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Vol. 19, Issue 5, 2101-2112, May 2008

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Cholesterol Loss Enhances TrkB Signaling in Hippocampal Neurons Aging in Vitro

Mauricio G. Martin^*,, Simona Perga^*,, Laura Trovò, Andrea Rasola, Pontus Holm^||, Tomi Rantamäki^¶, Tibor Harkany^#, Eero Castrén^¶, Federica Chiara^*,, and Carlos G. Dotti

VIB and Department of Human Genetics, Catholic University of Leuven, B-3000 Leuven, Belgium; Cavalieri Ottolenghi Scientific Institute, Università degli Studi di Torino, 10043 Orbassano (Torino), Italy; Department of Biomedical Sciences, Università degli Studi di Padova, 35122 Padova, Italy; ^#Laboratory of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, and ^||Department of Neuroscience, Karolinska Institute, S-171 77 Stockholm, Sweden; and ^¶Sigrid Juselius Laboratory, Neuroscience Center University of Helsinki, FIN-00014 Helsinki, Finland

Submitted September 13, 2007; Revised February 6, 2008; Accepted February 11, 2008
Monitoring Editor: Robert Parton

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Binding of the neurotrophin brain-derived neurotrophic factor (BDNF) to the TrkB receptor is a major survival mechanism during embryonic development. In the aged brain, however, BDNF levels are low, suggesting that if TrkB is to play a role in survival at this stage additional mechanisms must have developed. We here show that TrkB activity is most robust in the hippocampus of 21-d-old BDNF-knockout mice as well as in old, wild-type, and BDNF heterozygous animals. Moreover, robust TrkB activity is evident in old but not young hippocampal neurons differentiating in vitro in the absence of any exogenous neurotrophin and also in neurons from BDNF –/– embryos. Age-associated increase in TrkB activity correlated with a mild yet progressive loss of cholesterol. This, in turn, correlated with increased expression of the cholesterol catabolic enzyme cholesterol 24-hydroxylase. Direct cause–effect, cholesterol loss–high TrkB activity was demonstrated by pharmacological means and by manipulating the levels of cholesterol 24-hydroxylase. Because reduced levels of cholesterol and increased expression of choleseterol-24-hydroxylase were also observed in the hippocampus of aged mice, changes in cellular cholesterol content may be used to modulate receptor activity strength in vivo, autonomously or as a way to complement the natural decay of neurotrophin production.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
During development, neurotrophins are mandatory for the survival, differentiation, and growth of different neuronal populations (Reichardt, 2006). In the mature nervous system, neurotrophins are important for the modulation of neuronal connectivity and activity-dependent plasticity (Conover and Yancopoulos, 1997; Blum and Konnerth, 2005). Neurotrophins bind and activate receptor tyrosine kinases (RTKs), in turn leading to multiple intracellular signaling pathways, most notoriously those involving mitogen-activated protein kinases and phosphatidylinositol 3-kinase (PI3K) (Kaplan and Miller, 2000; Reichardt, 2006). In the hippocampus, a region of the brain critically involved in certain types of learning and memory, the most prominently expressed neurotrophin receptor is TrkB (Tokuyama et al., 1998), whose cognate ligand is BDNF (brain-derived neurotrophic factor). In agreement with a role in memory-associated processes, loss-of-function studies of both TrkB and BDNF result in changes in affective and cognitive states in mammals and humans (Minichiello et al., 1999; Pozzo-Miller et al., 1999; Xu et al., 2000; Egan et al., 2003; Yeo et al., 2004). Although there is no doubt that BDNF is the main modulator of TrkB activity, a number of evidences indicate that certain roles mediated by the activation of TrkB may occur independently from BDNF. For instance, TrkB conditional knockout mice present clear defects in pre- and postsynaptic morphogenesis in the hippocampus (Luikart et al., 2005), yet this is not the case in BDNF conditional knockout mice (Gorski et al., 2003; Hill et al., 2005). The last observations are in turn consistent with the lack of an overt effect on the development of the hippocampus in BDNF knockout animals (Ernfors et al., 1994; Jones et al., 1994). Furthermore, the absolute need for BDNF for all and every TrkB-mediated activity in the adult hippocampus is challenged by reports showing that the levels and activity of BDNF in the hippocampus start to decay in early adulthood in rats, reaching lowest levels at the end of the first year of life (Gooney et al., 2004; Shetty et al., 2004; Hattiangady et al., 2005; see however Katho-Semba et al., 1998). One simple conclusion from these results is that mechanisms alternative or complementary to BDNF binding have evolved during cellular/neuronal differentiation, to guarantee TrkB activity in the adult brain. The best characterized of such mechanisms is the promiscuity of the lower affinity ligands NT3/NT4 to bind to TrkB (Yan et al., 1993; Davies et al., 1995). Paradigmatically, in vivo studies demonstrated that the lack of BDNF is not always compensated by NT4 and vice versa (Conover et al., 1995; Stenqvist et al., 2005). Robust TrkB signaling in the mature brain can in principle also occur through the increased expression of TrkB subunits, and/or of its p75 coreceptor (Zaccaro et al., 2001; Hartmann et al., 2004; Marshak et al., 2007). Contradicting this possibility, recent studies have shown the down-regulation of full-length TrkB in the hippocampus, starting 2 mo after birth (Silhol et al., 2005). It also appears possible that TrkB signaling in the adult brain could occur via the up-regulation of ligands like adenosine or gangliosides, which have been shown to act as potent inducers of TrkB activity in cells in vitro (Lee and Chao, 2001; Duchemin et al., 2002).

Irrespective of the possibilities of TrkB binding to a combination of classical or noncanonical ligands, one can also envision that cells have evolved non-ligand–mediated mechanisms to guarantee the most efficient transduction of this important signaling receptor. One such mechanism may be the modulation of the content/ratios of different membrane lipids, most notoriously cholesterol. Cholesterol plays a crucial role in the generation of ordered domains in the plasma membrane that laterally segregate certain proteins, thus reducing their rate of lateral diffusion and, by virtue of this, increasing clustering and consequently signaling strength (Edidin, 2003; Hancock and Parton, 2005; and Hancock, 2006). In fact, the number of studies in this regard is steadily increasing, also for RTKs (Paratcha and Ibanez, 2002; Suzuki et al., 2004; Nicolau et al., 2006; Hanzal-Bayer and Hancock, 2007; Jacobson et al., 2007; Pereira and Chao, 2007). These observations prompted us to address the hypothesis that the activity of TrkB in the differentiated hippocampus may be controlled via a physiological modulation of cholesterol levels in the neuronal plasma membrane.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Cell Culture and Reagents
Primary cultures of embryonic rat hippocampal neurons were prepared as described (Kaech and Banker, 2006). Hippocampal neurons from BDNF knockout mice were obtained from crossing BDNF +/– mice (Ernfors et al., 1994). The two hippocampi from each 14–15-d embryo were put in separate tubes; a piece of cortex from each embryo, for later determination of genotype with BDNF specific primers, was also put in separate tubes. The hippocampi were then trypsinized and dissociated as for the rat hippocampal cultures, followed by plating in individual dishes. Each dish was later classified as BDNF +/+, +/–, or –/–, according to the PCR analysis of the corresponding cortical specimens. 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, cells were incubated with particular inhibitors for 2 h to assess target inhibition or for 36 h to measure cell death. The monoclonal anti-Flotillin 1 antibody, clone 18, was from Transduction Laboratories (Franklin Lakes, NJ); the polyclonal chicken anti-BACE 1 antibody, raised against Fc-Asp 2-fusion protein, and the monoclonal anti-transferrin receptor antibody were from Zymed Laboratories (San Francisco, CA). Antibodies used to detect pTrk were polyclonal rabbit either from Santa Cruz Biotechnology (Santa Cruz, CA) or from Cell Signaling (Beverly, MA). Antibodies used to detect TrkB were polyclonal rabbit either from Santa Cruz Biotechnology or from BD Transduction Laboratories (Lexington, KY). Mouse monoclonal anti-tubulin were all from Cell Signaling, and the rabbit antiserum anti-p85 was from Upstate Biotechnology (Lake Placid, NY). CYP46A1 antibody was provided by Dr. Russell (University of Texas Southwestern Medical Center, Dallas, TX) and described previously (Lund et al., 1999).

Stimulation by Ligand and Neurotrophin Inhibition
Predifferentiated 10 days in vitro (10 DIV) hippocampal neurons were cultured in a serum-free medium and stimulated in independent experiments with 100 ng/ml BDNF for 30 min, 50 ng/ml nerve growth factor (NGF) for 10 min, or with a conditioned medium derived from 26 DIV neurons for 120 min. Neurotrophin activities were neutralized in 10 DIV cell culture medium by adding agonist antibodies raised against BDNF, NGF, NT3, NT4/5 (anti-neurotrophin antibody sample pack 1; Chemicon, Temecula, CA) at a concentration of 5 mg/ml for 120 h.

Plasma Membrane Purification
Mature rat hippocampal neurons were lysed by osmotic swelling in 25 mM MES Buffer, pH 7.0, 5 mM dithiothreitol, 2 mM EDTA and protease inhibitor cocktail (Roche, Indianapolis, IN), in ice for 15 min. Extracts were homogenized with a syringe (22 gauge) and centrifuged for 5 min at 700 x g and at 4°C. Supernatants were brought to a final concentration of 1.6 M sucrose, put on the bottom of an SW50 rotor centrifuge tube (Beckman Instruments, Fullerton, CA), overlaid with a continuous sucrose gradient from 1.6 to 0.4 M, and subsequently centrifuged at 12,0000 x g for at least 12 h at 4°C. Eight fractions were collected from the top to the bottom of the tube. The fractions that contain plasma membrane were subjected to centrifugation at 10,0000 x g for 18 h at 4°C. The fractions were finally analyzed by Western immunoblot using antibodies against specific markers of membrane compartments.

Separation of DRMs
Detergent-resistant membranes (DRMs) were prepared from hippocampal membranes (see above) by Triton X-100 extraction, and soluble and insoluble (DRMs) fractions were separated as described previously (Tansey et al., 2000). For some experiments, total plasma membrane extracts were brought to 60% sucrose in MES Buffer Saline (MBS), and a sucrose step gradient was overlaid (35 and 5% sucrose). After centrifugation at 100,000 x g for 18 h at 4°C, fractions were collected from the top of each tube. Fractions 4 and 5 were identified as the DRM fractions by the presence of the DRM marker Flotillin 1.

Western Blotting, Immunoprecipitations, and Antibodies
Rat or mouse hippocampal tissues were homogenized in PBS containing 9% sucrose, protease inhibitors (CLAP: pepstatin, antipain, and chymostatin, each at a final concentration of 25 mM) 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 x g, and supernatants were considered as total extracts. A further centrifugation step was performed at 10,000 x g for 1 h at 4°C to pellet the membrane fraction.

Total and membrane pellets of hippocampal neurons were extracted with two different detergents depending on the aim of the experiment: Nonidet P-40 (1% Nonidet P-40, 10% glycerol, 100 mM NaCl, 2 mM EDTA, 10 mM Tris/HCl, 500 mM sodium orthovanadate, and protease inhibitors) or Triton X-100 buffer (100 mM MES, pH 7, 150 mM NaCl, 1% Triton X-100) in the presence of 1 mM sodium orthovanadate and protease inhibitors. Extracts were clarified by centrifugation, and the protein concentrations were quantified by the BCA method (Bio-Rad Laboratories, Hercules, CA). Proteins were then transferred onto nitrocellulose membranes and probed with primary antibodies for 16 h. Species-specific peroxidase-conjugated secondary antibodies were subsequently used to perform enhanced chemiluminescence detection (Amersham, Little Chalfont, United Kingdom).

For immunoprecipitations, hippocampal neurons were extracted as described above. The soluble and insoluble fractions were diluted with the appropriate buffer to equalize detergent amount (1% Triton and 0.1% SDS). For TrkB and p85 immunoprecipitations, 1 µg of polyclonal antibody/sample was added to protein A–Sepharose beads, and samples were rotated at 4°C overnight. Beads were washed with cold lysis buffer. Immunoprecipitated complexes were separated by 10% PAGE-SDS electrophoresis and subjected to Western blot analysis. Quantification was done by densitometry of the autoradiograms using the NIH Image J software package (http://rsb.info.nih.gov/ij/).

Lipid Extraction and Thin-Layer Chromatography
Lipids were extracted from plasma membranes obtained from rat hippocampal tissues or cultured neurons according to Bligh and Dyer (1959). Extracted lipids (cholesterol, ceramide, and sphingomyelin) were subsequently analyzed by thin-layer chromatography (TLC) on silica gel 60 HPTLC plates using a double-step system (hydrophilic running solvent: chloroform/acetone/acetic acid/methanol/water 50:20:10:10:5) and hydrophobic solvent: hexane/ethyl acetate (5:2) for cholesterol and ceramide. The plate was then dried and developed by spraying with 7% sulfuric acid and heating at 150°C in an oven.

Membrane Cholesterol Quantification, Reduction, and Replenishment
Total cholesterol was measured in membrane extracts from hippocampal neurons in vitro or whole hippocampus as previously described (Abad-Rodriguez et al., 2004). For the induction of membrane cholesterol reduction, 0.4 µM mevilonin and 0.5 mM β-methyl-cyclo-dextrin were added daily to 5-d-old neurons during 4 d (96 h). At the end cholesterol was measured, to ascertain that the treatment did not result in more than 25% reduction. Only validated cases membranes were utilized for further experimentation. Cells were scraped in MES Buffer-CLAP at 4°C, and centrifuged at 1000 x g. Postnuclear supernatants were further centrifuged at 10,0000 x g for 1 h at 4°C to get the membrane pellet. Protein and cholesterol concentrations were measured after resuspension in MES Buffer-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 0.3 mM together with 2 mg/ml free cholesterol. The treatment was performed for 15 min, and then cells were washed and scraped with MES Buffer-CLAP 0.1% Triton X-100 at 4°C

Analysis of Gene Expression
Total RNA samples were prepared from 5 x 10⁵ hippocampal neurons at 10, 15, 20, and 25 DIV using the ABI Prism 6100 Nucleic Acid PrepStation (Applied Biosystems, Foster City, CA). One hundred nanograms of total RNA were first incubated with poly-T oligonucleotides and subjected to reverse transcription using the Superscript First-Strand Synthesis System (Invitrogen, Carlsbad, CA), following the manufacturer's instructions. cDNA was used as a template for the real-time PCR analysis based on the SYBR Green PCR Master Mix assay with the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA). Real-time PCR primers were designed using primer-Express software (Applied Biosystems). Expression of genes encoding TrkA, TrkB, and BDNF and of genes involved in cholesterol synthesis and metabolism was analyzed, and transcriptional expression was normalized using the housekeeping gene encoding for the β Actin (actb) as reference in order to avoid differences due to possible RNA degradation or different reverse transcription efficacy. Relative expression levels of the targets were calculated with the comparative cycle threshold (C_T) method according to the following formula: sample C_T = (gene C_T – actbC_T). To follow expression during ageing, results were normalized to a calibration standard sample of RNA (10 DIV sample). This control was assigned the normalization ratio = 1 and the normalization ratio of each sample was calculated according to the formula: 2^–CT = 2^{–(sample CT – calibrator CT)}.

Immunofluorescence Microscopy
For the light microscopy detection of Cyp46, neurons on glass coverslips were washed in PBS (0.01M), fixed with 4% paraformaldehyde, and processed for immunofluorescence by using anti-CYP46. For the analysis of phospho-TrkB on the surface of young and old cells, cells were first incubated with the corresponding antibody (see above) at 15°C for 15 min to minimize loss by internalization, and excess antibody washed away and then fixed and processed for immunofluorescence microscopy. The surface distribution of the ganglioside GT1b was detected with anti-GT1b mAb (Seikagaku, Tokyo, Japan). Samples were analyzed on an Olympus IX81 fluorescence microscope (Melville, NY). Quantification was performed using the NIH ImageJ software, and signal intensities were normalized by area.

Plasmid Construction
The cyp46A1 targeting sequence was designed by using the RNA interference design algorithm at www.dharmacon.com/DesignCenter/DesignCenterPage.aspx, and 46-sense and 46-antisense oligos encoding cyp46A1-short hairpin RNA (shRNA) were designed as reported by Rubinson et al. (2007). To generate pSi46 plasmid, these oligonucleotides were annealed and cloned into the HpaI and XhoI sites from pLentiLox 3.7 (Rubinson et al., 2007): 46-sense oligo: 5'-TAGATGTACCGTGCGATTCATTCAAGAGATGAATCGCACGGTACATCTTTTTTTC-3'; 46-antisense oligo: 5'-TCGAGAAAAAAAGATGTACCGTGCGATTCATCTCTTGAATGAATCGCACGGTACATCTA-3'.

The full ORF expression clone p46+ including the full cyp46A1 mouse open reading frame was purchased from RZPD (German Resource Center for Genome Research).

Transfection
Primary dissociated hippocampal cells isolated from rat embryos (on embryonic day 18) were transfected using the Rat Neuron Nucleofector Kit from Amaxa Biosystems (Köln, Germany).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Robust TrkB Activity in the Hippocampus of Old Mice Is Not Reliant on High BDNF Expression
Consistent with previous work showing a decay in BDNF levels in the mature and aged brain (Hattiangady et al., 2005; Silhol et al., 2005), the mRNA levels were lower in 10- and 21-mo-old mouse hippocampi as compared with those in 1-mo-old mice (Supplementary Figure 1A). To test whether decreased BDNF levels during aging were paralleled by a decreased activity of TrkB (the main Trk receptor in the hippocampus; Merlio et al., 1992; Cellerino, 1996), phosphorylated TrkB levels were measured in hippocampal membranes of mice as they grew old. Quite paradoxically, Western blotting revealed weak yet noticeable levels of phosphorylated TrkB in the hippocampi of 21-d-old mice and much higher in 10- and 20-month-old animals (Figure 1A). In support of the levels of receptor phosphorylation leading to activation of downstream survival effectors, a similar curve was observed for the downstream target Akt (Figure 1B). To further test that hippocampal cells can potently activate the TrkB pathway in low-BDNF situations, we performed Western blot analysis of hippocampal membranes from BDNF heterozygous (+/–) and knockout (–/–) mice, which have, respectively, half or no BDNF compared with wild-type (see Ernfors et al., 1994; Agerman et al., 2003). Supporting the above prediction, the membranes of 21-d-old BDNF –/– mice and the membranes from 10-mo-old BDNF +/– and +/+ mice all presented a highly phosphorylated TrkB signal (Figure 1, C and D).

View larger version (38K): [in this window] [in a new window]   Figure 1. TrkB signaling robustness in vivo is independent of the levels of BDNF. (A) Western blot of hippocampal membranes from postnatal day 21 (P21), postnatal month 10 (10 m) and postnatal month 20 (20 m), with antibodies against active (pTrk) and total (TrkB) Trk receptors. The active form of the receptor increases dramatically with age, reaching highest levels in 20-mo-old mice. (B) A similar curve was observed for the downstream target Akt, suggesting that the levels of receptor phosphorylation lead to activation of survival effectors. (C) Western blot of hippocampal membranes from 21-d-old BDNF knockout (BDNF –/–) and wild-type animals (BDNF +/+) animals, with antibodies against active (pTrk) and total (TrkB) receptor. Note that the absence of ligand (BDNF –/–) does not affect the levels of receptor expression nor activity (compare with BDNF +/+). The relative amount of pTrk/TrkB present in KO mice respect to the wild-type case is shown on the right. (D) Western blot of hippocampal membranes from post natal day 21 (P21) and postnatal month 10 (10 m) wild-type (BDNF +/+) and BDNF heterozygous mice (+/–). Levels of the active receptor (pTrk) increase with age, in both types of animals.

To determine if hippocampal aging in vitro is accompanied by increased TrkB phosphorylation, as in the in vivo situation (see Figure 1A), the levels of phosphorylated TrkB were measured in membranes from hippocampal neurons maintained in culture for 10, 15, and 26 DIV, representative of immediate terminally differentiated, fully mature, and aging neurons, respectively. This study revealed weak levels of TrkB phosphorylation in 10 DIV neurons, 10% higher in 15 DIV neurons, and a further and a most significant increase in aged 26 DIV neurons (Figure 2A). In agreement with the biochemical data, immunofluorescence microscopy analysis of phospho-TrkB distribution revealed more and more peripherally distributed structures in aged (20 DIV) than in 10 DIV neurons (Figure 2B). Because 20 DIV neurons also presented fully active TrkB at the biochemical level, identical to 26 DIV neurons, 20 or 26 DIV were indistinctly taken as representative of the in vitro aged situation.

View larger version (43K): [in this window] [in a new window]   Figure 2. TrkB activity increases during in hippocampal neurons in vitro. (A) Immunoblotting (IB) of membranes from hippocampal neurons maintained for 10, 15, and 26 d in vitro (DIV). Increased receptor activity (pTrk) occurs with days in vitro, despite equal levels of receptor expression. Protein loading was assessed with anti-TrkB (TrkB) antibody. Quantification of blots from different experiments (n = 5) shows the increase of the pTrk/TrkB levels in aged cells. (B) Immunofluorescence microscopy analysis of phospho-TrkB of the surface of young and old cells. Distribution revealed more and more peripherally distributed structures in old neurons as compared with young neurons. This analysis reveals a high degree of colocalization between active Trk (green) and the ganglioside GT1b (red) in the aged neurons (insets). (C) Immunoblot analysis of Akt activity in cellular extracts from 10, 15, and 26 DIV neurons. Akt activity is low in 10 DIV neurons in comparison with 15 and 26 DIV neurons. This age-dependent increase in activity is not due to differences in the amount Akt protein expression. Quantitative analysis is shown on the right. (D) Effect of addition of the Trk inhibitor K252a on Trk phosphorylation and Akt activity. Addition of the inhibitor to 26-d-old cells (+, treated; –, untreated) result in the great reduction of Trk and Akt phosphorylation after 24-h treatment. These results suggest that the high levels of Trk activity found in old cells promote survival.

To further strengthen the notion that hippocampal neurons in vitro is a good system to study the possible mechanisms behind robust TrkB activity in low neurotrophin conditions, the activity of the receptor was next assessed in hippocampal neurons in vitro from BDNF knockout embryos (obtained by crossing BDNF +/– mice; see Materials and Methods), grown in the absence of any exogenous neurotrophin. In these cells, rate of hippocampal neuron survival and the degree of morphological differentiation was, as observed in vivo (Ernfors et al., 1994), undistinguishable from wild-type neurons (Figure 3A). More relevant for the purpose of this work, 15 DIV neurons from BDNF –/– embryos presented a phosphorylated TrkB signal, not different from that of neurons from wild-type littermates at the same age in vitro (Figure 3B). This last result conclusively demonstrates that hippocampal neurons have mechanisms to elicit TrkB activity independently from the presence of the receptor natural ligand. To test whether or not such activity could be attributed to excess production/release of low-affinity ligands, we shifted back to the rat embryo hippocampal neurons from wild-type animals, which present robust TrkB activity in the absence of exogenous neurotrophins (see Figure 2A). For this purpose, the culture medium of 26 DIV neurons, in which low-affinity ligands would be highest if responsible for the presence of high receptor phosphorylation at this stage, was added to 10 DIV cells, that present only weak TrkB activity (see Figure 2 A). This treatment did not induce TrkB activity in 10 DIV cells (Figure 3C), suggesting that activation in the old neurons was not due to increased production of NT3/NT4 or adenosine (see Introduction). In further agreement, incubation of 15 DIV neurons with a cocktail of neurotrophin function-blocking antibodies neither prevented nor reduced the spontaneous appearance of TrkB activity in these neurons (Figure 3D). On the other hand, the antibody cocktail was efficient to inhibit the activity induced by supraphysiological amounts of BDNF (Figure 3E), ruling out that their inefficacy to block spontaneous activation in old neurons (Figure 3D) was due to lack of inhibitory activity or the capacity to reach the sites of receptor location on the neuronal surface (synaptic cleft). Surely, these data do not exclude that activation of TrkB during aging may be due to binding to as yet unidentified or nonsoluble ligands, which might have become up-regulated with aging. On the other hand, these data, together with the previous in situ results, clearly demonstrate that differentiated hippocampal neurons posses mechanisms that guarantee TrkB activity even under conditions of minimal availability of canonical neurotrophins.

View larger version (51K): [in this window] [in a new window]   Figure 3. TrkB activity in fully differentiated hippocampal neurons in vitro is ligand-independent. (A) Phase-contrast images of hippocampal neurons from BDNF +/+ and BDNF –/– embryos, maintained in culture for 1, 4, and 15 DIV: cell number and degree of morphological differentiation is similar. (B) Immunoblotting of membranes from hippocampal and cortical neurons, from BDNF –/– and BDNF +/+ embryos, maintained for 15 DIV: the intensity of phosphorylated TrkB signal in the –/– cells is comparable to that of +/+ cells. Protein loading was assessed with anti-TrkB (TrkB) antibody, and the relative amount of pTrk/TrkB present in –/– cells with respect to the wild-type case is shown below. (C) Neurons from wild-type rat embryos maintained in vitro for 10 d (10 DIV) were treated for 30 min with the medium of neurons maintained in vitro for 26 d (26 DIV medium): this treatment does not elicit any TrkB activation in these cells, despite abundant levels of receptor. (D) Western blot analysis of TrkB activation of 15 DIV neurons incubated or not with a cocktail of neurotrophin-blocking antibodies (Anti-NT Abs), starting on day 10 in vitro (see Materials and Methods). Note that the antibody mix did not perturb the increase in TrkB signal, whether for total Trk (TrkB) or its phosphorylated form (pTrk). (E) Control of antibody-mix efficacy as neurotrophin-blocking TrkB activation. Neurons kept in vitro for 10 DIV neurons were incubated with 100 mg/ml BDNF; effect on TrkB activity (pTrkB) was measured 30 min later. Treatment triggers intense TrkB activity (pTrkB; BDNF +) in nonneurotrophin antibody-mix–incubated neurons (Anti-NT Abs, –); TrkB activity is absent in cells coincubated with the antibody mix (BDNF +/Anti-NT Abs, +).

View larger version (44K): [in this window] [in a new window]   Figure 4. Moderate still continuous loss of cholesterol from the plasma membrane of differentiated hippocampal neurons in vitro determines TrkB DRM partitioning. (A) Colorimetric measurement of plasma membrane cholesterol from hippocampal neurons kept for 10, 15, and 26 DIV. Mean ± SD values (nmol/mg protein, n = 4) were 0.61 ± 0.029, 0.55 ± 0.047, and 0.43 ± 0.023 in 10, 15, and 26 DIV neurons, respectively. (B) TLC analysis of the membrane cholesterol content in DRMs of 10, 15, and 26 DIV neurons; cholesterol is clearly and progressively reduced in DRMs from 15 and 26 DIV neurons. Percentages of cholesterol reduction in DRMs (fractions 4 and 5) and non-DRMs (fractions 6–8) were measured by densitometry and normalized for sphingomyelin values of different sucrose fractions (n = 2). In DRMs, cholesterol was decreased to 43.29% in 15 DIV cells, and to 25.92% in 26 DIV cells. The migration of the cholesterol standard is indicated (cholesterol). (C) Western blotting with anti-total Trk, anti-phospho Trk, and anti-prion protein (PrPc) antibodies, of 4°C detergent-extracted, sucrose-gradient centrifuged membranes from 10 and 26 DIV hippocampal neurons in vitro. Note that receptor activity is exclusively present in the DRM domains (fractions 4 and 5) of 26 DIV neurons. The PrPc lane demonstrates that the presence in fractions 4 and 5 truly reflect DRM partitioning. (D) Immunoprecipitations of TrkB from the DRMs and non-DRMs of 10 and 26 DIV neurons, probed with antibodies against phospho Trk and the anti-p85 subunit of PI3K. Note that the downstream survival effector of TrkB is precipitated exclusively from the DRM domains of 26 DIV neurons. (E) Control for correct separation between DRMs and non-DRMs fractions. Lysates utilized in C were hybridized with antibodies against the non-DRM protein Transferrin receptor (IB: TfR) or the DRM protein PrPc (IB: PrPc).

View larger version (36K): [in this window] [in a new window]   Figure 5. (A) Sucrose gradient membrane fractions from cold detergent-solubilized plasma membranes from 10, 15, and 26 DIV hippocampal neurons followed by immunodetection for the protein BACE 1. Note that BACE1 segregation to DRMs progressively decreases with time. Percentages of BACE1 distribution in DRMs (fractions 4 and 5, ) and nonDRMs (fractions 6–8, ) were measured by densitometry and reported in the graphics to the right of the Western lot (mean ± SD, n = 4). In exact numbers, BACE 1 was 35.3 ± 5.8% in the DRMs of 10 DIV neurons, decreasing to 25.3 ± 4.1% in 15 DIV neurons and further decreasing to 15.8 ± 6.4% in senescent cells. (B) Sucrose gradient fractions of cold detergent-solubilized plasma membranes from 10 and 26 DIV neurons followed by immunodetection for the protein Fyn. Percentages of Fyn distribution in DRMs (fractions 4 and 5, ) and non-DRMs (fractions 6–8, ) were measured by densitometry and reported in the graphics on the right (mean ± SD, n = 4). In 10 DIV neurons 34.75 ± 2.8% of Fyn is in DRMs, increasing to 58.9 ± 8.6% in 26 DIV neurons.

View larger version (24K): [in this window] [in a new window]   Figure 6. Pharmacological-induced changes in cholesterol levels in young neurons modulate pTrk activity. (A) Western blot of hippocampal membranes from 10, 15, and 26 d in vitro (DIV) control neurons as well as from 10 d in vitro (10 DIV) neurons with cholesterol levels reduced to the values of untreated neurons 26 DIV neurons (see Figure 2A; Chol) and 10 DIV neurons with cholesterol levels replenished after reduction, to reach 10 DIV levels (Chol). Note how cholesterol reduction in the 10 DIV cells triggers intense TrkB activity, whereas its replenishment to their natural values makes receptor activity disappear. (B) Sucrose gradient fractions of cold detergent-solubilized plasma membranes from 10 in vitro (DIV) with control (ctrl), pharmacological reduction of membrane cholesterol ( Chol, 25% less than in control cells) or with cholesterol levels replenished after reduction to control levels (Chol). The 25% decrease in membrane cholesterol induces the displacement of Flotillin-1 away from DRMs (Chol), yet replenishment restores DRM-preferential partitioning (Chol, compare with Chol). Percentages of Flotillin-1 distribution in DRMs (fractions 4 and 5, ) and nonDRMs (fractions 6–8, ) were measured by densitometry (graphics on the right, mean ± SD, n = 4). Flotillin 1 was 31.3 ± 8% in the DRM domains of 10 DIV neurons. Cholesterol depletion reduced Flotillin-1 in DRMs to 14 ± 3.0%; cholesterol replenishment of the latter cells increased Flotillin 1 in DRMs to 50.4 ± 8.0%.

View larger version (36K): [in this window] [in a new window]   Figure 7. Differentiation-occurring increase in cholesterol-24-hydorxylase is sufficient and necessary for TrkB activation. (A) Compared with 10 DIV neurons, and after normalization for actin mRNA, mRNA expression levels of cholesterol-24-hydroxylase (CYP46) increase at 26 DIV. Variations related to 10 DIV (value of 1) are indicated. (B) Immunofluorescence analysis of the expression levels of cholesterol-24-hydroxylase in hippocampal neurons maintained for 10 and 26 DIV. Antibody concentration and image exposure were identical for all three time points. Higher reactivity occurs in the 26 DIV neurons, consistent with the higher expression of the messenger (see A). (C) Hippocampal neurons in suspension were transfected either with the plasmid expressing cyp46a1 (p46+) or scrambled vector (pLL3.7) or with the plasmid expressing the cyp46a1-siRNA (pSi46). TrkB activity was measured at 7 DIV or 10 DIV. Note how the increased expression of the cholesterol-24-hydroxylase (CYP46) resulted in high levels of active TrKB in these cells, and reduction of the cholesterol 24-hydroxylase by siRNA resulted in lower levels of Trk phosphorylation. The amount of pTrk/tubulin either in pSi46 or p46+ transfected cells respect to the control is shown. Quantification of the Western blottings performed using anti-cholesterol-24-hydroxylase antibody reveals the efficacy of the knockdown strategy. (D) Immunofluorescence microscopy with anti phospho-Trk antibody (p-Trk) reveals that reduction in cholesterol-24-hydroxylase results in reduced levels of active Trk (IF:pTrk). The efficacy of the knockdown strategy was analyzed with anti-cholesterol-24-hydroxylase antibody reveals (see IF: CYP46).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

View larger version (20K): [in this window] [in a new window]   Figure 8. In vivo reduction of membrane cholesterol, changes in membrane DRM partitioning characteristics and increased levels of cholesterol-24-hydroxylase. (A) Colorimetric analysis of membrane cholesterol levels in hippocampal membranes at different postnatal times: 10 d (p10), 1 mo (1 m), 3 mo (3 m), 10 mo (10 m), and 21 mo (21 m). Cholesterol is most abundant in the membranes of early postnatal hippocampus (p10) undergoing significant loss with time, reaching the lowest levels at month 21 (21 m). (B) Western blot analysis with an antibody against the canonical DRM protein Flotillin 1 in detergent extracted, sucrose gradient separated, DRM and non-DRM fractions from 1- and 21-mo-old mice hippocampi. Note the clear DRM partitioning in the membranes from young animals and the more homogeneous distribution in the old animals. (C) Western blotting (left) and quantification (right) of the expression levels of cholesterol-24-hydroxylase in hippocampal extracts from 10 d (p10) and 1-, 3-, 10-, and 21-mo-old mice. CYP46 levels were normalized for tubulin. (D) Real-time PCR analysis of expression levels of cholesterol-24-hydroxylase. Total RNA was obtained from hippocampus from 10 d (p10) and 1-, 3-, 10-, and 21-mo-old mice. As shown in C and D, time-associated increased expression of the enzyme parallels both loss of cholesterol (see above, A) as well as the increased partitioning of TrkB in DRMs in this structure (see Figure 3C).

Cholesterol loss in differentiated neurons, both in vivo and in vitro, seems to occur via up-regulation of the catabolic enzyme cholesterol-24-hydroxylase. This brain specific enzyme is responsible for the conversion of cholesterol into 24S-hydroxycholesterol, the excreted form of cholesterol (Lund et al., 1999, 2003), an event that progressively increases with age (Lutjohann, 2006). Although this does not rule out that other events contribute to brain cholesterol reduction during ageing, such as a decay in the activity of synthesizing enzymes or a reduction in cholesterol transport from astrocytes, overexpression of cholesterol24-hydroxylase induced TrkB activation in young neurons and its knockdown prevented its occurrence in the old neurons, proving that modulation of this enzyme can act as a sufficient and necessary stimulus for receptor activation. Although the finding that the activity of the enzyme is also high in the differentiated hippocampus in situ makes us feel confident that this pathway might operate in vivo, direct validation is needed. Quite encouraging that this may be the case, the only (up to now) event demonstrated to increase the expression of cholesterol-24-hydroxylase activity is oxidative stress (Ohyama et al., 2006). Unfortunately, the use of cholesterol-24-hydroxylase knockout mice cannot be envisioned for this purpose, as these mice compensate decreased cholesterol excretion by a reduction in de novo synthesis (Lund et al., 2003).

The observation that up-regulation of cholesterol-24-hydroxylase can activate TrkB makes unavoidable to discuss its relevance for Alzheimer's disease (AD). In fact, although still controversial, several groups have found a significant association between intronic mutations in this gene and increased risk of AD (Kolsch et al., 2002; Papassotiropoulos et al., 2003; Borroni et al., 2004; see however, Desai et al., 2002; Wang and Jia, 2007). Although we here report that cholesterol loss, triggered by the up-regulation of cholesterol-24-hydroxylase, leads to TrkB activation, we have previously demonstrated that cholesterol loss of more than 30% can lead to apoptosis (Ledesma et al., 2003; Abad-Rodriguez et al., 2004). In this context, and considering that TrkB activity is important for survival of differentiated cortical neurons under stress (Gates et al., 2000), it is quite conceivable that intronic mutations could predispose to AD through a "gain-of-function" process, i.e., premature and constant loss of cholesterol leading to cell death upon the occurrence of further loss (beyond 30%) triggered when age-associated (i.e., cumulative) stress passes the physiological threshold. But it is also possible that the intronic mutations found in certain cases of AD may predispose to disease by a "loss-of function" mechanism, whereby this enzyme would not respond to physiological stress and therefore prevent the activation of an important antiapoptotic pathway like that of TrkB. To distinguish between these possibilities is another interesting derivation from this work.

	ACKNOWLEDGMENTS

 
We thank Outi Nikkila for preparing the BDNF +/– crosses; Kristel Vennekens (University of Leuven) for the hippocampal cultures; Francesca Gilli and Fabiana Marnetto for help with the real-time PCR experiments; and Laura Doglio, Jose Abad-Rodriguez, and Maria D. Ledesma for discussions and technical advice. The following institutions provided financial support: Swedish Medical Research Council and the Alzheimer's Association (T.H.), Sigrid Juselius Foundation and Academy of Finland (E.C.), and Fund for Scientific Research Flanders (FWO), Federal Office for Scientific Affairs (IUAP P6/43/), and Flemish Government (Methusalem grant; C.G.D.).

	Footnotes

 
This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09-0897) on February 20, 2008.

* These authors contributed equally to this work.

Address correspondence to: Federica Chiara (fchiara{at}bio.unipd.it) or Carlos G. Dotti (carlos.dotti{at}med.kuleuven.be).

	REFERENCES

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