme CYP46 (Lund et al., 1999) was knocked down in hippocampal neurons in vitro by specific siRNA, thus blocking the natural occurrence of cholesterol loss. The effect on survival in knocked down cells was measured by the Tunel assay. Fig. 6 shows that CYP46 knockdown resulted in a remarkable increase of apoptosis rate in 17 DIV neurons. On the contrary, CYP46 knockdown did not have a significant effect on the rate of apoptosis in neurons at 10 DIV (Fig. 6B), indicating (i) that the increased apoptosis was not due to a non-specific effect of the siRNA and (ii) that cholesterol loss is fundamentally required for the survival of aged neurons. To test the validity of the last conclusion, lentiviral particles designed to knockdown CYP46 were injected in the hippocampal CA1 region (see Section 2), in which this enzyme is strongly expressed (Ramirez et al., 2008). As expected, survival of hippocampal neurons was not affected by the lack of Cyp46 in young (4 months, data not shown) animals. Surprisingly however, also in 20-month-old mice hippocampal CYP46 knockdown
Fig. 5. Membrane cholesterol loss induces Akt activation and promotes survival in old hippocampal neurons. (A) Effect of pharmacological cholesterol reduction on Akt activity. 10 DIV neurons were treated with mevilonin/MCD (↓ Chol), in order to induce a 25% membrane cholesterol loss. This leads to Akt phosphorylation reaching levels comparable to levels observed in aged cells at 15 and 26 DIV. Replenishment of cholesterol into the same cells (↑ Chol) abrogated activity of Akt. (B) The cholesterol addition (↑ Chol) to 26 DIV neurons results in the loss of Akt phosphorylation. (C) Hippocampal neuron cell death rate, assessed by cytofluorimetric analysis of subG0/G1 hypodiploid nuclei, in 26 DIV untreated (Ctrl) or after cholesterol addition as described in (B). (D) Plot showing the percentage of hypodiploid nuclei in control and treated cells. Note the significant increase in death rate in cultures where cholesterol was added compared to control neurons. Bars represent mean ± s.d. of the percentages of hypodiploid nuclei (n = 4) (T-test, p < 0.05).
did not increase neuronal death (Fig. 7A). Because Cyp46 knockdown leads to apoptosis in old neurons in vitro, the lack of a similar effect in hippocampal neurons in vivo indicated that the Cyp46-cholesterol loss pathway would be of survival usefulness only when exogenous stress is added to the basal situation of metabolic stress accumulation. Alternatively, a reduction in cholesterol synthesis may have occurred in vivo,
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Fig. 6. CYP46 enhances survival strength in aged hippocampal neurons in vitro. Hippocampal neurons were nucleofected with a plasmid expressing a GFP marker and a shRNA designed to knockdown CYP46 (pSi46). Apoptosis rate was analyzed in 10 and in 17 DIV neurons by TUNEL. (A) Fluorescence microscopy image showing pSi46 transfected cells (green) and Tunel staining (red). The white arrows indicate apoptotic nuclei in Cyp46 knockdown cells. Some apoptotic nuclei are also evident in non-transfected cells, however these are a vast minority compared to Cyp 46 knockdown cells (see B). (B) Percentage of apoptotic cells, assessed by TUNEL staining, of pSi46 transfected or control cells at 10 and 17 DIV (results obtained from three experiments as shown in A). The plot shows a significant increase in death rate, compared to control cells, at 17 DIV when CYP46 was knocked down. At 10 DIV both, control and pSi46 transfected cells showed a very low number of apoptotic cells. Bars represent mean ± s.d. from three different experiments (T-test, p < 0.01).
to prevent cholesterol accumulation. In fact, synthesis inhibition prevents cholesterol increase in neurons from Cyp46 knock-out mice (Lund et al., 2003). One way to distinguish between these possibilities was to test the effect of CYP46 knockdown after the intraperitoneal injection of kainic acid (KA) (see Section 2). This is an established procedure to trigger seizures and excitotoxic neuronal injury (Bettler and Mulle, 1995; Fraker et al., 1995; Kure et al., 1991) and would therefore distinguish if Cyp46 participates in survival under extreme stress. In this experiment, neurons transduced with control virus did not show signs of cell damage or death, neither in young (data not shown) nor in old animals (Fig. 7B), indicating that the stress paradigm utilized was not cytotoxic at this time. Differently, neurons transduced with Si46 lentivirus presented clear signs of damage, demonstrated by
Fig. 7. Effect of CYP46 knockdown in vivo in control and in Kainic Acid injected rats. (A) Immunofluorescence images of hippocampal sections of 20 months old animals injected with empty (control) or siCYP46 lentivirus and stained to detect apoptotic nuclei by TUNEL. Transduced cells were identified by expression of GFP. Note that no differences were observed between a control and Si46 virus. (B) Detection of damaged cells in hippocampal slices from 20 months old rats treated intraperitoneally with kainic acid (KA) after viral injection. Note that only CYP46 knocked down cells present signals of cell damage, indicated by the presence of cytoplasmic membrane degradation, beaded neurites, and accumulation of high amount of intracellular aggregates. A magnified picture of a neuron from each case is shown in the insets.
the presence of cytoplasmic membrane degradation, distorted aspect of the cell architecture, i.e. beaded neurites, and accumulation of high amount of intracellular aggregates (Fig. 7B). As we expected considering the results shown in Fig. 6, no cell damage was identified in neurons transduced with Si46 lentivirus in young (KA treated) animals. 4. Discussion It is widely held that oxidative stress increases in the aging brain. Brain cells are particularly prone to gain
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oxidative metabolites because of this organ’s high oxygen consumption per weight unit. Moreover, the brain is not particularly enriched in antioxidant protective defenses (Floyd and Hensley, 2002), clearly pointing out that neuronal survival can be only accomplished if these cells turn on other most efficient survival mechanisms. Because neurotrophins provide with essential survival information during embryonic and early post-natal life, it was commonly thought that they were also involved in the survival of the differentiated brain. However, it was recently shown that both sympathetic and sensory adult neurons live in the absence of trophic factors, suggesting that these cells activate survival pathways that do not require growth factors (Benn and Woolf, 2004). We have recently demonstrated that hippocampal neurons in vitro and in vivo activate the TrkB/Akt survival pathway in a ligand-independent manner, by virtue of the loss of membrane cholesterol levels as neurons age due to increased levels of the cholesterol-hydroxylating enzyme Cyp46 (Martin et al., 2008). The existence of BDNF alternative mechanisms for TrkB activation are also supported by previous reports showing that BDNF-induced TrkB phosphorylation is impaired in the brain of aged rats compared to young counterparts (Gooney et al., 2004), and by our recent study using old, wild type and BDNF knock-out mice (Martin et al., 2008). All these results, together with recent demonstrations that phosphorylation of TrkB in response to supra-physiological concentrations of exogenous BDNF requires normal cholesterol levels (Pereira and Chao, 2007) demonstrate: (i) that BDNF is not a major determinant of TrkB-mediated survival during aging, (ii) that cholesterol loss has a preponderant role in TrkB survival at this stage of life and (iii) that BDNF does not cooperate with cholesterol loss for the occurrence of TrkB activation. Still, despite its low levels, BDNF in the old brain might contribute to survival by activating Trk receptors, which are not affected by cholesterol loss. This possibility and the whole role of BDNF in normal, low cholesterol, aged neurons, need to be thoroughly investigated. In this work we have evaluated the real contribution of this cholesterol loss-induced TrkB/Akt activation to survival of aging neurons. Our results showed that TrkB knockdown in old neurons results in a clear increase of apoptosis rate, even in the neurotrophin depleted conditions in vitro (see Fig. 4). Further contributing to this conclusion, the results showed in Fig. 3 show that inhibition of Akt phosphorylation also resulted in 50% increased rate of apoptosis. Then, we showed that, impairing cholesterol loss either by increasing cholesterol levels in old cells by use of pharmacological treatment or by knocking down CYP46, resulted in the shutting down of the Akt kinase leading to significant increased levels of apoptosis. The discrepancy in the rate of apoptosis obtained when either TrkB or CYP46 were knocked down in vitro (onefold increase for TrkB, Fig. 4; fivefold increase when CYP46 was knocked down, Fig. 6) could be due to differences in knockdown efficiency, considering that different methodology was
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used in each case (see Section 2). However, it is also possible that the differences are due to Cyp46-mediated activation of more than one survival pathway. This possibility needs to be investigated. In agreement with previous results showing that neurons with normal cholesterol levels present low to undetectable TrkB activity (Martin et al., 2008), CYP46 knockdown in young neurons at 10 DIV did not have any effect on neuronal survival. This implies that these cells do not require pro-survival activation, due to low stress levels at this stage (see Fig. 2). Also, lentiviral-mediated CYP46 gene knockdown in vivo did not result in neuronal death in young animals even after treatment with KA, further indicating that cholesterol-loss mediated activation of survival is only required when levels of intracellular stress exceed the physiological threshold. This would explain the situation in hippocampal neurons maintained for long periods of time in vitro, under the chronic presence of uncontrolled excitatory neurotransmission and constant fluctuations in temperature, humidity, concentrations of gases and nutrients. Consistently, cholesterol-mediated survival pathway in vivo was required in old animals (20 month) only after the induction of additional stress, by kainic acid. These data, together with the previous demonstration that ROS are the only factors capable to increase CYP46 transcriptional activity (Ohyama et al., 2006), strongly indicate that stress by-products accumulated during aging are a key determinant of Cyp46 activation and, by virtue of this, of cholesterol loss from the neurons’ membrane. Furthermore, our results show that the levels of stress by-products are a critical step to define if cholesterol loss leads to survival or not. Further work is required to define the type of activities that become modified by the loss of membrane cholesterol when survival is not yet required. This presented data conduce to discuss the relevance of the cholesterol loss pathway in the pathogenesis of Alzheimer’s disease (AD), because it is well established that neuronal populations in AD cases are subjected to particularly high stress conditions (Perry et al., 2002). In fact differential expression of CYP46 has been described AD cases: decreased levels of the enzyme in neurons and increased levels around neuritic plaques (Bogdanovic et al., 2001; Brown et al., 2004). The decrease of CYP46 levels observed in AD neurons could indicate decreased ability of these neurons to activate TrkB/Akt survival pathway and, in the same direction, the high levels around neuritic plaques may reflect the response of the neighbor cells to the high insult provided by the deposition of amyloid aggregates. Furthermore, although still controversial, several groups have found a significant association between single nucleotide polymorphisms (SNPs) in cyp46A1 gene and increased risk of Alzheimer’s disease (Borroni et al., 2004; Kolsch et al., 2002; Papassotiropoulos et al., 2003; see however Desai et al., 2002; Wang and Jia, 2007). It remains to be elucidated if these mutations may predispose to disease by a “loss-of function” mechanism, where impossibility to rise enzyme levels under physiological stress would impair the activation of an important anti-apoptotic
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pathway like that of TrkB. Irrespective of this possibility, the main message from our work should be that cholesterol loss is a physiological process that neurons put to work to fight against stress. We have directly proven that cholesterol loss protects neurons against high stress (i.e. in vitro neurons or after KA injection) therefore it remains to be established what is the true physiological significance of cholesterol loss in the non-stressed brain, as it occurs in normally aged mice and rats. The relatedness of cholesterol loss to AD and whether or not cholesterol loss is activated for survival in other cell types see Cartagena et al., 2008, perennial and cycling, are important questions for future research.
Conflict of interest There are no actual or potential conflicts of interest.
Disclosure statement The appropriate institutional approval for all animal experiments was obtained.
Acknowledgements We thank, Kristel Vennekens and Tatiana Estrada Hernandez for technical assistance. The following institutions provided financial support: Fund for Scientific Research Flanders (FWO), SAO-FRMA Foundation (2006), Federal Office for Scientific Affairs (IUAP P6/43/) and Flemish Government (Methusalem Award).
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Cyp46-mediated cholesterol loss promotes survival in stressed hippocampal neurons Mauricio G. Martin a,∗,1 , Laura Trovò a,1 , Simona Perga b , Agniezska Sadowska a , Andrea Rasola c , Federica Chiara c,d , Carlos G. Dotti a,∗ a
VIB Department of Developmental Molecular Genetics and Katholieke Universiteit Leuven Department of Human Genetics, Heerestraat 49, 3000 Leuven, Belgium b Cavalieri Ottolenghi Scientific Institute, Università degli Studi di Torino, A.O. San Luigi Gonzaga, Regione Gonzole 10, 10043 Orbassano, TO, Italy c Department of Biomedical Sciences, Università degli Studi di Padova, 35122 Padova, Italy d Department of Environmental Medicine and Public Health, Università degli Studi di Padova, 35122, Padova, Italy Received 26 February 2009; received in revised form 23 April 2009; accepted 30 April 2009 Available online 3 June 2009
Abstract Aged neurons constitute an outstanding example of survival robustness, outliving the accumulation of reactive oxygen species (ROS) derived from various physiological activities. Since during aging hippocampal neurons experience a progressive loss of membrane cholesterol and, by virtue of this, a gradual and sustained increase in the activity of the survival receptor tyrosine kinase TrkB, we have tested in this study if cholesterol loss is functionally associated to survival robustness during aging. We show that old neurons that did not undergo the cholesterol drop, upon knockdown of the cholesterol hydroxylating enzyme Cyp46, presented low TrkB activity and increased apoptotic levels. In further agreement, inducing cholesterol loss in young neurons led to the early appearance of TrkB activity. In vivo, Cyp46 knockdown led to the appearance of damaged hippocampal neurons in old mice exposed to exogenous stressful stimuli. Cholesterol loss seems therefore to contribute to neuronal survival in conditions of prominent stress, either acute or chronic. The relevance of this pathway in health and disease is discussed. © 2009 Elsevier Inc. All rights reserved. Keywords: Cholesterol; TrkB; Akt; Survival; Stress; CYP46
1. Introduction Ageing is characterized by a decline in cognitive functions such as reduced learning ability and memory. In early reports these deficits were linked to neuronal loss in the hippocampus, one of the brain regions where task memory centres are located (Burke and Barnes, 2006; Driscoll et al., 2006; Morrison and Hof, 1997). These observations were consistent with subsequent data suggesting that neuronal death during senescence could be due to the decrease in the availability ∗ Corresponding authors at: VIB/KUL, Heerestraat 49, 3000 Leuven, Belgium. Tel.: +32 163 30 519; fax: +32 163 46 522. E-mail addresses: [email protected] (M.G. Martin), [email protected] (C.G. Dotti). 1 Equal contributing authors.
0197-4580/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2009.04.022
of growth factors (Hattiangady et al., 2005; Mattson and Magnus, 2006; Shetty et al., 2004; Silhol et al., 2005). Conversely, a plethora of recent evidences show that neuronal density in the hippocampus is unaffected during ageing in both, animal models and humans, despite the decrease in neurotrophin concentration (Burke and Barnes, 2006). This last scenario indicates that post-differentiated neurons must possess a robust and long-term survival strategy to outlive the noxious effects of trophic factor deficiency under the prolonged pressure of multiple stress insults, which act as major determinants of ageing (Andersen, 2004; Balaban et al., 2005; Benn and Woolf, 2004; Schmitt, 2003). The mechanisms behind survival under such adverse conditions remain largely undefined. The Trk family of receptor tyrosine kinases is one of the better characterized survival-promoter pathways in
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hippocampal neurons, especially during development, through activation of the PI3K/Akt signal transduction pathway (Airaksinen and Saarma, 2002; Huang and Reichardt, 2003; Zheng and Quirion, 2004). Receptor tyrosine kinases are activated either following cognate growth factor stimulation, and/or after receptor clustering within cholesterol-rich plasma membrane micro-domains, also known as DRMs (detergent resistant membrane domains) or lipid rafts. In previous work we have demonstrated that under conditions of ligand paucity, the age-associated increase in TrkB activity is correlated with a mild yet progressive loss of cholesterol and this, in turn, is correlated with the increased expression of the cholesterol catabolic enzyme cholesterol 24-hydroxylase. Direct cause–effect, cholesterol loss-TrkB activity, was demonstrated by pharmacological means and by manipulating the levels of cholesterol 24-hydroxylase (Martin et al., 2008). It remains undetermined however, if cholesterol loss is a mechanism employed by cells to promote survival. This was tackled in the current work.
2. Materials and methods 2.1. Cell culture and reagents Primary cultures of rat embryo hippocampal neurons were prepared as previously described (Goslin et al., 1991). For biochemical analysis, 105 cells were plated into 3 cm plastic dishes coated with poly-l-lysine (0.1 mg/ml) and containing minimal essential medium with N2 supplements (MEM-N2). Neurons were kept under 5% CO2 at 37 ◦ C. Where indicated, TrkB kinase inhibitor K-252a (Alexis Biochemicals, San Diego, CA) was added to medium to a final concentration of 50 nM (Lee and Chao, 2001). Cells were incubated with the inhibitor for 2 h to assess target inhibition or for 36 h to measure cell death. Monoclonal anti-p53 was from SantaCruz Biotechnology (Santa Cruz, CA); polyclonal rabbit anti-Akt, polyclonal rabbit anti-phospho-Akt (Ser473) polyclonal anti-phospho-SAPK/JNK and anti-SAPK/JNK kinase, mouse monoclonal anti-Tubulin were all from Cell Signaling (Beverly, MA).
ferred to nitrocellulose membranes and probed with primary antibodies for 16 h. Species-specific peroxidase-conjugated secondary antibodies were subsequently used to perform enhanced chemiluminescence (Amersham, Little Chalfont, UK). Images were recorded by use of a FujiFilm LAS 3000 CCD camera and quantification of the images was performed using the NIH-image software. 2.3. Membrane cholesterol quantification, reduction and replenishment Cholesterol was measured in samples containing equal amount of protein using Ecoline 25 (Merck, Darmstadt, Germany). The Ecoline assay relies on the production of H2 O2 by cholesterol oxidase. H2 O2 is converted into a colored quinonimine in a reaction with 4-aminoantipyrine and salicylic alcohol catalyzed by peroxidase. The optical density was measured at 500 nm and we used pure cholesterol (Sigma) solutions as standards. To reduce membrane cholesterol levels, 0.4 M mevilonin and 0.5 mM M-cyclodextrin (MCD) were added daily to 5-day-old neurons during 4 days (96 h). At the end cholesterol was measured (∼25%), to confirm that the treatment did not result in more then 25% of reduction. Cells were scraped with MBS Buffer (25 mM Hepes pH 7.0; 5 mM DTT; 2 mM EDTA) supplemented with CLAP at 4 ◦ C, and centrifuged at 1000 × g. Post-nuclear supernatants were further centrifuged at 100,000 × g, 1 h at 4 ◦ C to get the membrane pellet. Protein and cholesterol concentrations were measured after resuspension in MBS-CLAP with 0.1% Triton-X 100. Cholesterol–MCD inclusion complexes were prepared as described (Klein et al., 1995). These complexes, containing 0.3 mM cholesterol, were added to the medium at a final concentration of 0.03 mM together with 2 mg/ml of free cholesterol. The treatment was performed for 15 min and then cells were washed and scraped with MBS-CLAP 0.1% Triton-X 100 at 4 ◦ C. 2.4. Cell death determination by cytofluorimetric analysis
2.2. Western blotting Mouse hippocampal tissues were homogenized in PBS containing 9% sucrose, protease inhibitors (CLAP: pepstatin, antipain, chymostatin, leupeptin) each at a final concentration of 25 mM/ml) and 1 mM sodium orthovanadate using a dounce homogenizer and 10 passages through a 22-gauge syringe. Samples were centrifuged for 10 min at 2500 × g and supernatants were considered as total extracts. A further centrifugation was performed at 100,000 × g for 1 h at 4 ◦ C to pellet the membrane fraction. Extracts were clarified by centrifugation and protein concentration was quantified by the BCA method (Bio-Rad Laboratories, Hercules, CA). Proteins were then trans-
Hippocampal neuron cell death was measured 18 h after treatment by assessing the number of hypodiploid nuclei with the DNAcon3 kit (ConsulTS, Rivalta, Italy). Briefly, cells were gently lysed with 0.1% TritonX100 in the presence of RNAse and of a chromatin stabilizer, and DNA was stained with 50 mg/ml propidium iodide. Samples were kept for 1 h in the dark at room temperature and the DNA index was then measured by cytofluorimetric analysis on a FACSCalibur flow cytometer (Becton Dickinson, San Josè, CA) using CellQuest software. Hypodiploid, subG0/G1 nuclei were defined as those displaying a PI staining value lower than that of cells in the G0/G1 cell cycle phase (diploid DNA peak, Fraker et al., 1995).
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Fig. 1. Age-associated increase of stress by-products in the mouse hippocampus. (A) ROS levels detected by DHR staining. Fields with comparable cell density were chosen as indicated by DAPI staining in (B). Note the dramatic increase of ROS as mice age from 1 to 20 months, both in the number of cells revealing ROS and in the amount of ROS within cells. All images were taken with identical exposure and light intensity parameters. (C) Lipofuscin auto-fluorescent granules (yellow) in the mouse hippocampus. Same cells are labeled with the DAPI dye, to reveal the cells’ nuclei. Note that lipofuscin accumulation with age parallels the increase in ROS. (D) Western blot on whole hippocampus extracts from 1, 10 and 20 months old mice. The presence of stress in aging brain in situ is evidenced by the up-regulation phospho-Jnk, P53 and P21. The relative amount of pJnk/Sap, p53 and p21 corrected for tubulin is shown on the right.
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Fig. 2. Age-associated increase of stress by-products in hippocampal neurons in vitro. (A) Cells of 10, 15 and 26 DIV were stained with dihydrorhodamine-123 (DHR-123), converted into fluorescent RH-123 by hydroxyl radicals. The fluorescence microscopy images (lower row) make evident the increase in ROS with age in vitro. This is however shown in quantitative terms in the graph bar at the right of the images (mean ± s.d., n = 3). The phase-contrast images in the upper row (phase) demonstrate that ROS accumulation is not accompanied by cell death. (B) Lipofuscin granules in the lysosomes of aged neurons. Auto-fluorescent lipofuscin granules (taken in the red channel) are only found in old neurons, 26 DIV (arrow). (C) Western blot of whole cell extracts from hippocampal neurons maintained in vitro for 10, 15 and 26 days. The time-accumulation of stress is indicated by the increased levels of phospho-Jnk, p53 and p21. The relative amount of pJnk/Sap, p53 and p21 corrected for tubulin is shown on the right.
2.5. SiRNAs and plasmids The cyp46A1 targeting sequence was designed by using the RNAi design algorithm at www.dharmacon.com/ DesignCenter/DesignCenterPage.aspx and 46-sense and 46antisense oligos encoding cyp46A1-shRNA were designed as reported by Rubinson et al. (2007). pSi46 plasmid was constructed by cloning the 46-sense/antisense duplexes in pLentiLox 3.7, as previously described (Martin et al., 2008). 2.6. Lentiviral preparation and rat injections Lentiviral particles were produced using the pSi46 plasmid and the packaging system described by Rubinson et
al. (2007). Cells were transfected using FuGene 6 reagent (Roche) in 3.5 ml medium and viral particles were collected from filtered medium by centrifugation during 2 h at 25,000 RPM after transfection. Viral particles were resuspended in 50 l of 1× PBS, quickly frozen in liquid nitrogen and stored at −80 ◦ C. Rats were injected into C1 region of the hippocampus with 2 l of the viral suspension, following the coordinated described in the stereotaxic Rat Brain Atlas (Paxinos and Watson) and animals were sacrificed 10 days after injection. Kainic acid (15 mg/kg) was injected intraperitoneally 9 days after viral injection and animal were sacrificed 24 h later. Rats were perfused via the left ventricle with 4% neutral buffered PFA.
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Animals were anaesthetized by intraperitoneal administration of a mixture of ketamine (75 mg/kg) and xylazine (10 mg/kg). 2.7. Brain sections After perfusion in 4% PFA, brains were removed and equilibrated in successively in 10, 20 and 30% sucrose. Finally brains were frozen in OCT medium and cut in 10 or 20 m thick slices. 2.8. Neuronal transfection Primary dissociated hippocampal cells isolated from rat embryos (on embryonic day 18) were transfected using the Rat Neuron Nucleofector Kit from Amaxa biosystems. SiRNA duplexes were delivered to neurons by use of Lipofectamine LTX (Invitrogen). 2.9. Immunofluorescence microscopy
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levels notoriously increased in 10-month-old mice, reaching a maximum at 20 months (Fig. 1A). Fields with comparable cell density were chosen, as can be assessed by DAPI staining in Fig. 1B. Accordingly, lipofuscin deposits were not identified in the hippocampus of young mice but they were evident in slices obtained from 10-month-old mice. Again, further increase was observed between 10 and 20 months (Fig. 1C). The accumulation of free radicals and oxidized cellular residues during aging in vivo is paralleled by the age-associated increase in the activity of death and stressrepairing pathways mediated by phospho-Jnk, P53 and P21 (Fig. 1D). Embryonic hippocampal neurons in primary culture undergo a most stereotyped process of differentiation: axonal and dendritic differentiation takes place in the first 7 days in vitro (DIV), synapse formation by about 10–12 DIV and death around 25–30 DIV (Goslin et al., 1991). To establish if this system could be used as experimental model to investigate the mechanisms involved in survival during aging, the levels of the above, in situ, stress/anti-stress parameters were analyzed in hippocampal neurons maintained in vitro for dif-
Neurons on glass coverslips were washed in PBS (0.01 M), fixed with 4% paraformaldehyde, and processed for immunofluorescence. Samples were analyzed on an Olympus IX81 fluorescence microscope. Quantification was performed using the NIHImageJ software and signal intensities were normalized by area. Apoptotic nuclei were identified by use of In Situ Cell Death Detection Kit, TMR red (Roche). 2.10. Measurement of hydrogen peroxide and peroxynitrite levels in mitochondria Cells were treated with 10 M DHR (Molecular Probes, Eugene, OR) for 20 min and then washed. The fluorescent product, rhodamine-123, was measured by fluorescence detection with excitation and emission wavelengths of 500 and 536 nm, respectively. Images were captured under Olympus IX81 fluorescence microscope.
3. Results 3.1. Signs of aging in hippocampal neurons in vitro and in situ Aging cells present numerous changes, morphological, biochemical and functional. Simple parameters to identify aging cells are the presence of cumuli of reactive oxygen species (ROS) in mitochondria (Chen et al., 1998), lipofuscin granules (Gray and Woulfe, 2005; Szweda et al., 2003), high activity of the Jnk MAP kinase (Pham et al., 2005) and up-regulation of p53 and p21 (Wu et al., 2004). Fig. 1 shows that similar changes occur in the aging hippocampus: ROS are undetectable in the brains of 1-month-old mice but the
Fig. 3. TrkB/Akt pathway promotes survival in aged neurons in vitro. (A) Effect of Trk inhibitor K252a on Akt activity in 26 DIV cells, detected by immunoblot analysis with anti-pAkt antibodies. Protein loading was assessed with anti-Akt antibody. (B) Hippocampal neuron cell death rate, assessed by cytofluorimetric analysis of subG0/G1, hypodiploid nuclei, in 15 DIV neurons untreated or treated with K252a for 36 h. K252a significantly increases death rate of treated cells (C). Bars represent mean ± s.d. of the percentages of hypodiploid nuclei from four different experiments (T-test, p < 0.05).
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ferent lengths of time. As shown in Fig. 2A, the levels of ROS increased with aging in vitro, from 10 to 26 DIV and the formation of lipofuscin granules was exclusive of old neurons (Fig. 2B). Again, increased levels of phospho-Jnk, P53 and P21 were observed as neurons age from 10 to 26 DIV (Fig. 2C). Since stress/death signaling begins at around 15 DIV (Fig. 2C) and apoptosis only reaches significant levels after 25 DIV, this was the time window utilized to dissect the possible mechanisms of survival under stress. 3.2. TrkB/Akt activity is required for survival of aging hippocampal neurons in vitro In a previous work we have shown that one characteristic of neuronal aging is the progressive increase in the levels of phosphorylated TrkB and its downstream kinase Akt (Martin et al., 2008). Therefore, we assessed the relevance of Trk/Akt pathway on the survival of hippocampal neurons of more than 15 DIV, time at which stress products start accumulating (see above). We started by inhibiting TkrB phosphorylation by adding to cells the K252a kinase inhibitor (Lee and Chao, 2001) and then determined cell death rate by
cytofluorimetric analysis of subG0/G1, hypo-diploid nuclei (see Section 2). Fig. 3A shows that incubation of 15 DIV cells with this inhibitor markedly reduced Akt phosphorylation, directly proving inhibitor efficacy (see below for specificity test). Furthermore, the treatment significantly increased cell death rate compared to untreated neurons in which the Trk activation is left unaffected (Fig. 3B and C). Because K252a has more targets than TrkB, we next assessed the contribution of TrkB/Akt to survival of aged neurons by knocking down TrkB by siRNA methodology. Fig. 4 shows that TrkB knockdown resulted in a 100% increase of apoptosis compared to cells treated with a negative control siRNA. These results leave little doubt of the relevance of TrkB/Akt activity for survival of hippocampal neurons under the stressful environment of the tissue culture dish. 3.3. Cholesterol loss is required for TrkB survival activity of aging hippocampal neurons in vitro Since TrkB activity in aging neurons is triggered by cholesterol loss, independently of BDNF (Martin et al., 2008), we next investigated whether or not cholesterol loss at
Fig. 4. TrkB is required for survival of aged hippocampal neurons in vitro. siRNA duplexes designed to knock down TrkB (siTrkB) were delivered to neurons and apoptosis rate was measured in 17 DIV neurons by the TUNEL assay. In the control, scrambled siRNA duplexes (siNeg) were used. (A) Phase contrast and fluorescence microscopy overlay images showing apoptotic (red) nuclei in siTrkB transfected neurons (green). Lower images is the enlarged view of the cells in the area boxed in the upper phase/fluorescence images, to render more evident the apoptosis in TrkB siRNA neurons. (B) Neuronal death rate, assessed by TUNEL assay, in control cells and in cells where TrkB was knocked down. The plot shows a significant (100%) increase in death rate in siTrkB-treated cells compared to control neurons. Bars represent mean ± s.d. from three different experiments (T-test, p < 0.05).
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this stage of life participated actively in the survival of neurons with accumulated stress, either in old neurons in vivo (Fig. 1) or in post-differentiation neurons in vitro (Fig. 2). The first (in vivo stress accumulation) reflect the normal metabolic stress situation whereas the second (in vitro stress accumulation) may reflect a supra-physiological/pathological, exogenous plus endogenous, stress condition. To analyze the role of cholesterol loss we directly reduced membrane cholesterol in 10 DIV neurons, time at which membrane cholesterol content is highest (Martin et al., 2008). For this, the cholesterol synthesis inhibitor mevilonin and a cholesterol removal drug (-methyl-cyclodextrin, MCD) were added 10 DIV neurons (see Section 2). Fig. 5A (↓chol), shows that cholesterol reduction resulted in the clear activation of Akt. To prove that this effect is a true consequence of the loss of cholesterol and not secondary to any other possible effect of the drugs, cholesterol was added back to the drugtreated 10 DIV cells (see Section 2). This treatment restored the low basal levels of p-AKT to levels of control 10 DIV cells (Fig. 5A; ↑chol). From here we conclude that cholesterol loss is a sufficient stimulus to activate the survival branch of TrkB. Next, we analyzed if cholesterol loss is required for survival of older neurons, which present abundant intracellular stress (see Fig. 2). For this, membrane cholesterol levels of 26 DIV hippocampal neurons were raised to levels of younger cells by adding cholesterol–MCD mix (see Section 2). Survival was then analyzed by cytofluorimetric analysis. As shown in Fig. 5B, the addition of cholesterol (↑chol) to old neurons led to reduced Akt activity, to almost undetectable levels. Consistent with a true functional link, cholesterol addition to these cells increased the rate of neuronal apoptosis in comparison to their untreated counterparts (Fig. 5C and D). 3.4. Cyp46 activity is required for the survival of aging hippocampal neurons, in vitro and in vivo To further test the importance of cholesterol loss in neuronal survival, the cholesterol-hydroxylating enzyme CYP46 (Lund et al., 1999) was knocked down in hippocampal neurons in vitro by specific siRNA, thus blocking the natural occurrence of cholesterol loss. The effect on survival in knocked down cells was measured by the Tunel assay. Fig. 6 shows that CYP46 knockdown resulted in a remarkable increase of apoptosis rate in 17 DIV neurons. On the contrary, CYP46 knockdown did not have a significant effect on the rate of apoptosis in neurons at 10 DIV (Fig. 6B), indicating (i) that the increased apoptosis was not due to a non-specific effect of the siRNA and (ii) that cholesterol loss is fundamentally required for the survival of aged neurons. To test the validity of the last conclusion, lentiviral particles designed to knockdown CYP46 were injected in the hippocampal CA1 region (see Section 2), in which this enzyme is strongly expressed (Ramirez et al., 2008). As expected, survival of hippocampal neurons was not affected by the lack of Cyp46 in young (4 months, data not shown) animals. Surprisingly however, also in 20-month-old mice hippocampal CYP46 knockdown
Fig. 5. Membrane cholesterol loss induces Akt activation and promotes survival in old hippocampal neurons. (A) Effect of pharmacological cholesterol reduction on Akt activity. 10 DIV neurons were treated with mevilonin/MCD (↓ Chol), in order to induce a 25% membrane cholesterol loss. This leads to Akt phosphorylation reaching levels comparable to levels observed in aged cells at 15 and 26 DIV. Replenishment of cholesterol into the same cells (↑ Chol) abrogated activity of Akt. (B) The cholesterol addition (↑ Chol) to 26 DIV neurons results in the loss of Akt phosphorylation. (C) Hippocampal neuron cell death rate, assessed by cytofluorimetric analysis of subG0/G1 hypodiploid nuclei, in 26 DIV untreated (Ctrl) or after cholesterol addition as described in (B). (D) Plot showing the percentage of hypodiploid nuclei in control and treated cells. Note the significant increase in death rate in cultures where cholesterol was added compared to control neurons. Bars represent mean ± s.d. of the percentages of hypodiploid nuclei (n = 4) (T-test, p < 0.05).
did not increase neuronal death (Fig. 7A). Because Cyp46 knockdown leads to apoptosis in old neurons in vitro, the lack of a similar effect in hippocampal neurons in vivo indicated that the Cyp46-cholesterol loss pathway would be of survival usefulness only when exogenous stress is added to the basal situation of metabolic stress accumulation. Alternatively, a reduction in cholesterol synthesis may have occurred in vivo,
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Fig. 6. CYP46 enhances survival strength in aged hippocampal neurons in vitro. Hippocampal neurons were nucleofected with a plasmid expressing a GFP marker and a shRNA designed to knockdown CYP46 (pSi46). Apoptosis rate was analyzed in 10 and in 17 DIV neurons by TUNEL. (A) Fluorescence microscopy image showing pSi46 transfected cells (green) and Tunel staining (red). The white arrows indicate apoptotic nuclei in Cyp46 knockdown cells. Some apoptotic nuclei are also evident in non-transfected cells, however these are a vast minority compared to Cyp 46 knockdown cells (see B). (B) Percentage of apoptotic cells, assessed by TUNEL staining, of pSi46 transfected or control cells at 10 and 17 DIV (results obtained from three experiments as shown in A). The plot shows a significant increase in death rate, compared to control cells, at 17 DIV when CYP46 was knocked down. At 10 DIV both, control and pSi46 transfected cells showed a very low number of apoptotic cells. Bars represent mean ± s.d. from three different experiments (T-test, p < 0.01).
to prevent cholesterol accumulation. In fact, synthesis inhibition prevents cholesterol increase in neurons from Cyp46 knock-out mice (Lund et al., 2003). One way to distinguish between these possibilities was to test the effect of CYP46 knockdown after the intraperitoneal injection of kainic acid (KA) (see Section 2). This is an established procedure to trigger seizures and excitotoxic neuronal injury (Bettler and Mulle, 1995; Fraker et al., 1995; Kure et al., 1991) and would therefore distinguish if Cyp46 participates in survival under extreme stress. In this experiment, neurons transduced with control virus did not show signs of cell damage or death, neither in young (data not shown) nor in old animals (Fig. 7B), indicating that the stress paradigm utilized was not cytotoxic at this time. Differently, neurons transduced with Si46 lentivirus presented clear signs of damage, demonstrated by
Fig. 7. Effect of CYP46 knockdown in vivo in control and in Kainic Acid injected rats. (A) Immunofluorescence images of hippocampal sections of 20 months old animals injected with empty (control) or siCYP46 lentivirus and stained to detect apoptotic nuclei by TUNEL. Transduced cells were identified by expression of GFP. Note that no differences were observed between a control and Si46 virus. (B) Detection of damaged cells in hippocampal slices from 20 months old rats treated intraperitoneally with kainic acid (KA) after viral injection. Note that only CYP46 knocked down cells present signals of cell damage, indicated by the presence of cytoplasmic membrane degradation, beaded neurites, and accumulation of high amount of intracellular aggregates. A magnified picture of a neuron from each case is shown in the insets.
the presence of cytoplasmic membrane degradation, distorted aspect of the cell architecture, i.e. beaded neurites, and accumulation of high amount of intracellular aggregates (Fig. 7B). As we expected considering the results shown in Fig. 6, no cell damage was identified in neurons transduced with Si46 lentivirus in young (KA treated) animals. 4. Discussion It is widely held that oxidative stress increases in the aging brain. Brain cells are particularly prone to gain
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oxidative metabolites because of this organ’s high oxygen consumption per weight unit. Moreover, the brain is not particularly enriched in antioxidant protective defenses (Floyd and Hensley, 2002), clearly pointing out that neuronal survival can be only accomplished if these cells turn on other most efficient survival mechanisms. Because neurotrophins provide with essential survival information during embryonic and early post-natal life, it was commonly thought that they were also involved in the survival of the differentiated brain. However, it was recently shown that both sympathetic and sensory adult neurons live in the absence of trophic factors, suggesting that these cells activate survival pathways that do not require growth factors (Benn and Woolf, 2004). We have recently demonstrated that hippocampal neurons in vitro and in vivo activate the TrkB/Akt survival pathway in a ligand-independent manner, by virtue of the loss of membrane cholesterol levels as neurons age due to increased levels of the cholesterol-hydroxylating enzyme Cyp46 (Martin et al., 2008). The existence of BDNF alternative mechanisms for TrkB activation are also supported by previous reports showing that BDNF-induced TrkB phosphorylation is impaired in the brain of aged rats compared to young counterparts (Gooney et al., 2004), and by our recent study using old, wild type and BDNF knock-out mice (Martin et al., 2008). All these results, together with recent demonstrations that phosphorylation of TrkB in response to supra-physiological concentrations of exogenous BDNF requires normal cholesterol levels (Pereira and Chao, 2007) demonstrate: (i) that BDNF is not a major determinant of TrkB-mediated survival during aging, (ii) that cholesterol loss has a preponderant role in TrkB survival at this stage of life and (iii) that BDNF does not cooperate with cholesterol loss for the occurrence of TrkB activation. Still, despite its low levels, BDNF in the old brain might contribute to survival by activating Trk receptors, which are not affected by cholesterol loss. This possibility and the whole role of BDNF in normal, low cholesterol, aged neurons, need to be thoroughly investigated. In this work we have evaluated the real contribution of this cholesterol loss-induced TrkB/Akt activation to survival of aging neurons. Our results showed that TrkB knockdown in old neurons results in a clear increase of apoptosis rate, even in the neurotrophin depleted conditions in vitro (see Fig. 4). Further contributing to this conclusion, the results showed in Fig. 3 show that inhibition of Akt phosphorylation also resulted in 50% increased rate of apoptosis. Then, we showed that, impairing cholesterol loss either by increasing cholesterol levels in old cells by use of pharmacological treatment or by knocking down CYP46, resulted in the shutting down of the Akt kinase leading to significant increased levels of apoptosis. The discrepancy in the rate of apoptosis obtained when either TrkB or CYP46 were knocked down in vitro (onefold increase for TrkB, Fig. 4; fivefold increase when CYP46 was knocked down, Fig. 6) could be due to differences in knockdown efficiency, considering that different methodology was
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used in each case (see Section 2). However, it is also possible that the differences are due to Cyp46-mediated activation of more than one survival pathway. This possibility needs to be investigated. In agreement with previous results showing that neurons with normal cholesterol levels present low to undetectable TrkB activity (Martin et al., 2008), CYP46 knockdown in young neurons at 10 DIV did not have any effect on neuronal survival. This implies that these cells do not require pro-survival activation, due to low stress levels at this stage (see Fig. 2). Also, lentiviral-mediated CYP46 gene knockdown in vivo did not result in neuronal death in young animals even after treatment with KA, further indicating that cholesterol-loss mediated activation of survival is only required when levels of intracellular stress exceed the physiological threshold. This would explain the situation in hippocampal neurons maintained for long periods of time in vitro, under the chronic presence of uncontrolled excitatory neurotransmission and constant fluctuations in temperature, humidity, concentrations of gases and nutrients. Consistently, cholesterol-mediated survival pathway in vivo was required in old animals (20 month) only after the induction of additional stress, by kainic acid. These data, together with the previous demonstration that ROS are the only factors capable to increase CYP46 transcriptional activity (Ohyama et al., 2006), strongly indicate that stress by-products accumulated during aging are a key determinant of Cyp46 activation and, by virtue of this, of cholesterol loss from the neurons’ membrane. Furthermore, our results show that the levels of stress by-products are a critical step to define if cholesterol loss leads to survival or not. Further work is required to define the type of activities that become modified by the loss of membrane cholesterol when survival is not yet required. This presented data conduce to discuss the relevance of the cholesterol loss pathway in the pathogenesis of Alzheimer’s disease (AD), because it is well established that neuronal populations in AD cases are subjected to particularly high stress conditions (Perry et al., 2002). In fact differential expression of CYP46 has been described AD cases: decreased levels of the enzyme in neurons and increased levels around neuritic plaques (Bogdanovic et al., 2001; Brown et al., 2004). The decrease of CYP46 levels observed in AD neurons could indicate decreased ability of these neurons to activate TrkB/Akt survival pathway and, in the same direction, the high levels around neuritic plaques may reflect the response of the neighbor cells to the high insult provided by the deposition of amyloid aggregates. Furthermore, although still controversial, several groups have found a significant association between single nucleotide polymorphisms (SNPs) in cyp46A1 gene and increased risk of Alzheimer’s disease (Borroni et al., 2004; Kolsch et al., 2002; Papassotiropoulos et al., 2003; see however Desai et al., 2002; Wang and Jia, 2007). It remains to be elucidated if these mutations may predispose to disease by a “loss-of function” mechanism, where impossibility to rise enzyme levels under physiological stress would impair the activation of an important anti-apoptotic
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pathway like that of TrkB. Irrespective of this possibility, the main message from our work should be that cholesterol loss is a physiological process that neurons put to work to fight against stress. We have directly proven that cholesterol loss protects neurons against high stress (i.e. in vitro neurons or after KA injection) therefore it remains to be established what is the true physiological significance of cholesterol loss in the non-stressed brain, as it occurs in normally aged mice and rats. The relatedness of cholesterol loss to AD and whether or not cholesterol loss is activated for survival in other cell types see Cartagena et al., 2008, perennial and cycling, are important questions for future research.
Conflict of interest There are no actual or potential conflicts of interest.
Disclosure statement The appropriate institutional approval for all animal experiments was obtained.
Acknowledgements We thank, Kristel Vennekens and Tatiana Estrada Hernandez for technical assistance. The following institutions provided financial support: Fund for Scientific Research Flanders (FWO), SAO-FRMA Foundation (2006), Federal Office for Scientific Affairs (IUAP P6/43/) and Flemish Government (Methusalem Award).
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