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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 62:117-125 (2007)
© 2007 The Gerontological Society of America

Adult Neurogenesis in the Hippocampus of Long-Lived Mice During Aging

Liou Y. Sun and Andrzej Bartke

1 Department of Pathology, University of Michigan Medical School, Ann Arbor.
2 Geriatrics Research, Department of Internal Medicine and Department of Physiology, Southern Illinois University School of Medicine, Springfield.

Address correspondence to Liou Sun, PhD, Room 3008 BSRB, Box 2200, 109 Zina Pitcher Place, University of Michigan, Ann Arbor, MI 48109-2200. E-mail: leeosun{at}gmail.com


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Ames dwarf mice live considerably longer than normal animals, exhibit apparently normal cognitive functions, and maintain them into advanced age. Neurogenesis occurs throughout adult life span in the dentate gyrus of mammalian hippocampus and has been suggested to play an important role in cognitive function. We now report that the total number of bromodeoxyuridine (BrdU)-labeled cells in this brain region in aged Ames dwarf mice was not different from that in aged normal mice, whereas the fraction of newly generated neurons was significantly increased by monitoring BrdU labeling and cell marker expression. Evidence of activation of antiapoptosis signal transduction cascade was also found in the hippocampus of aged dwarf mice. Together with previous findings, the results may suggest that an increase in hippocampal insulin-like growth factor-I protein expression and subsequent activation of antiapoptotic signaling might contribute to survival of newly born neurons and subsequently to the delay of cognitive loss during aging in these long-lived dwarf mice.

ONE of the most interesting topics in neuroscience is the birth of new neurons that occurs in discrete regions of adult mammalian brain, namely, the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ) (1–3). In these regions, regulation of adult neurogenesis is often demonstrated by systemic injection of an exogenous synthetic (S)-phase marker such as bromodeoxyuridine (BrdU), the thymidine analogue, which is taken up by dividing cells during the S phase (4). This technique has shown that neural progenitor cells arising from the SGZ migrate into the granule cell layer (GCL) where they differentiate into new neurons and integrate into the local network, receiving afferents and sending out functional efferents (5), whereas SVZ-born neurons are destined for the olfactory bulb (6). The process of neurogenesis in the adult dentate gyrus can be divided into different phases including proliferation, differentiation, and migration.

These newly born neurons may have a physiological role, because blockade of hippocampal neurogenesis is reported to inhibit hippocampus-dependent learning (7), and because reducing the population of new interneurons in the olfactory bulb impairs odor discrimination (8). Although a growing number of pharmacological and environmental manipulations have been shown to influence adult neurogenesis, the functional implication of the newly born neurons remains poorly understood. Hippocampus is one of the brain regions most susceptible to aging (9). The aging hippocampal formation is affected by a number of structural and functional changes, including reduced expression of growth factor and steroid receptors (10) and impairment of hippocampal long-term potentiation (LTP) induction under certain conditions (11,12). Neurogenesis in the dentate gyrus occurs throughout the life span, but decreases with increasing age in rats, mice, monkeys, and humans (13–15). Decreased hippocampal neurogenesis may be involved in age-related cognitive deficits because of its proposed role in learning and memory function (7). In contrast, hippocampal neurogenesis is reported to be increased in aged mice living in an enriched environment (16). Enhanced neurogenesis is accompanied by improved learning, exploratory behavior, and locomotor activity (16). Therefore, it is suggested that restoring hippocampal neurogenesis may be a strategy for reversing age-related cerebral dysfunction. In addition, growth factors stimulate proliferation of neuronal precursors from adult brain in vitro and in vivo. For example, fibroblast growth factor (FGF)-2 is expressed in adult rodent SVZ and SGZ (17) and intracerebroventricular (ICV) infusions of FGF-2 upregulate dentate neurogenesis significantly in the aged brain and also increase neurogenesis in SVZ and neuronal migration to the olfactory bulb (18,19). Insulin-like growth factor (IGF)-I is another interesting factor, given its pattern of regulation across the life span and its ability to stimulate neurogenesis (20). In addition, although IGF-I levels decline in aged rats, their restoration increases neurogenesis in aged rat brain (21).

The aim of the present study was to investigate the effects of aging and dwarfism on neurogenesis in the dentate gyrus of Ames dwarf mice. The Ames dwarf mouse was selected because of its special phenotypic characteristics of delayed aging. Ames dwarf mice are homozygous for a loss-of-function mutation at the prop-1 locus (Prop1df) (22). The mutation impairs the development of the anterior pituitary resulting in dramatic depletion or absence of growth hormone-, thyroid-stimulating hormone-, and prolactin (PRL)-producing cells in the adenohypophysis and primary deficiency of the corresponding hormones (23). Circulating IGF-I is undetectable in Ames dwarf mice because of the primary growth hormone deficiency (24,25). However, these mice have a significantly increased life span and delayed onset of age-related changes including brain aging, and maintain physiological function at more youthful levels (26). Recently, we found that old Ames dwarf mice had elevated levels of growth hormone and IGF-I in the hippocampus, and increased neurogenesis was found in the dentate gyrus of young adult dwarf mice (27,28).


   MATERIALS AND METHODS
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Animals
Young adult (3-month-old) and aged (20-month-old) male Ames dwarf mice and age-matched normal male mice (n = 8 from each group) were used in the study. Normal mice were the siblings of the dwarfs. Animal protocols were reviewed and approved by the Southern Illinois University Animal Care and Use Committee. All animals had access to water and food ad libitum and were housed under a 12-hour light/dark cycle.

Antibodies
Primary antibodies and their final dilutions were: Rat-BrdU, 1:500 (Accurate Chemical, Westbury, NY); polyclonal goat antigrowth hormone immunoglobulin G (IgG), 1:300 (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal rabbit anti-IGF-I IgG, 1:200 (Santa Cruz Biotechnology); polyclonal rabbit antibodies against phospho-Bad (Ser-136), against Bad, against Bcl-2, 1:250 (Santa Cruz Biotechnology); mouse monoclonal anti-ß-actin IgG, 1:2000 (Sigma, St. Louis, MO); mouse anti-NeuN IgG, 1:100 (Chemicon, Temecula, CA); anti-DCX, 1:100 (Santa Cruz Biotechnology); mouse anti-ß-III tubulin, 1:1000 (Promega, Madison, WI); rabbit antiglial fibrillary acid protein (GFAP) IgG, 1:300 (Dako, Carpinteria, CA); and rabbit anti-Akt, phospho-Akt, caspase-3, and caspase-9, 1:500 (Cell Signaling, Danvers, MA). The following secondary antibodies from Jackson ImmunoResearch (West Grove, PA) were used: FITC-conjugated donkey-mouse IgG, 1:500; FITC-conjugated donkey-rabbit IgG, 1:500; rhodamine Red-X-conjugated donkey-goat IgG, 1:500; rhodamine Red-X-conjugated donkey-mouse IgG, 1:500; rhodamine Red-X-conjugated donkey-rat IgG, 1:500; or biotin-conjugated donkey-rat IgG, 1:500.

BrdU Injections
Cell proliferation was measured by the incorporation of the thymidine analogue BrdU, which is incorporated into the DNA of dividing cells in immunohistochemically detectable quantities during the S phase of cell division (3). Each animal received intraperitoneal injections of BrdU (Sigma) (50 mg/kg at a concentration of 10 mg/mL in 0.9% NaCl) daily for 7 consecutive days, and all animals were then killed 24 hours after the last BrdU injection. The animals were anesthetized by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (10 mg/kg) and perfused with 0.9% NaCl followed by 4% paraformaldehyde.

Histology
After perfusion, brains were removed and post-fixed overnight in 4% paraformaldehyde at 4°C. The brains were then equilibrated in 30% sucrose for an additional 24 hours. Sections 40 µm thick were then prepared in the coronal plane using a freezing microtome.

Immunostaining
Diaminobenzidine (DAB) histochemical staining for BrdU was used for stereological quantification of labeled cells. Free-floating sections were treated with 0.6% H2O2 in phosphate-buffered saline (PBS) for 30 minutes to block endogenous peroxidase. For DNA denaturation, sections were incubated for 2 hours in 50% formamide/2x standard saline citrate (SSC) (0.3 M NaCl and 0.03 M sodium citrate) at 65°C, rinsed for 5 minutes in 2x SSC, incubated for 30 minutes in 2N HCl at 37°C, and rinsed for 10 minutes in 0.1 M boric acid, pH 8.5. Several rinses in PBS were followed by incubation in PBS/0.1% Triton X-100/3% normal horse serum (PBS-Ths) for 30 minutes and incubation with mouse anti-BrdU antibody in PBS-Ths overnight at 4°C. After being rinsed in PBS, sections were incubated for 1 hour with biotinylated horse antimouse antibody. Tissues were washed in PBS and then incubated for 30 minutes in pre-assembled biotin–avidin–horseradish peroxidase complex according to the manufacturer's recommendations (ABC Elite; Vector Laboratories, Burlingame, CA). Sections were then washed and incubated in DAB solution for sufficient time to develop intense brown staining in BrdU-labeled nuclei. Rinsed sections were then mounted on uncoated Superfrost slides (Fisher Scientific, Santa Clara, CA), dried, dehydrated through a graded alcohol series into xylene, and cover-slipped with Permount mounting medium (Fisher Scientific).

Double-Labeling Immunofluorescence
Sections were treated for DNA denaturation as described above, followed by several rinses in PBS and incubation in PBS/0.1% Triton X-100/3% normal donkey serum (Jackson ImmunoResearch) for 30 minutes. For labeling of BrdU and cell specific markers, sections were incubated with monoclonal rat anti-BrdU and anti-NeuN or anti-GFAP at 4°C overnight followed by antirat rhodamine Red-X IgG and antimouse or antirabbit FITC IgG for 2 hours at room temperature and after thorough removal of primary antibodies. Sections were washed and wet mounted, then dried in the dark. Fluorescent mounting medium was applied prior to placing coverslips onto the slides. For visualization and photography, specimens were observed under a confocal microscope.

Stereology and Quantification of BrdU-Labeled Cells
Stereology was performed on tissues stained for BrdU using DAB histochemistry as described above. From all animals, every sixth section (240 µm apart) of the series was stained for BrdU using the peroxidase method. Positive cells were counted using a 40x objective (Leica, Exton, PA) throughout the rostrocaudal extent of the GCL. Stereological principles and analyses were conducted as described by Williams and Rakic (29), and the optical dissector method was modified in that cells appearing sharp in the uppermost focal plane were not counted. Resulting numbers were multiplied by 6 to obtain the estimated total number of BrdU-positive cells per GCL. BrdU-positive counts were limited to the hippocampal granular cell layer and adjacent hilar SGZ margin using the fractionator probe (Stereo Investigator software; MBF Bioscience, Williston, VT). Fluorescently labeled tissue sections were evaluated using a Zeiss 510 confocal laser-scanning device attached to a Zeiss-Axiovert microscope using LSM510 software. Appropriate gain and black-level settings were determined on control tissues stained with secondary antibodies alone. Upper and lower thresholds were set using the range indicator function to minimize saturation in positive cells. To assess the phenotype of BrdU-labeled cells, the numbers of BrdU- and/or NeuN-positive cells were scored within the dentate GCL including the adjacent hilar margin or SGZ in the sections taken from the rostral, mid, and caudal hippocampus.

Western Blot Analysis
Hippocampi were homogenized in 10 vol of solubilization buffer (1% Triton, 100 mM HEPES, 100 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride [PMSF], and 0.035 TIU/mL aprotinin [pH 7.4]) at 4°C. Protease and phosphatase inhibitors and 1% of Triton-X 100 (Sigma) were added. After mixing, homogenates were centrifuged at 13,000 g for 30 minutes, and the supernatant was removed. Total hippocampal proteins were determined by the colorimetric method using a bovine serum albumin (BSA) protein assay reagent (Pierce, Rockford, IL). Protein levels were quantified by Western blotting using antibodies specific for the respective proteins. In brief, homogenate proteins (40 µg/well) from mouse hippocampus were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and were then transferred to nitrocellulose membrane by electroblotting at 80 V for 80 minutes. After the transfer, membranes were washed using Tris-buffered saline (TBS) and blocked with 3% BSA in TBS for 1 hour at room temperature. After blocking, the membrane was incubated in appropriately diluted primary antibody (see reagents for different dilution) overnight at 4°C. After incubation and three washes, the membrane was probed with the specified horseradish peroxidase-linked second antibody for 2 hours at 20°C. Blots were processed using enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Piscataway, NJ), and immunoreactive bands were viewed on an image analysis system consisting of a monochrome video camera (Dage MTI CCD-72; Dage-MTI, Michigan City, IN) connected to an Inter Focus Ltd. image analysis system (MCID Imaging Research Inc. [now part of GE Healthcare], St. Catharines, Canada). The signals of the proteins were normalized to ß-actin signals. Quantification of immunoblot signals was performed using GeneTools ImageQuant software (Syngene Inc., Frederick, MD).

Statistical Analyses
Results are presented as mean ± standard error of the mean (SEM). The statistical evaluation was performed using two-factor analysis of variance (ANOVA; phenotype and age), followed by Fisher's protected least significant difference test as a post hoc test. Student's t test was used when two groups were analyzed. Values of p <.05 were considered significant. All statistical analyses were performed using StatView 5.0 software (Abacus Concepts, Berkeley, CA).


   RESULTS
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Neurogenesis and Aging
To study the impact of aging on neurogenesis in the dentate gyrus, young adult (3 months old) and aged (20 month old) dwarf and normal mice were given BrdU for 7 consecutive days and killed 24 hours later, and the number of BrdU-labeled cells in the dentate gyrus of the hippocampus between different groups was compared by immunohistochemistry and cell counting. BrdU-labeled cells were detected in dentate gyrus (Figure 1) in both young adult and aged dwarf and normal mice. Cell proliferation was predominantly confined to the subgranular layer (Figure 1). The BrdU-positive nuclei were often clustered in the subgranular layer and exhibited variable shapes (Figure 1E). However, the majority of BrdU-labeled cells were located inside the GCL and characterized by large round nuclei (Figure 1F).


Figure 01
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  		Figure 1. Age-dependent proliferation and survival of bromodeoxyuridine (BrdU)-labeled cells in the dentate gyrus. Ames dwarf (A and C) and normal mice (B and D) received BrdU injections on 7 consecutive days and were analyzed 24 hours later. Although BrdU-positive cells exhibit a variety of shapes (E), most cells are predominantly aligned and clustered at the hilar-granule cell border revealing large round nuclei with a chromatin structure similar to mature granular cells (F). Note the dramatic decrease in BrdU-positive cells in 20-month-old mice (C and D) compared to 3-month-old mice (A and B). Scale bars are 60 µm (A–D) and 10 µm (E and F)

 
Qualitative assessment indicated a dramatic decrease in total number of BrdU-labeled cells in the aged mice when compared to young mice (in both dwarf and normal animals) (Figure 2). The results were consistent with previous evidence that there is an age-related decline in proliferation of newly born cells in the dentate gyrus of mouse hippocampus (30). Consistent with our previous report (28), there is a robust increase in numbers of newly generated cells (BrdU positive) in the dentate gyrus of young adult Ames dwarf mice compared with young normal mice. However, there was no significant difference of total number of BrdU-labeled cells in the dentate gyrus area between aged dwarf and aged normal mice (Figure 2).


Figure 02
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  		Figure 2. Stereological estimation of the total number of bromodeoxyuridine (BrdU)-positive cells in the dentate gyrus during aging. Results revealed that there was an age-related decrease in the number of newly born cells in the granule cell layer (GCL) in both dwarf and normal mice. More BrdU-labeled cells were found in young Ames dwarf GCL than in normal mice. Interestingly, however, in the GCL region, no difference was seen between aged Ames dwarf and aged normal mice. Asterisks indicate significant differences from young normal mice (*p <.05; **p <.005)

 
To determine whether the BrdU-labeled cells that we observed expressed phenotypic neuronal features, we double-labeled brain sections with antibodies against BrdU and against the cell-type marker proteins NeuN or GFAP, or other specific markers such as Doublecortin. A considerable decrease was observed in the fraction of BrdU/NeuN-colabeled cells between the young and old groups in both dwarf and normal animals. Compared with that of young normal mice, dentate gyrus of young Ames dwarf mice has more newly born neurons (BrdU/NeuN double-labeled). Interestingly, more BrdU/NeuN-positive cells were detected in the dentate gyrus area of aged Ames dwarf mice than in that of the aged normal animals (Figure 3). For both normal and dwarf mice, a very small number of BrdU-positive cells in the GCL colocalized with GFAP, whereas many BrdU-positive cells remaining in the hilus were GFAP-positive. In addition, BrdU-labeled cells were seen also in several other brain areas including hippocampal regions CA1 and CA3 and cerebral cortex, but these cells were not immunoreactive with the cell markers in current study.


Figure 03
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  		Figure 3. Cellular phenotyping of bromodeoxyuridine (BrdU)-positive cells. Top: BrdU immunofluorescence (red) was combined with the neuronal marker NeuN (green), and colocalization was assessed with confocal scanning microscopy. In both aged normal (A) and aged dwarf mice (B), BrdU-labeled cells were positive for the neuronal marker NeuN (arrow). Note the variety of BrdU labeling, ranging from dense stained neurons to only partly labeled neurons, indicating a dilution effect of repeated stem cell divisions. Scale bar = 30 µm. Bottom: Results of quantification of BrdU/NeuN double-positive cells in the granular cell layer (GCL) of the dentate gyrus of aged normal mice and aged dwarf mice. Asterisks indicate significant differences (*p <.05)

 
Survival or Death
Consistent with a previous study (27), aged Ames dwarf mice had elevated levels of IGF-I and increased phosphorylation of Akt locally in the hippocampus (Figures 4 and 5) despite having virtually no IGF-I in peripheral circulation. One reported mechanism of action of IGF-I is to increase the phosphorylated state of Akt, a protein kinase activated by insulin and various growth factors that is involved in blocking proapoptotic pathways through receptor-mediated phosphatidylinositol 3-kinase signaling (31).


Figure 04
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  		Figure 4. Hippocampal insulin-like growth factor (IGF)-I protein expression of aged Ames dwarf and normal mice. Top: Autoradiography of Western blot. Bottom: Densitometric analysis of Western blots. ß-Actin signal was used to normalize data. Each lane of the blots represents materials from a different normal or dwarf mouse. *p <.05. Representative blot is shown

 

Figure 05
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  		Figure 5. Increased activation of phosphoinositide 3-kinase-Akt (P-AKT) pathway in the hippocampus of aged Ames dwarf and aged normal mice. Phosphorylation of Akt in the hippocampus of Ames dwarf and normal mice. Top: Autoradiography of Western blot of phosphorylated Akt and total Akt protein. Bottom: Densitometric analysis of Western blots. Values are expressed as means ± standard error of the mean. Asterisk indicates significant differences (*p <.05)

 
Bcl-2 protein has been proposed to serve as a signaling receptor for intracellular organelles in which serine phosphorylation of both apoptotic and antiapoptotic family members are hypothesized to affect cell survival. Numerous studies have reported on the ability of enhanced Bcl-2 expression to promote the survival of various cell types, as well as the ability to block apoptotic cell death (32–34). Western blot analysis of immunoreactive Bcl-2 protein with a molecular mass of 26 kd from proteins extracted from aged dwarf and normal mice hippocampus indicated that that the Bcl-2 protein expression was significantly (p <.05) more abundant in dwarf mice relative to that in normal mice (Figure 6).


Figure 06
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  		Figure 6. Bcl-2 protein expression in the hippocampus of aged Ames dwarf and aged normal mice. Top: Autoradiography of Western blot of Bcl-2 protein. Bottom: Densitometric analysis of Western blots. Values are expressed as means ± standard error of the mean. Asterisk indicates significant differences (*p <.05). ß-actin signal used to normalize the data

 
Bad is a pro-apoptotic member of the Bcl-2 family of proteins. Phosphorylation of Bad is a key step in the signaling events that come into play in the rescue of cells from apoptosis. As shown in Figure 7, phosphorylation of Bad (at SEM136) was significantly increased in the hippocampus of old dwarf mice compared with old normal mice. The total Bad protein did not differ between the two groups. Both IGF-I and phosphorylated Akt have been shown to prevent cleavage of caspase-9, thereby inhibiting apoptosis. Here we found that there is a significantly (p <.05) reduced amount of caspase-9 cleavage and subsequently decreased cleaved 37 kd and 39 kd subunits in the dwarf hippocampus (Figure 8), indicating that caspase activation involved in the apoptotic pathway was also attenuated. Thus the results suggest that the presence of a greater number of newly born neurons in the dwarf mice compared with normal mice may be due to survival effects of IGF-I and subsequent activation of antiapoptotic pathway.


Figure 07
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  		Figure 7. Phosphorylation of Bad in the hippocampus of aged Ames dwarf and normal mice. Top: Autoradiography of Western blot of phosphorylated Bad and total Bad protein. Bottom: Densitometric analysis of Western blots. Values are expressed as means ± standard error of the mean. Asterisk indicates significant differences (*p <.05)

 

Figure 08
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  		Figure 8. Cleavage of caspase-9 in the hippocampus of aged Ames dwarf and normal mice. The amount of caspase-9 cleavage was significantly reduced in dwarf hippocampus. The cleaved 37 kd and 39 kd subunits were decreased in dwarf mice by more than 50% compared to those in the normal mice (densitometric analysis of Western blot) (*p <.05)

 

   DISCUSSION
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we have investigated the impact of aging and dwarfism on neurogenesis in the dentate gyrus of long-lived Ames dwarf and normal mice. It has been demonstrated that the incorporation of BrdU into hippocampal granule cells might reflect the number of proliferating progenitor cells. The thymidine analog BrdU is incorporated into DNA and can be detected immunohistochemically in cell progeny (35). Bioavailability of BrdU after injection has been estimated to last ~2 hours, and BrdU labels DNA only during the S phase, which has been estimated to last ~8 hours (36,37). Thus, the regimen used in this study is not likely to cause an overestimation of the number of proliferating cells, because one injection cannot label all dividing cells during a 24-hour period.

Age-Related Changes in Neurogenesis
Neurogenesis persists in the hippocampal formation of old mice. In all four groups, proliferating cells (Figure 1) were located in the SGZ, and differentiating cells migrated into the GCL. BrdU-labeled cells expressed the neuronal marker NeuN 1 week after injection of BrdU (Figure 3). Our stereological findings of a significant age-dependent decline in neurogenesis in the GCL parallel previous findings in mice (30) and rats (13,15). The reduction of neurogenesis with aging was possibly caused by an age-related decrease in the number of newly generated cells. Several mechanisms could be responsible for the reduction in hippocampal neurogenesis during aging. Neuronal precursor cells could either change their cell-specific fate, leading to their becoming glial cells, or alter their mitotic activity, leading to a reduction of the actual number of newly born cells. Also, the newly born cells could die before differentiating into granule neurons.

The progressive decline of precursor cell proliferation during aging raises the question of whether the precursor cells become unresponsive to environmental cues or whether the environment does not provide the stimuli for further proliferation. The newly born cells either die or lose their appropriate signaling mechanism (such as growth factors) for the mitotic stimulus. Moreover, during aging the local environment for the precursor cell changes so that the mitotic stimulus is no longer provided. Several factors that regulate neuronal birth in the adult dentate gyrus were recently described (38–40). Mayo and colleagues (41) reported that infusions of the neurosteroid pregnenolone sulfate (PREG-S) will reverse memory impairments in aged rats. The possible role of oxidative stress in increasing neuronal vulnerability in aged hippocampal neurons has also been discussed (42). Interestingly, the effects of dietary restriction on neural progenitor/stem cells have been described (43). Dietary restriction induced an increase in newly generated neural cells in the adult brain, an increase in expression of neurotrophic factors such as brain-derived neurotrophic factor (BDNF), and an increase in the resistance of neurons to dysfunction and apoptosis (43). Glucocorticoids have also been shown to inhibit neurogenesis in the dentate gyrus (38,44). Interestingly, corticosterone levels were highest in young normal female mice and lowest in young normal male mice, with the levels in Ames dwarf mice falling in the middle (45). However, plasma corticosterone levels in old Ames dwarf mice did not differ from the values measured in normal animals (45). Further studies will be needed to relate the levels of glucocorticoids to the degree of neurogenesis in Ames dwarf mice. Glutamatergic afferents to the dentate gyrus also limit adult neurogenesis, as does psychosocial stress (46). Peripheral administration of IGF-I increases cellular proliferation in the dentate subgranular proliferative zone (47).

Interestingly, in current studies we found that newly born cell differentiation and survival were not changed in parallel with proliferation in aged Ames dwarf mice; the total number of BrdU-labeled cells was not different from that in the aged normal mice, whereas the fraction of newly generated neurons (BrdU and NeuN double-labeled) was significantly increased. This finding might indicate that the progression rate of neural stem cells and/or neuron precursors to mature neurons is significantly increased in Ames dwarf mice. However, the total number of newly born neuronal precursors is similar in both normal and dwarf mice. There is also a possibility that an effect on cell survival/apoptosis could be responsible for the increase in newly born neurons observed in dentate gyrus of aged Ames dwarf mice. Little is known about the role of apoptosis in the regulation of adult neurogenesis during aging, although it is plausible that the decrease in BrdU-positive cells after injection is attributable to elimination by programmed cell death. Quantitatively, the loss of BrdU-positive cells might reflect an overestimation of apoptosis, because BrdU itself could damage some labeled cells. However, cumulative labeling with BrdU over a number of days might reduce the temporal resolution in terms of distinguishing between proliferation and survival effects. So with our current BrdU injection protocol, the number of newly generated cells or neurons represents a combination of the proliferation rate, the differentiation rate, and net cell survival/apoptosis. It will be important to distinguish proliferation from survival/differentiation effects by using different BrdU strategies (48,49). Future studies will focus on these issues.

Activation of Antiapoptotic Pathway: Survival of the Newly-Generated Neurons
Cell birth and cell death appear closely associated in the dentate gyrus as continuous cell turnover takes place. Consequently, the dentate gyrus consists of a diverse and heterogeneous group of mature and developing cells. Furthermore, this turnover is highly sensitive to various hormonal and environmental stimuli (50,51). For example, removal of steroid hormones by adrenalectomy induces apoptosis in the dentate gyrus, but at the same time increases the division of immature cells (52). In contrast, stress reduces new cell birth (46,53).

Apoptosis is an important biological mechanism through which tissues shape normal developmental patterns and adapt to new environmental changes. The occurrence of apoptosis is regulated by serial alterations of intracellular molecules in response to extracellular signaling changes. A key event in early stages of apoptosis is mitochondrial permeability transition, which may release cytochrome c and, in turn, activate caspase 3 (54). Homodimers of Bcl-2 associate with mitochondrial membrane and stabilize membrane permeability (55). The protective effect of Bcl-2 on mitochondria permeability is lost when Bcl-2 homodimers are sequestered by the formation of Bcl-2/Bax heterodimer (55,56). Bad, which is regarded as a heterodimeric partner for Bcl-XL and Bcl-2, displaces Bax and promotes cell death (57). In the current study, we showed an increased level of Bcl-2 protein, increased phosphorylation of Bad, and inhibition of caspase-9 cleavage (a pattern of activation of antiapoptosis signal transduction) in the hippocampus of dwarf mice compared with normal mice.

Recent evidence indicates that the Akt-phosphoinositide 3-kinase pathway plays an important role in IGF-I promotion of neuronal survival by increasing the expression of the Bcl-2 family proteins and decreasing caspase activity (58–60). Interestingly, recent reports indicate that IGF-I could attenuate apoptosis in hippocampal neurons after ischemia (61), and suggest that the observed increase in the fraction of newly born neurons after IGF-I treatment is in part due to an effect on cell survival. In summary, the above results may suggest that increase in hippocampal IGF-I protein expression and subsequent activation of Akt-phosphoinositide 3-kinase and antiapoptosis signal transduction cascade might contribute to a delay of the age-related cognitive loss in these long-lived mice compared with normal animals.

Taken together, we speculate that aging would influence the balance between neurogenesis and apoptosis in the mouse hippocampus as well as the process of the regulation of the neuronal progenitors. Locally produced IGF-I might work primarily as a promoting factor to increase neurogenesis in the dentate gyrus area of the hippocampus in early adulthood; however, it might function mainly as a survival factor to inhibit neuronal death during aging in the central nervous system.


   Acknowledgments
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
This work was supported by National Institutes of Health Grant AG019899 and the Ellison Medical Foundation Grant (to A.B.), by Glenn Foundation/AFAR Scholarships (to L.S.), and by the Southern Illinois University Geriatrics Medicine and Research Initiative.


   Footnotes
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 
Decision Editor: Huber R. Warner, PhD

Received March 9, 2006

Accepted July 18, 2006


   References
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 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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H. R. Warner
2006 Kent Award Lecture: Is Cell Death and Replacement a Factor in Aging?
J. Gerontol. A Biol. Sci. Med. Sci., November 1, 2007; 62(11): 1228 - 1232.
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