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Published as doi: 10.1096/fj.08-109306.
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(The FASEB Journal. 2008;22:3823-3835.)
© 2008 FASEB

Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the basic fibroblast growth factor signaling cascade and reducing extracellular regulated kinase 1/2-dependent cyclin E expression

Luis G. Rabaneda*,1, Manuel Carrasco*,1, Miguel A. López-Toledano*, Maribel Murillo-Carretero*, Félix A. Ruiz*,{dagger}, Carmen Estrada* and Carmen Castro*,2

* Area de Fisiologia, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain; and

{dagger} Hospital Universitario Puerta del Mar, Cádiz, Spain

2Correspondence: Plaza Falla, 9, 11003 Cádiz, Spain. E-mail: carmen.castro{at}uca.es


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Hyperhomocysteinemia (HHcy)—abnormally elevated plasma levels of homocysteine (Hcy)—has been associated with the development of neurodegenerative dementia and mild cognitive impairment. This association suggests that HHcy might facilitate memory loss in the elderly. As memory loss can occur through a deteriorated neurogenic capacity, we have studied the effects of Hcy on neural progenitor cells (NPCs) both in vitro and in vivo. We show that Hcy exerts an antiproliferative effect on basic fibroblast growth factor (bFGF) -stimulated NPCs isolated from the postnatal subventricular zone (SVZ), accompanied by inactivation of the extracellular signal-regulated kinase (Erk1/2) and inhibition of Erk1/2-dependent expression of cyclin E. Using a mice model we show that, under normal folate conditions, HHcy exerts an inhibitory effect on adult brain neurogenesis. This inhibition occurs in the caudal areas of the dentate gyrus (DG) of the hippocampus, a neurogenic area mainly involved in learning and memory performance, and in the SVZ, recently implicated in olfactory learning performance. In both areas reduced number of proliferative neuroblasts were found. Since neuroblasts are primarily bFGF-responsive progenitors already committed to a neuronal phenotype, our results strongly suggest that excess Hcy inhibits neurogenesis in the DG and SVZ by inhibiting the bFGF-dependent activation of Erk1/2 in these cells.—Rabaneda, L. G., Carrasco, M., López-Toledano, M. A., Murillo-Carretero, M., Ruiz, F. A., Estrada, C., Castro, C. Homocysteine inhibits proliferation of neuronal precursors in the mouse adult brain by impairing the bFGF signaling cascade and reducing Erk1/2-dependent cyclin E expression.


Key Words: hyperhomocysteinemia • neurogenesis • subventricular zone • dentate gyrus • dementia


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
HOMOCYSTEINE (HCY) IS A sulfur-containing intermediate of methionine metabolism. Hcy accumulates in peripheral blood, and induces hyperhomocysteinemia (HHcy) when Hcy metabolism is impaired due to defects in crucial enzymes that participate in either Hcy recycling through transmethylation to methionine, or in Hcy elimination through the trans-sulfuration pathway. Dietary deficiencies (such as folate) in the substrates and cofactors that participate in the above reactions may also lead to HHcy.

Over the past 5 years, HHcy has been linked closely to memory loss and development of dementia in the elderly. HHcy and low dietary folate are now known as independent risk factors for the development of Alzheimer’s disease and other neurodegenerative dementias (1 , 2) . Likewise, HHcy is an independent risk factor for memory deficit and development of cognitive impairment without dementia (3 , 4) . In addition, it has lately been shown in aged adults of the Dutch population that dietary folate supplement improves cognitive capacity (5) , which indicates that excess Hcy, low folate, or low folate-induced HHcy may weaken memory function in the brains of aged people.

Attempts to understand the molecular mechanisms implicated in HHcy-induced memory loss have focused mainly on studying the toxic effects of Hcy on mature neurons (6 , 7) . However, memory loss can also result from deteriorated neurogenic capacity. It is now well established that, in the mammalian adult brain, new neurons emerge within specific regions. One of these regions is the dentate gyrus (DG) of the hippocampus, a brain region that plays a significant role in cognitive performance (8 , 9) . Throughout adulthood, new neurons are generated from dividing precursors in the subgranular zone and reach the granular cell layer of the DG (10 , 11) , where they become integrated into existing circuits of the hippocampus (12 , 13) . Results from several studies imply that these new neurons in the DG of the adult brain play an important role in memory tasks (14 15 16) . The SVZ, in the lateral wall of the lateral ventricles, is another area with strong neurogenic activity in adult rodents (17) . Newly generated neuroblasts produced within the SVZ proliferate and migrate tangentially through the rostral migratory stream (RMS) and develop into inhibitory interneurons in the granular and periglomerular layers of the olfactory bulb (18) . Neurogenesis in the SVZ plays a role in olfactory learning performance (19) .

It has recently been reported that, in adult mice fed a folate-deficient diet, fewer cells undergo division within the DG, with the concomitant development of HHcy, and that in vitro inhibition of folate synthesis by methotrexate impairs embryonic neuroepithelial cell proliferation (20) . Thus, it would be reasonable to hypothesize that there must be a direct cause-effect association between HHcy and neurogenesis in the adult brain. However, folate 1-carbon units are essential for de novo purine and thymidine biosynthesis, and folate deficiency has been shown to reduce proliferation of various cell types, inducing S-phase cell cycle arrest (21 22 23) . Therefore, it is not yet clear whether HHcy is the cause of the observed reduction in neurogenesis or simply a reflection of the lack of folate. Furthermore, supposing that HHcy was directly responsible for the diminished neurogenesis, several questions would remain to be answered, such as whether the reported inhibitory action of Hcy on adult brain neurogenesis is restricted to the DG or also affects the SVZ, what types of molecular mechanism are involved in the inhibition of neural progenitor cell (NPC) proliferation by elevated Hcy, and whether the Hcy-induced antiproliferative effect is exerted on a specific type of neural precursor cell.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
For in vitro studies, a stock solution of 100 mM DL-Hcy was prepared from Hcy thiolactone, as described elsewhere (24) . In all in vitro experiments, double amounts of DL-Hcy were added to cell culture wells in order to obtain each concentration of L-Hcy used. For in vivo studies, DL-Hcy was purchased directly from Sigma-Aldrich (St Louis, MO, USA). U0126, also from Sigma-Aldrich, was used at 10 µM.

Animal subjects
CD1 mice were used throughout this study. Mice were housed individually under controlled conditions of temperature (21–23°C) and light (L:D 12:12) with free access to food and water. Mice were fed with AO4 standard maintenance diet (SAFE, Épinay-sur-Orge, France). Care and handling of animals were performed according to the Guidelines of the European Union Council (86/609/EU), following the Spanish regulations (RD 1201/2005) for the use of laboratory animals.

SVZ cell isolation and culture
NPCs were obtained from the SVZ of postnatal (P7) mice following the procedure reported by Doetsch et al. (18 , 25) and modified by Torroglosa et al. (26) . Neurosphere cultures were maintained in defined medium (DM) composed of DMEM/F-12 (1:1) plus the medium supplement B27 (Invitrogen; Carlsbad, CA, USA), 2 mM glutamine and 2 µg/ml gentamicin. Epidermal growth factor (EGF) (20 ng/ml) and bFGF (10 ng/ml; both from PeproTech, Frankfurt, Germany), were added unless otherwise indicated. To test the effects of Hcy on NPCs, neurosphere cells were mechanically disaggregated in DM. Single cells were seeded at a density of 20 cells/µl, in 96-well plates. DL-Hcy was added at the time of seeding. Triplicates of each condition were tested in every experiment. Results presented here are the average of at least 3 independent experiments performed in triplicate samples.

Neurosphere formation assays
The number of newly formed neurospheres per well was counted under phase microscopy 72 h after seeding. To measure neurosphere size, images of 10 fields/well were obtained, and the area of the newly formed neurospheres (at least 50 neurospheres/well were measured) was estimated using the Cell F software analysis system (Olympus, Hamburg, Germany).

Cell viability assays
Percentages of dead cells among the neurospheres were determined 24 and 72 h after seeding, by trypan blue exclusion after disaggregation of cells.

In vitro cell death
Cells were plated onto poly-L-ornithine (PLO) -coated 8-well glass slide chambers (Nalgene Nunc International, Naperville, IL). Hcy was added at the time of plating. At 24 and 72 h after exposure to Hcy, cells were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min. Apoptosis was estimated by counting pyknotic nuclei after staining with DAPI (Sigma-Aldrich). A total of 25 fields/well was counted.

Caspase-3 activity assay
Caspase 3/7 activity was detected using caspase-Glo reagent (Promega, Madison, WI, USA). Cells were disaggregated from neurospheres seeded onto 96-well plates and grown in the presence of EGF and bFGF for 4, 8, and 24 h in the presence and absence of 100 µM Hcy, which was added at the time of seeding. Next, 100 µl of caspase-Glo 3/7 reagent was added to each sample and the cells were incubated for 1 h and assayed according to the manufacturer’s instructions.

Immunocytochemistry
Cells dissociated from neurospheres were seeded onto PLO-coated 8-well glass slide chambers and maintained for 72 h in DM supplemented with the growth factors indicated. Hcy was added at the time of seeding and cells were fixed with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer. Immunodetection of the different antigens was performed essentially as described elsewhere (27) , and fluorescent signals were detected using a BX60 epifluorescence microscope (Olympus, Center Valley, PA, USA). As negative control, adjacent cultures were incubated without a primary antibody and similarly processed. Antibodies used were rabbit polyclonal anti-Ki67 (Vector, Burlingame, CA, USA), mouse monoclonal anti-β-tubulin-III (Promega, Madison, WI, USA), polyclonal antipErk1/2 Tyr 202 and Tyr 204 (Cell Signaling Technologies, Boston, MA, USA), anti {alpha}-tubulin (Sigma-Aldrich), and goat anti-mouse IgG (H+L) and goat anti-rabbit IgG (H+L) labeled with either AlexaFluor 568 or 488 (Invitrogen). Direct counts of positive cells for each antibody were performed under epifluorescence microscopy and expressed as the percentage of the total number of cells, as assessed by nuclear DAPI staining. A total of 12 predetermined visual fields was analyzed per well, and 2–4 wells/condition were analyzed in each experiment.

Immunoblot analysis
Cells from neurospheres were disaggregated and incubated for 1.5 h in the presence or absence of 100 µM L-Hcy; then the growth factors (EGF, bFGF, or a combination of both) were added, and the culture was processed at different time points afterward. Cells were lysed with ice-cold cell lysis buffer containing phosphatase inhibitors (Cell Signaling Technologies) with 1 mM PMSF. Supernatants were collected after centrifugation (16,000 g) and their protein concentration was measured. Equal amounts (50 µg) of total protein from each cellular extract were used for immunoblot detection. We used the following antibodies: anti-pErk1/2 Tyr 202 and Tyr 204, anti-Erk1/2, anti-pAkt (Ser-437), and anti-Akt (Cell Signaling Technologies); anti-cyclin E (Santa Cruz Biotechnologies, Santa Cruz, CA, USA); and anti-{alpha}-tubulin (Sigma-Aldrich). Peroxidase secondary antibodies were from Pierce (Rockford, IL, USA). Antibody concentrations were chosen following manufacturers’ instructions.

Animal model of HHcy
An animal model of HHcy was established by implanting Hcy-releasing osmotic minipumps into adult mice (38–42 g). Under general anesthesia (ketamine 120 mg/kg; xylacine 20mg/kg), an osmotic minipump (Alzet 2002; Alzet, Palo Alto, CA, USA) was placed subcutaneously in the subscapular fossa region. Minipumps were filled with 200 µl of either vehicle (sterile filtered dH2O) or 100 mM Hcy, to be released over 2 wk (equivalent to <1 mg Hcy/day).

Bromodeoxyuridine (BrdU) administration
Two groups of control and HHcy mice were injected intraperitoneally with the thymidine analog BrdU (120 mg/kg/day) for a total of 7 days either during the second week of Hcy treatment (28) (group 1: 5 control mice and 5 HHcy mice; see Fig. 7D ) or during the week before introduction of the Hcy minipumps (group 2: 5 control mice and 5 HHcy mice; see Fig. 7D ).


Figure 1
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  		Figure 1. Hcy treatment inhibits proliferation of NPCs in vitro. A, B) Phase microscopy image of the neurospheres formed from SVZ cells grown for 72 h in the combined presence of EGF and bFGF control (A) and treated with 100 µM Hcy (B). C) Average number of newly formed neurospheres in control cultures and treated with Hcy for 72 h. The average number of neurospheres in control cultures was 506 ± 49. D) Dose-response effect of Hcy on average size of newly formed neurospheres. Treatment with Hcy lasted for 72 h. E) Total number of cells after disaggregation of neurosphere cells in control and 100 µM Hcy-treated cultures. F) Percentage of nonviable cells in control and 100 µM Hcy-treated neurosphere cultures. G) Fluorescence microscopy images of NPCs in cultures adhered onto PLO and grown in the combined presence of EGF and bFGF. Cells were double-stained with DAPI and Ki67. Left panels: control cultures; right panels: cultures treated with Hcy at 100 µM. Scale bar = 50 µm. H) Percentage of Ki67+ cells in control and 100 µM Hcy-treated NPC cultures adhered onto PLO. I) Caspase 3/7 activity of NPCs stimulated with EGF and bFGF in the presence and absence of Hcy for 4, 8, and 24 h. Caspase-3 activity was determined by the caspase-Glo 3/7 luminescence assay. Assays were performed in triplicate for each treatment (RLUs of blanks without cells were <0.2% of control). J) Total number of pyknotic nuclei in control and 100 µM Hcy-treated cultures adhered onto PLO. Data are means ± SE of the results obtained in 3–4 independent experiments. *P < 0.05.


Figure 2
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  		Figure 2. Hcy inhibits bFGF-stimulated NPC proliferation. A) Phase microscopy image of the neurospheres formed from NPCs isolated from the SVZ and grown for 72 h in the presence of EGF alone or in the combined presence of EGF and bFGF, in two different conditions: control without Hcy (top panels) and NPCs treated with 100 µM Hcy (bottom panels). B) Normalized dose-response curves of average neurosphere size plotted against Hcy when cultured in the presence of EGF alone or in the presence of bFGF + EGF. The average area in untreated cultures was 2418 ± 90 µm2 with EGF + bFGF and 1300 ± 108 µm2 with EGF alone. C) Proportion of Ki67+ in control and in Hcy-treated (100 µM) cultures adhered onto PLO in the presence of bFGF alone (*P<0.05). The percentage of Ki67+ cells in similar cultures without growth factors was 55 ± 7.5% of the culture with bFGF alone. D) Proportion of Ki67+ cells in control and in Hcy-treated (100 µM) cultures adhered onto PLO in the presence of EGF alone. The percentage of Ki67+ cells in similar cultures without growth factors was 30 ± 4% of the culture with EGF alone.


Figure 3
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  		Figure 3. Inhibitory effect of Hcy on bFGF-induced ERK 1/2 phosphorylation. A) Representative autoradiography images obtained after immunodetection of pErk1/2, total Erk1/2 and {alpha}-tubulin, and pAkt and total Akt, in the culture conditions indicated. B) Quantification of Erk1/2 phosphorylation after 30 min in the presence of each combination of growth factors, with and without pretreatment with 100 µM Hcy for 1.5 h prior to the addition of growth factors. Density values are percentages of the values of pErk1/2 density obtained in cultures without growth factors. C) Fluorescence microscopy images of NPCs in cultures adhered onto PLO and grown in the presence of bFGF in control conditions (top panels) or after treatment with 100 µM Hcy (bottom panels). Cells were immunostained stained with pErk1/2 and {alpha}-tubulin. Merged images are shown. Scale bar = 50 µM.


Figure 4
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  		Figure 4. Inhibition of pErk1/2 by U0126 decreases NPC proliferation and cyclin E expression. A) Representative autoradiography images obtained after immunodetection of pErk1/2, cyclin E, and {alpha}-tubulin in cultures grown in the conditions indicated. Concentrations: Hcy, 100 µM; U0126, 10 µM. Treatment with Hcy or U0126 was added 1.5 h before addition of bFGF. Cells were lysed 4 h after addition of bFGF. Values obtained in cultures grown without bFGF and without treatment were used as baseline. B) Quantification of Erk1/2 phosphorylation in the presence of each combination of bFGF and Hcy or U0126 treatment. Density values are percentages of the values of pErk1/2 density obtained in cultures without treatment and growth factors. C) Quantification of cyclin E in the presence of each combination of bFGF and Hcy or U0126 treatment. Density values are percentages of the values of cyclin E density obtained in cultures without treatment and growth factors. D) Percentage of Ki67+ cells in control, 100 µM Hcy, or 10 µM U0126-treated NPC cultures adhered onto PLO. Data are means ± SE of the results obtained in 4 independent experiments. #P < 0.05 vs. control; *P < 0.05 vs. bFGF alone.


Figure 5
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  		Figure 5. Effect of Hcy on the proliferation of neuronal precursors. A, B) Fluorescence microscopy images of NPCs in cultures adhered onto PLO and grown in the presence of bFGF in control conditions (A) or after treatment with 100 µM Hcy (B). Cells were immunostained with DAPI, Ki67, and β-tubulin-III. Merged images are shown. Scale bar = 50 µm. C) Numbers of double-stained Ki67+/β-III-tubulin+ cells, as percentages of the total number of β-III-tubulin cells in control and 100 µM Hcy-treated cultures. D) Numbers of Ki67+ cells in control and 100 µM Hcy-treated NPC cultures, as percentages of the total number of cells. Data are means ± SE of the results obtained in 3 independent experiments. *P < 0.05.


Figure 6
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  		Figure 6. Effect of Hcy on the proliferation of neuronal precursor in the SVZ. A) Immunodetection of Ki67+ cells in control and Hcy-treated mice, same experiments. Photomicrographs show coronal sections processed for Ki67 immunohistochemistry to indicate the recently divided cells in the SVZ of control (left) or Hcy-treated mice (right). cc, corpus callosum; CPu, caudate putamen. B) Quantification of the lateral ventricle (LV). Graph represents the number of Ki67+ cells in control and Hcy-treated mice. C) Coimmunodetection of Ki67 and DCX in control and Hcy-treated mice; quantitative data from the same experiments. Confocal merged images of adult mouse brain coronal sections through the SVZ. Double immunolabeling for DCX (blue) and Ki67 (red) in the SVZ of a control mouse and a mouse treated with Hcy. D) Graph represents the number of Ki67+ cells costaining with DCX+ in control and Hcy-treated mice, as a percentage of total Ki67+ cells.


Figure 7
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  		Figure 7. Effect of HHcy on the proliferation of cells in the mouse DG. A, B) Photomicrographs show coronal sections processed for Ki67 immunohistochemistry to indicate recently divided cells in the DG of the hippocampus of control (A) or Hcy-treated mice (B). C) Quantification of the number of Ki67+ cells within the DG of the hippocampus in control and Hcy-treated mice. D) Hcy treatment and BrdU administration protocols in mice of group 1 and group 2. E, F) Photomicrographs show coronal sections processed for BrdU immunohistochemistry to indicate newly synthesized cells in the DG of the hippocampus of Hcy-treated (E) or control mice (F). Inset: magnification of the marked area. GCL, granular cell layer; Sgr, subgranular zone; mol, molecular layer. G) Quantification of the number of BrdU+ cells within the DG of the hippocampus of control or Hcy-treated mice. H) Same results as presented in G but showing values for caudal and rostral DG regions separately. I) Quantification of the hippocampus volume in control and Hcy-treated mice.

Brain processing and immunohistochemistry
Two weeks after minipump implantation and 24 h after the last BrdU injection in group 1 animals, mice were transcardially perfused with 4% (w/v) paraformaldehyde in 0.1 M phosphate buffer. The brains were removed, postfixed for an additional 2 h, and cryoprotected by immersion in 30% sucrose solution overnight. Serial coronal brain sections (30 µm) were obtained through the SVZ (0.38–1.42 mm rostral to bregma) and DG (1.22–3.80 caudal to bregma) using a cryostat and processed for immunohistochemical detection of BrdU, Ki67, or doublecortin (DCX) following a procedure described elsewhere (29) . Primary antibodies were mouse monoclonal anti-BrdU, (Dako, Hamburg, Germany), rabbit polyclonal anti-Ki67 (Vector), and goat polyclonal anti-DCX (Santa Cruz Biotechnologies). Secondary antibodies were fluorescein isothiocyanate -labeled anti-rabbit IgG and Cy5-labeled anti-goat IgG (Jackson ImmunoResearch Labs Inc., West Grove, PA, USA), Cy3-labeled anti-mouse IgG, and biotinylated anti-mouse IgG (both from GE Healthcare, Albany, NY, USA).

Quantification of proliferative cells within the SVZ and DG
Proliferating cells, detected by their BrdU+ or Ki67+ nuclei, were counted by optical microscopy (Nikon Alphaphot YS2, Nikon Corporation, Tokyo, Japan) using an x40 objective, by stereological unbiased methods, as described elsewhere (29) . For the SVZ, all Ki67+ nuclei observed within the lateral wall of the lateral ventricles were counted, in every fifth section of each brain (in 7–9 sections/brain), and expressed as number of nuclei per cubic millimeter. In the DG, all BrdU+ or Ki67+ cells within the subgranular zone were counted in every fifth section of each brain (in 14–16 sections/brain) and expressed as total number per cubic millimeter. Data from 5 control mice and 5 HHcy mice of each group were analyzed statistically. The DG and SVZ volumes were calculated from the areas measured in the same sections using the cellF software (Olympus). Double fluorescent immunostaining of Ki67 or BrdU with the immature neuron marker DCX was quantified using Leica Spectral confocal microscopy (Leica Microsystems GmbH, Wetzlar, Germany).

In vivo cell death measurements
Cell death in the DG was analyzed by quantifying the number of pyknotic nuclei in the granular layer after DAPI nuclear staining, as described elsewhere (30) . Apoptosis was also analyzed using the terminal deoxynucleotidyl transferase end labeling (TUNEL) technique, as described elsewhere (31) . More than 30 DG sections per group of animals were counted.

Measurement of total plasma Hcy levels (tHcy)
Immediately prior to perfusion, blood samples collected by puncture of the left ventricle were centrifuged and plasma was kept frozen at –20°C until Hcy measurement. tHcy levels were determined using an enzymatic assay (Axis-Shield Diagnostics, Dundee, UK) and measurements were recorded at 340 nm and 37°C in a Biotek PowerWave HT microplate scanning spectrophotometer (Biotek Instruments, Winooski, VT, USA). Control plasma samples with normal and abnormal ranges of Hcy were used to calibrate the assay.

Statistical analysis
Statistical analyses of both in vitro and in vivo experiments were performed using ANOVA and the Bonferroni posttest, when more than 1 treatment group was compared with the control group. The Student’s t test was used when only 1 treatment group was compared with the control. The difference was considered significant at values of P ≤ 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Hcy inhibits NPC proliferation in vitro
Neurosphere cultures were used to investigate whether Hcy exerts a direct effect on NPC proliferation. Cells isolated from the SVZ of postnatal (P7) mice divide in culture in the combined presence of EGF and bFGF, and form structures known as neurospheres (32) . At various time intervals, the number and average size of the new neurospheres formed can be taken as indicative of NPC proliferation and/or survival. Treatment of NPC cultures with 100 µM Hcy for 72 h significantly reduced the size of the neurospheres formed without affecting their number. The effect of Hcy on neurosphere size was concentration-dependent (Fig. 1 A–D). After disaggregation of the neurospheres, the total number of cells was significantly lower in Hcy-treated cultures (Fig. 1E ). However, the percentage of nonviable cells was not changed by treatment with Hcy for 24 (not shown) or 72 h (Fig. 1F ). These data suggest that Hcy may be affecting progenitor cell proliferation rather than compromising cell viability. To be sure that the smaller size of the neurospheres formed in the presence of Hcy was a consequence of the decreased proliferative capacity of the NPCs and not a consequence of an increased rate of apoptosis, we first analyzed the fraction of proliferating cells in the control and Hcy-treated (100 µM L-Hcy, 72 h) cultures. Proliferating cells were detected in NPCs adhered onto a PLO substrate by immunolabeling of Ki67, a nuclear protein associated with the cell cycle, which is expressed from the G1 phase through the end of the M phase. The percentage of Ki67+ cells was significantly lower in cultures treated with Hcy compared with control cultures (31 vs. 45%, respectively) (Fig. 1G, H ). Similar results were found when Ki67+ cells were detected among floating neurospheres (see supplementary materials). Finally early and late apoptosis was tested in these cultures. Caspase-3/7 activity, as an early indicator of apoptosis, was measured at 4, 8, and 24 h after seeding (Fig. 1I ), and the percentage of pyknotic nuclei at 24 h (not shown) and 72 h (Fig. 1J ) after seeding was determined as an indicator of late apoptosis. No differences in caspase 3/7 activity or in the percentage of pyknotic nuclei were found between control and Hcy-treated cultures (Fig. 1I, J ). Taken together, these results indicate that, in vitro, Hcy inhibits NPC proliferation.

Hcy-induced inhibition of NPC proliferation requires the presence of bFGF
In the experiments described above, neurospheres were maintained in the presence of both bFGF and EGF. In the absence of bFGF and presence of EGF, neurospheres were ~40% smaller in size (Fig. 2 A, B, 2418±90 µm2 in the presence of bFGF plus EGF vs. 1300±108 µm2 with EGF alone), whereas the total number of neurospheres was unchanged. The effect of Hcy was then tested in cultures grown in the absence of bFGF and therefore in the presence of EGF alone. As shown in Fig. 2B , Hcy had no effect on average neurosphere size under these conditions.

To dissect in more detail the effect of Hcy on cells stimulated by either bFGF or EGF separately, cultures of adhered NPCs were used and the percentage of Ki67+ cells in cultures grown with each growth factor separately was analyzed. The presence of bFGF or EGF in the culture media increased the percentage of Ki67+ cells by 1.3 and 1.6 times, respectively, compared to cultures not supplemented with any growth factor. The addition of Hcy to cultures grown in the presence of bFGF alone resulted in the percentage of Ki67+ cells being almost 50% lower (Fig. 2C ), whereas no effect of Hcy was observed on the percentage of proliferative cells when cells were grown in the presence of EGF alone (Fig. 2D ). These results indicate that growth stimulation by bFGF is essential for Hcy action and suggest that Hcy may interfere with some of the intracellular cell signaling cascades initiated by bFGF.

bFGF-induced activation of Erk1/2 is inhibited by Hcy
To investigate whether the addition of Hcy has functional consequences on the downstream effectors of FGF receptors, the activity of phosphoinositide-3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways was assessed by analyzing the phosphorylated state of Akt and Erk1/2. Phosphorylated Erk1/2 was detected in neurosphere cultures in the absence of growth factors, but the addition of bFGF or EGF increased the amount of pErk1/2 by ~4 to 5 times (Fig. 3 A, B). When the cultures were pretreated with Hcy before the addition of bFGF, the amount of growth factor-induced phosphorylated Erk1/2 found was 50% less, yet EGF-induced phosphorylation of Erk1/2 was not affected by Hcy (Fig. 3A, B ). Pretreatment of cultures with Hcy in the absence of growth factors had no effect on Erk1/2 phosphorylation (data not shown), which suggests that Hcy interferes with the bFGF signaling pathway.

The possible effect of Hcy on the phosphorylated state of Akt (p-Akt) was also analyzed. In control cultures without growth factors, Akt phosphorylation was almost undetectable. However, in cultures to which either EGF or bFGF had been added, relatively large amounts of p-Akt were found (Fig. 3A ). Pretreatment of these cultures with Hcy did not induce any significant change in Akt activation in any of the conditions tested.

Moreover, translocation of phosphorylated Erk1/2 into the nucleus was detected by immunocytochemistry in bFGF-treated NPC cultures adhered onto PLO, in both the presence and absence of Hcy. In cultures treated only with bFGF, phosphorylated Erk1/2 was detected in both the nucleus and the cytoplasm (Fig. 3C , top panels); the addition of 100 µM Hcy to these cultures significantly decreased Erk1/2 phosphorylation, thus also reducing the amount of phosphorylated Erk1/2 translocated into the nucleus (Fig. 3C , bottom panels). These results indicate that Hcy treatment indeed inhibits the MAPK pathway in NPCs.

Expression of cyclin E induced by bFGF-dependent Erk1/2 is inhibited by Hcy
Inhibition of the Erk1/2 pathway with specific inhibitors has been shown to block the EGF-dependent proliferation of mouse embryonic stem cells, and concomitantly to inhibit the expression of cyclin E, a cyclin involved in the transition from phase G1 to phase S (33) . Thus, in the light of the observed results, we tested whether, in postnatal NPCs, the expression of this cyclin was stimulated by bFGF and modulated by Hcy. In neurosphere cultures grown in the absence of growth factors, there was a basal expression of cyclin E. At 4 h after addition of bFGF, cyclin E expression increased 2-fold, concomitantly with an increase in Erk1/2 phosphorylation (Fig. 4 A–C). Under these conditions, pretreatment of cells with the Erk1/2 inhibitor U0126 prevented Erk1/2 phosphorylation and decreased cyclin E expression; these effects indicate that the expression of cyclin E induced by bFGF was a consequence of the MAPK pathway activation in these NPCs. After the addition of Hcy (100 µM) to neurosphere cultures in the presence of bFGF, only basal levels of Erk1/2 activation and cyclin E expression were detected (Fig. 4A-C ). These results strongly suggest that Hcy decreases NPC proliferation by interfering with the FGFR/Erk1/2/cyclin E pathway.

Inhibition of Erk1/2 decreases the percentage of proliferative cells
The next step was to test the effect of U0126 on the percentage of Ki67+ cells in bFGF responsive cultures. Addition of U0126 to cultures in the presence of bFGF reduced the number of Ki67+ cells by almost 75% (Fig. 4D ), indicating that the inhibition of Erk1/2 by Hcy and the resulting suppression of cyclin E were responsible for the lower levels of proliferating NPCs observed in cultures responsive to bFGF.

Hcy inhibits proliferation of neuronal progenitor cells in vitro
It has been reported that bFGF-responsive NPCs isolated from the SVZ are mainly restricted neuronal precursors (32) . Therefore, it was decided to analyze whether the proliferative capacity of this particular cell phenotype was specifically affected by the addition of Hcy in cultures grown with either EGF or bFGF. NPC cultures were double-labeled to detect simultaneously Ki67 and the neuronal progenitor marker β-tubulin-III. In cultures grown for 72 h in the presence of bFGF to which 100 µM Hcy had been added, the percentage of β-tubulin-III+ cells that were colabeled with the proliferation marker Ki67 (Ki67+/β-tubulin-III+) was much lower (by a factor of 5.4) than in similar cultures without Hcy (Fig. 5 A–C). No effect was found in cultures grown in the presence of EGF. In addition, in the bFGF-stimulated cultures with Hcy, the total percentage of Ki67+ cells found was also lower than in those without Hcy, but only by a factor of 2.3 (Fig. 5D ). This finding indicates that Hcy exerts a larger inhibitory effect on the bFGF-dependent proliferation of β-tubulin-III+ neuronal progenitor cells than on the proliferation of the entire NPC population.

In vivo effect of HHcy on the proliferation of NPCs within the SVZ
Our results so far had shown that in vitro Hcy exerts an inhibitory effect on SVZ NPC proliferation. Hence, we then investigated whether a similar inhibition was also found in vivo in the SVZ of an experimental model of HHcy. To induce HHcy in mice, we implanted subcutaneous osmotic minipumps containing Hcy. The treatment of adult mice with Hcy for 2 wk resulted in a plasma Hcy concentration (tHcy) that was notably higher than in control animals, by a factor of 2.8 (11.6±3.9 vs. 32.1±5.9 µM). Increments in body weight and daily food intake did not significantly vary between control and Hcy-treated groups.

The total number of cycling cells in the SVZ of mice with hyperhomocysteinemia and control mice was determined by immunodetection of Ki67+ cells. Chronic treatment with Hcy induced significant changes on the total number of newborn cells in this region (36025±4741 vs. 8984±1458 cells/mm3, P<0.05) (Fig. 6 A, B). Furthermore, in order to test whether Hcy affected the proliferation of neuronal precursors in vivo as well as in vitro, the percentage of Ki67+ cells with neuronal phenotype was detected by coimmunostaining with the immature neuron marker DCX. Our results showed that the percentage of Ki67+ cells that were DCX+ was significantly lower in Hcy-treated mice (Fig. 6C, D ) compared to control mice, thus indicating that Hcy affected proliferation of neuronal progenitors in the SVZ both in vivo and in vitro.

In vivo effect of HHcy on neurogenesis within the DG
A significantly lower number of proliferating Ki67+ cells was observed in the DG of mice with hyperhomocysteinemia compared to control mice (Fig. 7 A–C). Given the small number of cycling cells in the DG, cumulative proliferation was also studied by analyzing the incorporation of BrdU in DG cell nuclei after daily administration of the thymidine analog for 1 wk (Fig. 7D , group 1) Animals with HHcy presented significantly fewer BrdU+ cells in the DG (Fig. 7E-G ). Separate quantification of these cells in the rostral and caudal sections of the DG indicated that there were greater differences in the caudal than in the rostral areas (Fig. 7H ). Measurements of total hippocampal volume showed no differences between control and Hcy-treated animals (Fig. 7I ).

The lower number of BrdU+ nuclei in the DG of mice with hyperhomocysteinemia can be explained either by impaired NPC proliferation or decreased survival. However, TUNEL staining of hippocampal slices showed only the occasional presence of labeled cells in the DG of both control and Hcy-treated mice (Fig. 8 A), indicating a very low apoptosis rate in both groups. In addition, the total number of pyknotic nuclei counted in the DG was similar in control and Hcy-treated mice (Fig. 8B, C ), confirming that Hcy does not induce apoptosis.


Figure 8
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  		Figure 8. Analysis of newly generated neuroblasts, apoptosis, and cell survival within the DG of mice with hyperhomocysteinemia. A–C) Apoptosis rate. A) Representative fluorescence microscopy images of cells within the DG costained with TUNEL (green) and DAPI (blue). B) Representative fluorescence microscopy image of DAPI-stained pyknotic nuclei. Scale bars = 50 µm. C) Quantification of the total number of pyknotic nuclei within the DG. D) Cell survival: quantification of the total number of BrdU+ cells in control mice and in mice treated with Hcy for 2 wk after BrdU injections. E–G) Newly generated neuroblasts. Confocal images of adult mouse brain coronal sections through the hippocampus and quantitative data of the same experiments. Merged images of double immunolabeling for DCX (green) and BrdU (red) in the DG of a control mouse (E) and a mouse treated with Hcy (G). Graph (F) represents the quantification of the percentage of DCX+ cells costaining with BrdU+ in control and Hcy-treated mice.

Furthermore, in order to ascertain whether cell survival was affected by the 2-wk treatment period with Hcy, in a different set of experiments, we injected mice with BrdU during 7 days prior to the initiation of the minipump treatment with either Hcy or the vehicle (Fig. 7D ; group 2), and determined the number of BrdU+ nuclei in the DG. We found that, in control mice of both BrdU administration protocols, the total number of cells labeled with BrdU after the 7 day administration protocols (Fig. 7D ) were almost identical (irrespective of whether mice were sacrificed 24 h or 2 wk after the last BrdU injection; see Fig. 7D ) (3223±151 vs. 3385±524 cells/mm3, respectively), supporting the conclusion that, within the 2-wk time frame, significant cell death was not apparent. In addition, in those mice receiving Hcy for 2 wk after the administration of BrdU, the number of BrdU+ nuclei in the DG found was similar to that in controls (Fig. 8D ), thus indicating that HHcy did not affect cell survival. Taken together, the above results indicate that elevated levels of plasma Hcy decrease the rate of generation of new cells in the DG of the hippocampus by impairing their proliferating capacity.

Effect of Hcy on newly generated neuronal precursors within the DG
The percentage of neuronal precursors among newly generated cells (BrdU+) was detected by coimmunostaining with the immature neuron marker DCX and BrdU. Within the DG ~70% of the newly generated cells (BrdU+) expressed DCX in both control mice and those with hyperhomocysteinemia (76±24 and 68±11%, respectively), whereas less than 1% expressed the glial cell marker GFAP in the two groups (data not shown). Consequently, the percentage of neuronal progenitors within the DG that had incorporated BrdU was significantly lower in mice treated with Hcy compared to control mice (Fig. 8E-G ); this indicates that the generation of neuronal progenitors within the DG is significantly impaired by Hcy treatment.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we have shown that Hcy exerts an antiproliferative effect on NPCs isolated from the SVZ by inhibiting bFGF-induced proliferation, particularly of neuronal precursors. The analysis of the mechanisms involved reveals that Hcy interferes with the bFGF signaling pathway, leading to a decreased activation of Erk1/2 and cyclin E expression in bFGF-stimulated cells. We have also shown that, in vivo, under unmodified dietary folate conditions, HHcy impairs NPC proliferation in the SVZ and lowers the number of proliferative neuronal precursors. HHcy also affects NPC proliferation and the generation of neuronal precursors in the DG. Since neuron-specific progenitors are the source of newly generated DG neurons throughout adulthood (34) , our results strongly suggest that Hcy compromises neurogenesis in the DG and SVZ by impairing the bFGF signaling cascade.

Hcy exerts an antiproliferative effect on NPCs isolated from the SVZ by impairing the bFGF signaling cascade
Neurosphere cultures obtained from SVZ progenitor cells contain proliferative cells of different phenotypes (32) . Each neurosphere originates from 1 cell that could be either a stem cell or another type of progenitor with self-renewal capability. The number of neurospheres formed is an indirect measure of the initial number of precursor cells that enter the cell cycle and undergo cell division (35 , 36) , whereas the size of each neurosphere indicates the mitotic rate of the precursor cell progeny dividing inside the neurosphere (26) . In our neurosphere assay, Hcy reduced the size of neurospheres formed without affecting their number, indicating that the self-renewal capability of neurosphere-initiating cells was not affected in the presence of Hcy, whereas division within the initiating cells progeny was significantly reduced. The reduction of the number of cycling cells found on adhered NPC cultures or the smaller neurosphere size was not a consequence of increased apoptotic rate because caspase-3 activity as well as the percentage of pyknotic nuclei remained unaffected after Hcy treatment, nor was cell viability influenced by Hcy. Surprisingly, Hcy did not induce apoptosis in NPCs as it does in mature hippocampal neurons, in which Hcy exerts an apoptotic effect mediated by DNA damage (7) .

We have dissected the effects of Hcy under stimulation with different mitogens and have shown that Hcy impairs the bFGF-dependent proliferation of NPCs, because the effects of Hcy on neurosphere size or the percentage of Ki67+ cells require that NPC proliferation is being stimulated by bFGF. Signaling through FGFRs leads to the activation of two main pathways: the MAPK cascade that results in the phosphorylation of Erk1/2, and the PI3K/Akt cell survival pathway (37) . No differences in Akt phosphorylation were induced by Hcy treatment, whereas Hcy inhibited the bFGF-dependent activation of Erk1/2 as assessed by the presence of p-Erk and its translocation into the nucleus.

In addition, inhibition of the bFGF-dependent activation of Erk1/2 by the specific inhibitor U0126 resulted in a reduced number of cells that undergo cell cycle (Ki67+). Inhibition of the Erk1/2 pathway has been shown to block the proliferation of mouse embryonic stem cells in G1, leading to a reduced expression of the G1-related cyclin E (33) . The results presented here show that the inhibition of Erk1/2 in NPC cultures by either Hcy or the specific inhibitor U0126 results in the inhibition of cyclin E expression and a reduction of the percentage of Ki67+ cells. Furthermore, these results demonstrate that cyclin E expression can be modulated by growth factors in postnatal neural precursor cells and indicate that the antiproliferative effect induced by Hcy is mediated by its capacity to impair both the bFGF-dependent activation of Erk1/2 and the Erk1/2-dependent activation of cyclin E expression. This is not the first study to show that Hcy is related to the MAPK/ERK activation, as phosphorylation of Erk1/2 has been reported to be modulated by Hcy in vivo in hippocampal mature neurons (38) , and in vitro in various cell types (39 40 41) . Nevertheless, in all the above reports, Hcy induces the hyperactivation of this pathway, whereas we observed a down-regulation of the bFGF signaling cascade in NPC cultures.

Hcy significantly inhibits bFGF-stimulated proliferation of neuronal precursors
It has been shown previously that, within the hippocampus and the SVZ, the proliferation of neuronal precursors is mainly bFGF-responsive (32) , suggesting that a reduction in the bFGF-dependent proliferation of NPCs may result in the inhibition of neuroblast proliferation and, therefore, in a reduced neurogenic capacity. We have demonstrated here that, in vitro, after bFGF stimulation, almost 14% of all neuronal precursors were proliferating (Ki67+) whereas after treatment with Hcy this percentage was reduced to 2.6% (i.e., by a factor of 5.4). This effect is stronger than the effect exerted by Hcy on the entire NPC population, indicating that Hcy particularly impairs proliferation of neuronal precursors, probably by affecting the bFGF signaling cascade.

A previous report demonstrated a reduction on fetal neuroepithelial cell proliferation in vitro, also in the absence of apoptosis or cell death, when cells are cultured in the presence of bFGF and Hcy is elevated by treatment with methotrexate, an inhibitor of folate synthesis (20) . In that report it is not clear whether Hcy is inducing these effects or high Hcy is simply another effect of low folate concentration. The results presented in our study not only demonstrate that Hcy is sufficient to induce an antiproliferative effect on postnatal NPCs when folate levels have not been modified, but they also demonstrate that the particular target for Hcy is the neuronal precursors—those cells that proliferate in response to bFGF—and not those NPCs that proliferate when stimulated with EGF. In addition, this work shows Hcy-induced down-regulation of the bFGF signaling pathway in these cells, which leads to a reduction in the Erk1/2-dependent expression of cyclin E.

Moderate HHcy inhibits neurogenesis in the DG of mice, without affecting cell survival
To demonstrate that our in vitro findings also occurred in vivo, we developed a mouse model of moderate HHcy. Hcy-treated mice showed levels of tHcy slightly higher than that reported for a heterozygous MTHFR(–/+) mice model of moderate hyperhomocysteinemia (42) , and higher than that found in patients with dementia and Alzheimer’s disease (≥14 µM) (1 , 43 , 44) . By using this model, we found that proliferation of NPCs in the main neurogenic areas of the adult mouse brain, the DG and the SVZ, is inhibited by Hcy in vivo. Furthermore, according to our in vitro findings, Hcy significantly decreased the percentage of proliferative cells with neuronal precursor phenotype in the SVZ.

The antiproliferative effect of HHcy was also significant in the DG, leading to a decreased quantity of newly synthesized neuronal progenitors in this area. Differences within the DG were more notable in the caudal areas, which have been demonstrated to be involved in memory encoding and retrieval in humans (45 46 47) . HHcy did not induce apoptosis in the DG, as assessed by TUNEL staining or by pyknotic nuclei detection, and no differences in NPC survival between control and Hcy-treated animals were found during the course of the treatment. Thus, the Hcy-induced reduction of newly generated neuronal precursors was a consequence of the proliferative capacity of the NPCs in the DG being impaired. Unexpectedly, no Hcy-induced differences in hippocampal volume were found, whereas proliferation was reduced and cell survival remained unaffected. We hypothesize that the differences in the proliferation rate during the 2-wk treatment were not large enough to induce detectable changes in hippocampal volume.

Our results complement the findings of Kruman et al. (20) , in which low folate-induced HHcy compromises neurogenesis in the DG of mice fed a folate-deficient diet (20) , and also those of Chen et al. (48) , which show a reduced neurogenesis in the cerebellum of postnatal (p5) MTHFR(–/–) mice with hyperhomocysteinemia. However, we have been able to advance further and determine that, under normal folate conditions, HHcy induces similar antiproliferative effects specifically in the caudal areas of the DG and in the SVZ, significantly affecting the proliferation of neuroblasts and reducing the total number of newly synthesized neuronal precursors, which are the immediate precursors of mature neurons. This finding is consistent with our in vitro results, in which Hcy exerts its antiproliferative effect preferentially on bFGF-stimulated neuronal precursors and is also supported by a recent report in which FGFR-1(–/–) mice show impaired neurogenesis in the DG (49) .

Moderate HHcy and neurogenic capacity in the development of mild cognitive impairment
Epidemiological studies closely link HHcy to the development of dementia (1 , 43) , and even to mild cognitive impairment without dementia (4 , 50) . Cognitive impairment generally precedes the development of dementia and recent studies show that plasma levels of Hcy can be predictive of the latency period prior to the onset of dementia (4) . Moreover, daily Hcy injections reduce long-term potentiation (LTP) in adult rats and modulate spatial learning (51) . Memory loss and subsequent cognitive impairment might occur through diminished neurogenic capacity within the principal brain areas involved in memory and learning. Results from several laboratories suggest that hippocampal neurogenesis is functional and participates in learning and memory performance (12 , 52) . In mammals, increased neurogenesis is mediated by environmental factors such as exercise (53) or stimuli-enriched environments (19 , 54) that concomitantly improve learning and memory capacities. In the adult human hippocampus, both structural and functional changes have been demonstrated in individuals exposed to intensive spatial learning (55 , 56) . Inhibition of neurogenesis is known to impair learning and memory (14 , 57) and increased neurogenesis improves LTP of synaptic transmission (53) . Likewise, it has recently been shown that the FGFR-1(–/–) mice not only exhibit an impaired neurogenic capacity but also show significant alterations in memory consolidation and a decreased LTP, compared to control mice (49) . The results presented in our study strongly suggest that Hcy impairs neurogenic capacity within the more caudal areas of the DG of the hippocampus and the SVZ, probably by disrupting the bFGF signaling pathway of neuroblasts. Since it has been proposed that these regions are involved in memory retrieval (45 46 47) , it would be reasonable to hypothesize that the mechanisms described could lead to memory loss and thereby to cognitive impairment, a step known to precede the onset of dementia.


   ACKNOWLEDGMENTS
 
We thank Antonio Torres and José M. Martinez for technical assistance. This work was supported by grants from the Spanish Ministerio de Educación y Ciencia (SAF2005-05834). C.C. and F.R. are researchers in the Ramón y Cajal Program of the Spanish Ministerio de Educación y Ciencia. L.G.R. is the recipient of a fellowship from the Spanish Ministerio de Educación y Ciencia (AP 2006-3924).


   FOOTNOTES
 
1 These authors contributed equally to this work.

Received for publication March 19, 2008. Accepted for publication June 26, 2008.


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 RESULTS
 DISCUSSION
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