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Huffington Center on Aging and Department of Molecular and Cellular Biology and Department of Medicine, Baylor College of Medicine, Houston, Texas 77030
Correspondence: Address all correspondence and requests for reprints to: Roy G. Smith, Ph.D., Huffington Center on Aging, Baylor College of Medicine, One Baylor Plaza, M320, Houston, Texas 77030. E-mail: rsmith{at}bcm.tmc.edu
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Abstract |
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I. Introduction |
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It has been argued that the age-dependent decline in sex steroid, GH, and IGF-I production is nature’s way of protecting us from cancer and heart disease, but a far more likely scenario is that once we reach our reproductive capacity, nature begins programming us for death. This is clearly illustrated by the marked decline in immune function (5, 6) and by the increased production of glucocorticoids and cytokines that negatively impact metabolism, bone density, strength, exercise tolerance, cognitive function, and mood (3, 7, 8, 9, 10, 11); similarly, the production of sex steroids, dehydroepiandrosterone (DHEA), GH, and IGF-I that have positive impact on these functions declines (1, 2, 3, 4, 12). Hence, if we wish to maintain quality of life during aging we must oppose nature. However, simply replacing hormones pharmacologically does not recapture the endocrine profiles of young adults; therefore, an ideal method of intervention awaits a fundamental understanding of the underlying mechanisms causing age-dependent hormonal changes.
Altered CNS function appears to precede the metabolic, reproductive, and cognitive deficiencies associated with aging. We speculate that the underlying basis is a progression of neuroendocrine changes characterized by altered biological rhythms, reduced amplitude, altered frequency, and decreased orderliness of hormone, neuropeptide, and neurotransmitter release. Indeed, attenuation of overall functional activity in the CNS accompanies aging (13, 14, 15, 16, 17, 18, 19, 20, 21, 22) (Fig. 1
). For example, monoamine oxidase activity increases, causing a decrease in the concentrations of serotonin (5-HT) and dopamine (13), and this is paralleled by alterations in concentrations of receptors for hormones, neuropeptides, and neurotransmitters in the CNS. Reduced secretion of hypothalamic GnRH results in altered LH pulse amplitude, thus attenuating pulsatile gonadal steroid secretion (23). Similarly, a decrease in hypothalamic GHRH secretion causes reduced GH pulse amplitude and reduced IGF-I levels in GH target tissues (24, 25). Increases in amplitude of hormone release have also been noted and include ACTH and PTH (26, 27, 28).
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We favor a hypothesis of aging based on alterations in the dynamics of neuronal behavior. Such dynamic changes are consistent with a destabilizing effect on CNS function, which potentially increases the vulnerability of the aging brain to trauma. In this review, we address the significance of age-related changes in biological rhythms and the benefits of restoring normal rhythms. Age-associated changes in cognitive decline, which appear to be associated with disruption of endocrine pathways, are described. We also discuss the underlying age-dependent alterations in components of feedback pathways governing the release of hormones, neuropeptides, and neurotransmitters. Finally, because hormones signal by modulating gene transcription, we review age-related changes in factors involved in regulating the transcription of genes intimately involved in endocrine and CNS function. Although much excellent science has been done, the reductionist approach makes it impossible to clearly determine causality. Effects can be readily defined, but causes are likely multifactorial. Having made the reader cognizant of this caveat, we present an overview of selected topics of relevance to the molecular endocrinology of the aging CNS with the objective of providing the stimulus for continued investigation using whole systems approaches.
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II. Complex Behavior of Neurons in Aging |
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Reduced complexity could occur through loss or defect in a component and/or altered nonlinear coupling (feedback) between components of the system (45). A loss of neuronal components and coupling between components of neuronal networks is characteristic of aging. For example, a relative increase in the concentration of glucocorticoids compared with sex steroids, GH, and IGF-I is associated with shrinkage of the hippocampus, loss of neurons, and declining neurogenesis; loss of estradiol production is associated with fewer neuronal connections. The number of dopaminergic neurons also gradually declines during aging, producing deficits in the nigrostriatal dopamine system of rodents, monkeys, and humans (19, 46, 47). Such changes would predictably result in reduced complexity and efficiency of signaling within neural networks and reduced adaptive capability. An example of a decline in adaptive capacity of neurons during aging is the increased vulnerability of the brain to anoxia and ischemia, which in rats is associated with reduced glycolytic capacity of neurons (48). On this basis, we speculate that the onset of functional deficits associated with aging is a consequence of altered behavior of underlying regulatory pathways in the CNS.
B. Hormone pulsatility and aging
One of the most significant age-related events is an alteration in amplitude and pulsatile pattern of hormone release. The frequency of release of a hormone is as important, or more important in some cases, than the amount of hormone released. Target cells respond most effectively to exogenous hormonal stimulation when the frequency of stimulation approaches the endogenous frequency (49). Age-related changes in the endocrine system can appear superficially as apparent increases in complexity (45, 50, 51, 52, 53, 54). Veldhuis and colleagues (27, 55, 56, 57, 58) made extensive evaluations of age-related changes in the dynamics of pulsatile hormone release. They applied mathematical approaches to investigate the synchrony and pulsatility of GH, LH, testosterone, ACTH, cortisol, and insulin release during aging. By calculating the approximate entropy (ApEn) statistic as a measure of orderliness of synchronicity of hormone release, they showed that individual orderliness declined progressively during healthy aging. However, ApEn calculations do not directly distinguish between contributions of stochastic and deterministic behavior toward the observed regularity (45, 53). Therefore, the less ordered rhythmic patterns of hormone release observed during aging could result from a transition of the regulatory neuronal network controlling the ordered frequency of hormone release from adaptive complex behavior to stochastic behavior.
The ApEn calculations in concert with clinical data support the concept that aging is tightly associated with disruption of the time-delayed positive and negative feedback pathways controlling synchrony of hormone release. Therefore, application of nonlinear dynamics and mathematical analyses for analyzing the behavior of neurons that regulate the endocrine system and how this behavior changes as a function of age is important and reinforces our awareness of the limitations of reductionist methods.
C. Dopaminergic system as an example of age-related change in neuronal dynamics
In aging rats, dopamine production decreases as reflected by reductions in tyrosine hydroxylase mRNA (TH) in the pars compacta of the substantia nigra, and ventral tegmental area (59). The number of cells expressing TH mRNA is the same in young and old rats, but aging is associated with reduced TH gene expression per cell. Reduced production of dopamine during aging increases the susceptibility of neurons toward glutamate neurotoxicity, resulting in seizures and neuronal cell death (60). Dopaminergic neurons in the caudate-putamen, substantia nigra, and nigrostriatal pathway also show increased susceptibility to degeneration induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine treatment (61). This gradual loss of neurons from the neuronal network will be accompanied by progression toward reduced complexity in neuronal behavior.
Studies of the behavioral dynamics of the dopamine neurons are consistent with age-related progressive changes toward reduced complexity. When the electrophysiological behavioral characteristics of dopaminergic neurons were compared in the brains of young and old animals, two different firing modes (single-spike and bursting) that interweave to produce irregular interspike patterns were identified (62, 63, 64, 65). Mathematical analysis to discriminate nonlinear deterministic from either stochastic or linear oscillations showed that interspike intervals recorded from dopaminergic neurons exhibited a transition toward stochastic behavior during aging (65, 66). Although irregular stochastic behavior could also organize the irregular behavior of neurons, the rapid synchronization and processing of irregular input signals is less readily accommodated (41).
In summary, during aging there appears to be an increased susceptibility of physiological systems to trauma and stress. It has been speculated that this is a consequence of a transition of physiological systems from ordered adaptive complex behavior toward more stochastic behavior (40, 41, 42). The application of nonlinear dynamics to physiology is relatively new; therefore, until more work is done, the conclusions must be considered preliminary. Despite this caveat, the characterization of age-related changes in the electrophysiological dynamics of neuronal behavior, as observed with dopaminergic neurons, paves the way to test strategies designed to reverse or prevent age-related changes; furthermore, it allows us to determine whether experimental manipulation to improve adaptive capacity by restoring the behavioral complexity of the system will prevent increased vulnerability to trauma and stress. Hence, the application of dynamic measures of complexity offers the potential to quantitate physiological aging, to predict the outcome of molecular endocrine changes, and to provide a method for evaluating intervention strategies.
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III. Age-Dependent Changes in Biological Rhythms |
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Circadian rhythms exhibiting erratic firing and reduced amplitude are observed in aged rats and appear to be primarily controlled at the level of gene transcription in the SCN (69, 70). The effect of light pulses on modifying circadian rhythm differs in young vs. old animals. In young hamsters maintained under conditions of constant light, or after 6 h exposure to darkness, induction of phase advance and phase delay in circadian rhythm of locomotor activity is induced by treatment with the short-acting benzodiazepine, triazolam, whereas old hamsters are refractory (71).
In patients with Alzheimer’s disease (AD), day and nighttime levels of arginine vasopressin (AVP) mRNA in the SCN are identical, but in normal subjects, daytime levels are more than three times higher than at night (72). Liu et al. (72) speculated that the neuronal basis of the circadian rhythm disturbances in AD patients is located in the SCN, which perhaps explains the beneficial effect of light therapy on relieving restlessness at night. Clearly, these data do not establish a direct relationship between AVP circadian rhythms and AD; however, the results are intriguing and invite further clinical studies.
The fundamental mechanism underlying age-related alterations in biological rhythms of hormone release continue to be investigated. In the case of LH, the noradrenergic system is an important regulator of episodic release and provides an example of age-dependent changes in neurotransmitter action (73). Middle-aged rats show decreased levels of 
1-adrenergic receptors in the SCN. The diurnal rhythm of 1-adrenergic receptors expression, characteristic of young rats, disappears by middle age (73). Similarly, aging alters the rhythmic expression of vasoactive intestinal peptide (VIP) in the SCN (74). In young female rats, but not in middle-aged rats, VIP mRNA exhibits a 24-h rhythm. By contrast, the 24-h rhythm of AVP mRNA expression persists during aging. Thus, regulatory components of the SCN are differentially modified by aging.
B. Altered circadian rhythms modify sleep patterns
One of the most common features of aging is impairment in the quality of sleep with increased wakefulness and reduced slow-wave sleep (SWS) (75, 76). Biological clocks in the SCN of mammals are important regulators of sleep-wake cycles. SCN neurons produce VIP, AVP, and somatostatin. GHS-R is also expressed in the SCN, and the synthetic GHS-R ligand MK-0677 improves quality of sleep in healthy elderly subjects (77, 78).
Inputs to the SCN from retinal ganglion neurons and neurons of the lateral geniculate and raphe nuclei play an important role in entrainment and shift of circadian rhythms (79). Lesioning of the SCN causes a loss of circadian rhythms of hormone release and sleep-wakefulness (79, 80, 81). However, the SCN is not the only regulatory influence, because increases in SWS and slow-wave activity that follow sleep deprivation are not reduced in SCN-lesioned animals (82). There is strong evidence that regulation of sleep homeostasis involves adenosine activity in the basal forebrain (83). Indeed, adenosine may play a broader role in regulating sleep patterns, because it is an agonist for the GHS-R, which is also expressed in CNS and is involved in sleep regulation (78, 84, 85, 86).
To investigate a potential link among aging, circadian rhythms of hormone release, and sleep patterns, 24-h pulsatile profiles of cortisol, TSH, melatonin, prolactin, GH, and sleep patterns in healthy elderly men and young men were monitored (87). Mean cortisol levels were unaffected by age; however, the amplitude of circadian rhythm was reduced in elderly men. Daytime and nighttime levels of TSH and GH were markedly diminished according to age, whereas prolactin and melatonin concentrations were decreased only at nighttime. These age-dependent decreases were a result of reduced amplitude rather than a change in pulse frequency. The circadian increase of cortisol, TSH, and melatonin occurred 1-1.5 h earlier in the elderly men and was accompanied by a similar advance in rapid eye movement (REM) stage sleep. Healthy elderly subjects experience earlier clock time for melatonin circadian rhythms, body temperature, and cortisol peaks. Wake time is advanced relative to both clock time and internal circadian rhythms. The basis of such differences between young and old subjects remains to be elucidated, but it likely involves age-related changes in factors known to regulate sleep patterns. These include GHRH, adenosine, CRH, galanin, neuropeptide Y (NPY), vasopressin, and hypocretin or perhaps the endogenous ligand of the GHS-R, ghrelin (88, 89, 90, 91, 92).
C. Restoration of normal rhythms in aged animals
Age-dependent changes in biological rhythms can be reversed by implanting the SCN from rat or hamster fetus into the brain of the appropriately aged host (30, 31, 93). In young hamsters, repeated injection of benzodiazepines entrains the circadian clock to the exact injection period, whereas old hamsters are resistant to efficient entrainment (71). However, transplantation of fetal SCN into the hypothalamus of old hamsters partially rescues the aging phenotype by restoring phase shifts that are responsive to triazolam and restoring rhythmic c-fos expression in response to light (71). Similarly, fetal SCN transplantation modifies circadian rhythms of the CRH/ACTH axis in middle-aged rats to mimic those of young animals.
The demonstration that the young phenotype is restored in an old animal by transplanting fetal SCN tissue is fundamentally important because it shows that the aging SCN retains latent functional capacity. Furthermore, these results suggest that important factors regulating the temporal pattern of expression in the SCN are lost by the time rats reach middle age. Intriguingly, the fetal SCN either provides these factors or induces their expression in the host. Restoration of the host SCN can also be demonstrated when the transplanted fetal SCN cells are encapsulated, showing that an SCN rejuvenating factor(s) is secreted by the fetal cells (31, 94).
In addition to sex steroids that modulate dopamine signaling, catecholamine levels in the brain decline during aging (95, 96, 97, 98, 99, 100, 101, 102, 103). The aging hypothalamus has a reduced capacity to secrete dopamine and norepinephrine (104). Indeed, certain aspects of aging are induced by treating rats with drugs that reduce catecholamine levels in the hypothalamus, whereas drugs that elevate hypothalamic catecholamine levels reverse certain physiological aspects of aging (104). For example, when young hamsters are treated with reserpine to lower concentrations of 5-HT, norepinephrine, and dopamine in the hypothalamus, striatum, and pons/medulla, their circadian rhythms are altered and their responses to phase shifting stimuli are modified to produce a phenotype identical to that occurring spontaneously in old hamsters (71). Hence, reductions in monoaminergic activity in the brain probably contribute to the age-associated changes in the circadian clock system. Because this aging model can be manipulated by altering catecholamine levels, it allows experimental testing of the hypothesis that aging is coupled to decreased complexity of neuronal behavior.
D. Age-associated changes in circadian rhythms influence metabolism
The development of age-related reduced glucose tolerance, obesity, and peripheral insulin resistance accompanies alterations in the circadian rhythms of glucose regulation (68). Remarkably, when the daily rhythms of endogenous corticosterone and prolactin in old rats are modified by administering these hormones at times of the day corresponding to peak levels observed in young rats, the age-associated increases in insulin resistance and body fat are reversed (105). Resetting of the rhythms by appropriately timed hormone replacement restores the young phenotype. Most importantly, these experimental results emphasize the physiological importance of circadian rhythms on metabolism and are consistent with changes in behavioral complexity of regulatory neurons that produce altered rhythmicity of factors controlling glucose regulation.
Changes in circadian endocrine rhythms during aging are associated with altered carbohydrate and lipid metabolism, which causes increased deposition of fat at the expense of muscle. Neuroendocrine perturbations involving the hypothalamic-pituitary-adrenal (HPA) axis produce insulin resistance (106). Visceral fat (VF) accumulates and is an important contributing factor for age-associated insulin resistance and development of syndrome X (107, 108). VF is a rich source of 11ß-hydroxysteroid dehydrogenase type 1 (HSD1), which reduces 11-keto steroids to produce active glucocorticoids (109, 110). Hence, increased VF provides a rich source of the counterregulatory hormones for glucose homeostasis.
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IV. Aging, Memory, and Cognitive Decline |
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A. Age-related neuronal structural and functional changes
The endocrine system affects neuronal signaling and neuronal integrity; therefore, age-dependent endocrine changes influence structure and function of the CNS. Morphological studies of the hippocampus in young and old rats reveal that pyramidal neurons in old rats are smaller and contain fewer dendritic branches and spines (111). The density of presynaptic terminals per unit length of postsynaptic membrane is also lower (Fig. 2). Such changes are reminiscent of age-associated shrinkage of pyramidal neurons in the human brain (112, 113).
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-amino-butyric acid (GABA) inhibitory synapses and correlate with age-related reductions in GABAA receptor mRNA expression and GABAA receptor density (114, 115, 116). The reduced amplitude of excitatory serum PSCs in old rats is probably a result of desynchrony of neurotransmitter release from presynaptic terminals, which is likely exacerbated by the greater separation of presynaptic terminals in the aged brain (111). These observations support the hypothesis that aging results in a transition toward increased stochastic behavior of neurons that leads to less robust synchrony of neurotransmitter release. Surprisingly, aside from amplitude changes, it appears that compensatory mechanisms maintain comparable input to the pyramidal neurons despite significant synaptic loss during aging. Activation of the compensatory pathways may explain why cognitive impairment in normal aging is relatively modest compared with that observed in pathological conditions such as AD. As they age, rats and mice show changes consistent with age-dependent cognitive deficits in spatial memory and working memory (117, 118, 119, 120, 121). Spatial learning is dependent on the integrity of the hippocampal structures and is evaluated according to performances in the Morris water maze and Barnes maze. The early stage of memory is associated with early-phase long-term potentiation (LTP), which does not involve protein synthesis, whereas later stages that consolidate short-term to long-term memory are associated with late-term LTP requiring new mRNA and protein synthesis (120). Comparative studies in 3, 6, 12, and 18-month-old C57BL/B6 male mice showed that spatial memory was impaired in the majority of aged mice (12 and 18 months old) and was correlated with late-term LTP deficits in CA1 neurons of the hippocampus. Comparing performance in spatial tasks with neurobiological evaluation allows discrimination between a detrimental neurobiological change from compensatory adaptation (122).
B. Hippocampus and neurogenesis
Neurogenesis in the dentate gyrus (DG) of rats was first reported in 1965 (123), and recent results in primates extend these findings across species (124, 125, 126). The significance of the production of new neurons during adulthood is unknown, although studies in rodents suggest that neurogenesis plays an important role in learning (125, 127). By placing rats and mice in a stimulating environment, neurogenesis is induced (128, 129). Furthermore, training in a task that requires hippocampal function stimulates granule cell proliferation in the DG (127, 130). However, a decline in neurogenesis occurs during aging (131, 132, 133).
Production of neurons in the mature CNS is affected by trauma. New neurons are generated in the hippocampus after seizures (of variable amplitudes), stroke, and local lesions, suggesting that they may be involved in recovery from injury (134, 135, 136). Ischemia increased the production of neuronal cells in the subgranular zone of the DG that coexpress both markers of DNA replication and mature neurons. These results support a role for neurogenesis in what may be a process that leads to recovery after stroke (135). Excitotoxic and mechanical lesions of the granule cell layer performed in the adult rats showed an increase in proliferating cells on the lesioned side compared with the unlesioned side 24 h after surgery. There was also a significant positive correlation between the extent of damage and the number of proliferating cells. Three weeks after the lesion, the majority of cells produced as a result of this insult had morphological and immunohistochemical characteristics of mature granule neurons and were located in the granule cell layer (136).
C. Aging and neurogenesis
It is not surprising that attenuated neurogenesis is observed during aging because positive regulators such as the sex steroids, DHEA, GH, and IGF-I decline, and glucocorticoids, which inhibit neurogenesis, increase (137, 138, 139, 140, 141, 142, 143, 144, 145, 146). The important issue is whether these hormonal changes and reduced neurogenesis contribute to increased susceptibility of the CNS to irreversible damage and increased incidence of CNS-linked disorders. If cause and effect are linked, timely hormone replacement would be most beneficial.
D. Steroids and neurogenesis
Specific neural mechanisms alter the production of granule cells in the DG. The perforant path is the main excitatory afferent to the granule neurons and provides glutamatergic input, which appears to suppress the proliferation of granule cell precursors. Lesion of the entorhinal cortex, source of the perforant path, increases neurogenesis in the DG (147). In contrast to young rats, in old rats even acute stress produces an exaggerated release of glutamate in the hippocampus (148). Both corticosterone and glutamate N-methyl-D-aspartate (NMDA) receptor agonists inhibit neurogenesis. Treating adult rats with the NMDA-receptor antagonist dizocilpine maleate (MK-801) stimulates neurogenesis and increases the density of neurons in the granule cell layer. MK-801 also counters the corticosterone-induced decrease in cell proliferation. Hence, corticosterone and NMDA receptor activation appear to inhibit granule cell production in the rat DG through a common pathway, and NMDA-receptor activation is downstream of adrenal steroid effects (149). Overall, although the evidence remains associative, the collective findings in rodents and nonhuman primates argue that decreased neurogenesis is caused by elevations in plasma and locally produced corticosteroids. The effects of elevated glucocorticoids during aging are exacerbated by decreases in estradiol and IGF-I and likely contribute to age-related memory deficits observed in humans.
The stimulatory effects of estradiol and inhibitory effects of corticosterone on neurogenesis has been clearly demonstrated in rats. Regulation of neurogenesis by estradiol was tested by measuring the incorporation of bromodeoxyuridine (BrdU) into cell nuclei of the dentate granule cell layer at different estrus stages, and after ovariectomy with and without estradiol replacement (141). Figure 3 illustrates the beneficial effects of estradiol on stimulation of cell proliferation and cell survival in the DG of ovariectomized rats. Old age is accompanied by a marked decrease in production of hippocampal granule neurons (132, 150). Lowering corticosterone levels in old rats restores neurogenesis. Most importantly, this result shows that the neuronal precursor population is unaffected by old age, indicating that neurogenesis is inhibited by age-associated increases in basal corticosteroid levels, but the deficit can be rescued (132).
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Testosterone also plays a role in neurogenesis, and one of the best examples is from the avian system. Stimulation of neurogenesis in the adult canary incorporates neurons into the high vocal center, which is a nucleus in the adult canary brain that is important in the acquisition and presentation of learned song (151). This process occurs seasonally and is regulated by testosterone. Testosterone acts by stimulating production of brain-derived neurotropic factor (BDNF) in the female high vocal center. BDNF mimics the effect of testosterone, and infusion of a neutralizing antibody to BDNF blocks the testosterone-induced increase in new neurons. Hence, testosterone regulation of neuronal replacement in the adult canary brain is dependent on BDNF.
E. IGF-I and neurogenesis
Growth and development of neurons in the DG are modulated by IGF-I. Marked reductions in IGF-I and neurogenesis in the DG accompany aging (133). Stereological analysis of brain sections from transgenic mice that overexpress IGF-I postnatally in the brain shows increases in neuronal and synaptic density in the DG (146). To model somatic IGF-I, deficiency rats were hypophysectomized and maintained on glucocorticoid and T4 replacement (142). These rats were subjected to short-term (6 d) and long-term (20 d) infusion of IGF-I, and cell proliferation was monitored in the DG by incorporation of BrdU (142). Both short- and long-term IGF-I infusion maintained neurogenesis in the hypophysectomized rats. These results support the notion that IGF-I regulates neurogenesis in the dentate granule cell layer and suggest that age-associated decreases in IGF-I might be involved in age-related neurodegeneration.
Recent studies show that the rate of neurogenesis in the DG of the hippocampus declines as a function of age perhaps contributing to age-related cognitive changes. Intracerebroventricular (icv) IGF-I infusion ameliorates this age-associated decline. Lichtenwalner et al. (152) used BrdU labeling and multilabel immunofluorescence to evaluate age-dependent changes in neuronal production in the DG of adult Brown Norway (BN)/Fischer 344 rats. They found an age-dependent reduction in the generation of new cells in the adult dentate subgranular proliferative zone and a 60% reduction in the differentiation of newborn cells into neurons. Intracerebroventricular infusion of IGF-I to restore IGF-I levels in senescent rats restored neurogenesis to provide a 3-fold increase in neuronal production. This study highlighted the possibility that IGF-I is an important mediator of neurogenesis in the adult and suggested that the age-dependent decline in IGF-I-regulated neurogenesis could contribute to cognitive deficits.
Exercise is also beneficial for maintaining neurogenesis. In the adult mouse, running promotes neurogenesis in the DG, which is believed to be mediated by IGF-I (153). BDNF also increases in mice after running, which counteracts the negative impact of stress on BDNF production in the hippocampus (154, 155, 156). Indeed, exercise-induced increases in BDNF enhanced the rate of learning in the Morris water maze test (157, 158). Furthermore, BDNF mediates testosterone-induced survival of new neurons in the adult brain (151). However, neuroprotection requires IGF-I, because the protective effect of exercise is antagonized by the central infusion of IGF-I antibodies (159).
F. Neurosteroids and memory
Steroidogenic acute regulatory protein (StAR) controls adrenal and gonadal steroidogenesis. It was recently shown unequivocally that StAR mRNA and protein are expressed within glia and neurons in discrete regions of the mouse brain (160). Consistent with its role in de novo neurosteroidogenesis, StAR colocalizes with the cholesterol side chain cleavage enzyme P450(scc) in both mouse and human brains (160). These data support a role for StAR in the production of neurosteroids and identify potential sites of active de novo steroid synthesis in the brain (160). Neurosteroids synthesized in the CNS appear to attenuate age-related memory and learning impairments (161, 162, 163, 164, 165). For example, impairments induced by aging or by an NMDA receptor antagonist were inhibited by neurosteroids (164). When young (3 months old) and old mice (16 months old) were tested in two different behavioral models of long-term memory, the performance of aged mice in step-down passive avoidance and elevated plus-maze paradigms was markedly impaired compared with the performance of young mice; however, treatment with pregnenolone sulfate (PS) and DHEA sulfate attenuated the decline in performance. To determine whether the mechanism of attenuation was mediated through the nitric oxide (NO) synthase (NOS) signal transduction pathway, mice were pretreated with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) at doses that were predetermined to have no disruptive effect on cognition. L-NAME inhibited the beneficial and antiamnesic effects of PS and DHEA sulfate, and the effect of L-NAME was blocked by the competitive substrate for NOS, L-arginine. Hence, the beneficial effects of PS and DHEA sulfate on age-related learning and memory deficits appear to be mediated by inhibition of a NO-dependent pathway.
PS synthesis in the rat hippocampus declines during aging, and performance in two different spatial memory tasks (the Morris water maze and Y-maze) was found to correlate with levels of PS in the hippocampus (163). Old rats with the greatest memory deficits had the lowest hippocampal PS levels. A possible cause-and-effect relationship was suggested by showing that impaired memory was transiently improved by ip or bilateral intrahippocampal injection of PS (163, 166).
PS is a GABAA receptor antagonist and an allosteric activator of the NMDA receptor. Administration of PS into the CNS enhances acetylcholine (ACh) release in basolateral amygdala, cortex, and hippocampus and stimulates neurogenesis (166). ACh neurotransmission is involved in regulation of memory processes and modulation of the sleep-wake cycle and neurodegenerative diseases (166). These findings suggest that PS is at least partly involved in maintaining cognitive abilities, sleep patterns, and neurogenesis. It remains to be determined whether local neurosteroid synthesis declines during aging, or whether lower levels of neurosteroids in the CNS is a result of reduced peripheral steroid production. Studies are underway to address these questions through selective neurosteroid synthesis inhibition in the brain.
G. Gene expression in memory and learning
One caveat of drawing conclusions from behavioral tests on laboratory rodents is that the animals are housed in an artificial, sterile environment. If rats are exposed to an enriched environment during their youth, they perform better in memory tasks when they get older (167). Indeed, providing mice with toys, wooden blocks, spin wheels, and small houses produces biochemical and structural changes in the cortex, DG, and CA1 hippocampal structures (128).
Candidate genes that specifically contribute to memory and learning were identified by using high-density oligonucleotide microarray analysis to compare gene expression in mice maintained in standard housing with mice exposed to a stimulating environment (168). Many of the genes identified were known to play a role in neurotransmission, neuronal structure, and neuroplasticity. Certain of these genes are related to hormonally regulated genes (168). For example, the expression of estrogen-responsive finger protein was increased by 2.2-fold, and DNA methyl-transferase increased 26-fold; by contrast, retinoid X receptor- expression decreased by 26-fold. The expression of genes involved in apoptosis, such as Bcl-2 associated protein, Bax, caspase-6, and caspase-4 genes decreased by 3- to 4-fold. Expression of transcription factor X-box binding-protein, which interacts with cAMP response elements of genes to increase gene expression, increased 2.4-fold. Furthermore, expression of the cAMP-dependent protein kinase regulatory subunit was reduced 2.5-fold, which is relevant to aging because overexpression of this regulatory subunit compromises both hippocampal LTP activity and long-term memory (169). Interestingly, levels of apolipoprotein E (apoE) increased 2.3-fold (168). apoE signaling is involved in the dephosphorylation of 
protein and hyperphosphorylated is a component of the plaques and tangles characteristic of AD; hence, apoE may have neuroprotective properties. Although it must be remembered that these observations are merely associative, they provide important links with hormones and aging of the CNS. For example, IGF-I production declines during aging; however, increasing IGF-I levels in the brain mimics enrichment by protecting against apoptosis and neurodegeneration.
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V. GH Axis |
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It is important to note that GH replacement by bolus injection overrides the episodic physiological profile (179). Biochemical and biological data support the importance of the episodic profile. In the liver, different signal transduction pathways are activated when pulsatile vs. sustained administration of GH are compared (180). Male rats exhibit pronounced high amplitude pulses, whereas in females the profile is flatter and is reflected by distinctly different patterns of GH-regulated gene expression. In males, whole body pubertal growth rate is dependent on GH activation of signal transducer and activator of transcription 5b (STAT5b). In STAT5b knockout mice, males exhibit a female GH phenotype (181).
In humans, GH pulse amplitude and plasma IGF-I levels decline during aging (24, 38). Young adults exhibit a gender difference, in which women have the same pulse frequency as men but with a 2.4-fold increase in burst mass (182). However, these gender differences disappear during aging, and elderly men and women have equally low amplitude GH pulses and reduced IGF-I levels (24, 38). Based on the known properties of GH and IGF-I in vivo, this reduced amplitude, in combination with reduced sex steroid production, likely explains the observed age-dependent change in metabolism, increased fat/lean ratio, decreased muscle strength, reduced exercise tolerance, and increased bone loss. Hence, the functional deficits that result from aging are probably caused by suboptimal signaling from the hypothalamus. An ideal approach for modifying the aging phenotype would be to restore activity of the hypothalamic neurons that control GH pulse amplitude.
A recent study (183) describes the use of DNA microarray chip technology to relate the physiological decline in GH with molecular mechanisms underlying the aging process. Gene expression was compared in the liver of old rats, with or without GH replacement. Of 1000 genes detected in male rat liver, 47 transcripts were affected by aging and about 40% of the differentially expressed genes were normalized by GH treatment. This study is notable and refreshing because the authors evaluated gene expression in the animals after compensating for changes in GH. However, because of age-dependent changes in other hormones, such as sex steroids, and the difficulty of replacing hormones in a way that recaptures the physiology of a young animal, it is impossible to precisely differentiate hormone-dependent from hormone-independent age-related changes. Despite this caveat, studies using DNA microarray analysis of brain tissue from rats that show improvements in memory following GH replacement should be particularly informative.
B. Increase in longevity in GH-deficient rats and mice
It seems reasonable to speculate that restoration of GH release in a way that mimics the physiology of a young adult will provide functional benefits, not necessarily by increasing longevity, but by improving the quality of life. This is based on observations in GH-deficient humans showing that GH increases bone density and improves body composition, cognitive function, cardiac function, and exercise tolerance. However, despite this evidence, the likelihood of achieving beneficial effects by rejuvenating the GH/IGF-I axis physiologically is not universally accepted. One of the reasons is based on laboratory animal studies, where the data suggest that reducing GH, IGF-I, or insulin signaling increases longevity (184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194). However, these studies do not address quality of life in elderly subjects, which is more important than longevity. Indeed, although caloric restriction has also been shown to improve longevity in a variety of species, a recent, careful study and review of the literature shows that prolonged caloric restriction impairs cognitive function in rats (195).
The evidence for a negative impact of GH and IGF-I on longevity is based largely on studies in mutant mice that are either GH deficient or lack receptors for GH. All of these models exhibit dwarfism, reduced body temperature, and reduced fertility. Certain of these mouse models, such as the Ames dwarf (dw/dw) mouse are GH, IGF-I, prolactin, and thyroid hormone deficient. Although recent studies (188) indicate that the life span of hormone-deficient dwarf mice housed under stress-free laboratory housing conditions is 50% longer than their normal littermates, these observations are unlikely to predict survival in the natural environment. For example, earlier studies indicated that dw/dw mice had markedly reduced life span (45-60 d) and are immunocompromised (196, 197). The reduced longevity was suggested to be a result of more stressful animal housing conditions, typical of 30 yr ago, and poor adaptation to stress (198, 199). Indeed, it has been speculated that the negative effects of prolonged stress, which causes suppression of the immune system by glucocorticoids, are balanced in wild-type animals by the positive effects of GH, IGF-I, prolactin, and thyroid hormone (198, 200).
It is important to recognize the beneficial roles of the GH/IGF-I axis in human physiology. In addition to antagonizing the adverse effects of chronic stress on the immune system, GH and IGF-I may play a similarly protective role in the CNS, thereby potentially improving quality of life. Indeed, Koo et al. (201) reported that restoring GH levels produced beneficial effects on the immune system of old normal mice. They showed that when old mice are treated with an oral GH secretagogue, restoration of GH and IGF-I reversed the age-dependent shrinkage of the thymus and improved T-cell production. The advantage of rejuvenating the GH/IGF-I axis was illustrated by implanting aggressively growing tumors into the mice. Treatment with a GHS-R agonist reduced the rate of tumor growth, inhibited metastasis, and increased longevity (201).
C. GH in the CNS
The characteristics and significance of GH binding in the human brain have been reviewed by Nyberg and Burman (202). In addition to reduced GH release during aging, the concentration of GH receptors in the brain also declines. The highest density of GH binding is in the choroid plexus, with significant binding in the hippocampus, hypothalamus, amygdala, putamen, and thalamus (202). Although GH receptors are widely expressed in the CNS, and anecdotal reports claim that GH improves mood in the elderly, relatively few studies have investigated GH’s functional role in the brain (203). Indirect evidence suggests that GH plays an important role in CNS function. GH-deficient children have an increased incidence of anxiety, depression, and attention deficits, which may contribute to their observed learning disabilities in arithmetic, spelling, and reading compared with age-matched controls (202, 204). GH deficiency in adults is reported to be associated with reduced energy, unfulfilled personal life, low self-esteem, problems controlling emotional reactions, social isolation, impaired social function, mental fatigue, impaired general and mental health, and deficits in cognitive function (205, 206, 207, 208, 209, 210, 211). Markedly reduced GH levels, particularly the integrated nocturnal levels, have also been associated with major depressive illness (212). This may explain the increased incidence of depression and poor sleep quality in the elderly population.
The neuroprotective property of GH was documented in rats. Hypoxic-ischemic damage causes increased GH transport into the brain as manifested by an increase in the number of GH-immunopositive neurons (213). To demonstrate GH binding by immunostaining, the authors went to extraordinary lengths to document specificity. The immunohistochemical evidence showing that GH migrates to the sites of injury following hypoxic-ischemic injury is most persuasive. The authors also demonstrated that icv administration of GH was neuroprotective in the cortex and hippocampus (Table 1). Thus, the decline in the amplitude of GH secretion during aging likely attenuates GH-mediated neuroprotection.
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GH administration increases IGF-I gene transcription in the CNS. Whether a causal relationship between age-associated deficiencies in cognitive function and declining brain IGF-I levels exists is the subject of continuing debate. However, an association is supported by the observation that when IGF-I is administered icv to rats for 28 d, the age-dependent decline in spatial reference and working memory is reversed (216). GH studies in adults with childhood onset GH deficiency showed that GH replacement benefited CNS function (217, 218). Doses of GH that produced supraphysiological levels of IGF-I normalized memory function after 6 months of treatment. Lower doses selected to provide physiological IGF-I concentrations in the blood improved memory function more slowly, but normal function was restored after 12 months of treatment.
Reduced IGF-I levels are characteristic of aging (2, 215, 219, 220, 221). Endogenous IGF-I plays a significant role in recovery from insults such as hypoxia-ischemia (222). Neurons die within hours or days following initial injury because of activation of cell death pathways. However, IGF-I with its binding proteins and receptors is induced within damaged areas following brain injury, which suggests that IGF-I plays a neuroprotective role. Administration of IGF-I within a few hours after brain injury confers protection on gray and white matter; by contrast, IGF-I pretreatment is ineffective, probably because of limited intracerebral penetration into the uninjured brain. This important neuroprotective property of IGF-I argues for the maintenance of young adult IGF-I levels during aging.
It has been suggested that IGF-I deficiency could be involved in cognitive deficits seen with aging. In elderly humans (aged 65-86 yr), a correlation between a subject’s performance in the Mini Mental State Examination and plasma IGF-I was reported (223). To investigate this observation in more detail, cognitive functions known to decline during aging were compared with those insensitive to aging. The outcome was consistent with a protective effect of IGF-I on the onset of age-dependent cognitive deficiencies, particularly in speed of information processing (208, 224). Similarly, Dik et al. (225) investigated whether IGF-I was associated with cognitive performance and cognitive decline over a 3-yr period in 1318 subjects, aged 65-88 yr. Although cross-sectionally IGF-I was directly related to information processing speed, memory, fluid intelligence, and Mini Mental State Examination score, these statistics were not significant after adjusting for age and other factors. However, analysis in quintiles of IGF-I illustrated a threshold effect of low IGF-I on information processing speed, with lower speed in those subjects in the lowest quintile of IGF-I (
9.4 nmol/liter) vs. those in the other four quintiles. A low IGF-I threshold was also observed during a 3-yr decline in information processing speed. In conclusion, this study suggested that IGF-I levels below 9.4 nmol/liter are associated with the level and decline of information processing speed.
Serum IGF-I appears to regulate brain amyloid-ß (Aß) levels (226). During aging, IGF-I levels fall and Aß, which is involved in the pathogenesis of AD, accumulates in the brain. Elevations in brain Aß levels are found at an early age in mutant mice having low circulating IGF-I. Aß burden can be reduced in aging rats by increasing serum IGF-I, and it reflects the ability of IGF-I to induce the clearance of brain Aß. This is probably mediated by enhancing the transport of Aß carrier proteins such as albumin and transthyretin into the brain. The enhanced uptake is antagonized by TNF-. IGF-I treatment of mice overexpressing mutant amyloid markedly reduces their brain Aß burden; therefore, IGF-I appears to play an important role in modulating brain amyloid levels.
Studies on centenarians showed increased prevalence of dementia in those with lowest serum IGF-I levels (227). Collectively, these results are consistent with a causal link between the age-related decline of GH and IGF-I levels and cognitive deficits, which reinforces the need for continued investigation of IGF-I and CNS function. Ghrelin mimetics have been shown to normalize IGF-I levels in the elderly and to increase IGF binding protein 3; therefore, these compounds may prove beneficial as neuroprotective agents during aging (25, 38). The fact that IGF binding protein 3 levels are also increased suggests that the risk/benefit ratio regarding cancer risk may not be increased by such treatment.
E. Potential mechanisms of GH/IGF-I-mediated neuroprotection
The age-associated decline in GH and IGF-I is likely to cause deficits in functioning of the CNS because both hormones play an important role in vascular maintenance and remodeling. The cerebrovasculature is a source of IGF-I and nerve growth factor (NGF), which are known to play an important role in memory (216, 228, 229, 230). During aging, cerebral blood flow decreases and, together with reduced production of sex steroids, correlates with the age-related decline in plasma GH and IGF-I levels. In BN rats, arteriolar density and anastomoses decline markedly between the ages of 7 and 29 months. However, GH treatment produces increases in IGF-I, reverses the age-dependent changes, and increases the number of cortical arterioles (231). These data suggest that preventing the decline in GH and IGF-I during aging would help prevent age-related reductions in vascular density.
The continued viability of adult neurones requires neurotropic factors to support plasticity and provide neuroprotection. A decline in production of such factors probably contributes to the age-related functional deficits that occur in the aging brain. Through its property as a potent stimulator of myelination, IGF-I should protect against the demyelinating effects of increased levels of TNF- (232). In mouse glial cultures, TNF- increases apoptosis of oligodendrocytes, whereas IGF-I acts as a neuroprotectant by stimulating the differentiation and proliferation of oligodendrocyte precursors and inducing myelin-specific protein gene expression.
Production of specific NMDA receptor subtypes in the hippocampus of rats and mice falls during aging and appears to be regulated by IGF-I (233, 234); NMDA receptors have been implicated in memory and learning (235, 236, 237). Although NMDA1 receptor expression in the hippocampus is unaffected by aging, expression of receptor subtypes NMDAR2a (NMDA receptor 2a) and NMDAR2b decrease (233). In contrast to the hippocampus, in the cortex, an age-related decline of NMDAR2a and NMDAR2b is not evident, and IGF-I treatment does not influence the concentration of either receptor subtype. The reduced expression of specific NMDA receptor subtypes in the hippocampus, which is reversible by IGF-I treatment, probably affects cognitive function. By contrast, in a study of aging rhesus monkeys (6-26 yr), the levels of NMDAR2b were unchanged in the hippocampus but reduced in the prefrontal cortex and caudate nucleus (238). Hence, we must remain cognizant of the need for caution when extrapolating data from rodent models to humans.
F. GHRH and cognition
GHRH secreted from arcuate neurons activates somatotrophs in the anterior pituitary gland to elicit GH release, and GH stimulates increased production of IGF-I. Hence, administering exogenous GHRH to old animals restores GH and IGF-I levels. Indeed, chronic administration of a GHRH analog (D-Ala2-GHRH) prevents age-dependent decline in memory in rats (239). D-Ala2-GHRH or saline was injected daily into 9-month-old rats until the rats were 30 months old. At this stage, spatial learning and reference memory were compared in the treated and control groups using the Morris water maze. The performances of the aged rats were also compared with 6-month-old rats. The results confirmed that spatial memory declined during aging and that chronic D-Ala2-GHRH treatment prevented this decline. The authors hypothesized that GH and/or IGF-I mediated the beneficial effects on memory, because the age-related decline in GH and IGF-I was preventable by chronic D-Ala2-GHRH treatment. GHRH treatment also improved mental activity, psychomotor function, behavior, and humor in elderly human subjects (240). These results suggest that orally active GHS-R ligands would also prove beneficial because they act upstream of GHRH (25).
G. GH, GHRH, and sleep
The CNS effects of GH and GHRH are believed to regulate sleep. SWS and secretion of GH decrease proportionality during aging (241). The major peak of GH release associated with sleep is markedly reduced in elderly subjects, and the amplitude of the nighttime cortisol peak increases (68, 87, 241, 242, 243). The effect of fasting on the amplitude of GH release and on sleep patterns was investigated in a small group of elderly subjects (244). GH levels were increased to levels about 50% of that in young adults, SWS was unaffected, and REM sleep was decreased (244). Therefore, although age-associated hyposomatotropism was partially restored, fasting did not induce changes in SWS.
In addition to having stimulatory effects on GH release, GHRH promotes SWS (245, 246, 247, 248). However, the beneficial effect of exogenous GHRH on sleep has been questioned because of poor reproducibility. A possible reason for the disparities might relate to the modes of GHRH administration used in different studies. The route of administration is particularly relevant if the sleep-promoting property of GHRH is by direct action on the CNS. Bolus iv injections and intranasal administration are more efficient at delivering molecules rapidly to the CNS than slow iv infusion. Indeed, bolus and intranasal delivery of GHRH increased REM and SWS in old and young human subjects, whereas slow, continuous infusion was ineffective (245, 247, 249). Because GH secretagogues like ghrelin and its synthetic mimetics stimulate the release of GHRH from hypothalamic neurons (250, 251, 252, 253, 254), improvements in sleep quality elicited by the GH secretagogue MK-0677 are likely mediated by direct stimulation of hypothalamic GHRH neurons (78).
H. Somatostatin in the CNS
Increased somatostatin tone might cause the reduced amplitude of GH release observed in aging hypothalamus. However, although expression of somatostatin mRNA is reduced in frontal cortex, parietal cortex, striatum, and hippocampus, it is unchanged in the hypothalamus (255, 256). The age-related decline in somatostatin gene expression in the frontal and parietal cortex of rats paralleled impaired memory performance in a modified Morris water maze test (257).
To further investigate the consequences of reducing somatostatin in the CNS, somatostatin was depleted by treating rats with cysteamine (258). Cysteamine-treated rats exhibited significantly impaired performance in the Morris water maze, suggesting that somatostatinergic neurotransmission is important in brain functions that include learning and memory processes (258). Somatostatin-null mice have impairments in motor learning; however, because somatostatin and its receptors are present in the developing cerebellum, such impairments might be a consequence of developmental changes (259). Like rodents, an age-related decrease in somatostatin gene expression occurs in the CNS of the macaque monkey (Macaca fuscata) (255, 256). In macaques aged from 2 to over 30 yr, somatostatin mRNA levels decreased by 60-70% in the hippocampus, frontal cortex, temporal cortex, motor cortex, somatosensory cortex, and visual cortex. Although an association between declining somatostatin and impaired memory exists, causality remains to be established.
In the rat, administration of BDNF increases somatostatin expression in the CNS (260, 261). To determine whether the age-related decrease in somatostatin mRNA correlates with changes in BDNF in aging primates, BDNF mRNA was measured in macaque monkeys of different ages (256). Two BDNF transcripts (1.6 and 4.0 kb) are produced and expression of the 1.6-kb transcript was 60% lower in the hippocampus of old macaques (>30 yr old) compared with young macaques (2 yr old); the 4.0-kb transcript was unchanged. These results suggest a potential relationship between reduced BDNF and somatostatin expression during aging of primates; however, before entertaining the possibility of causal relationships, more detailed studies are needed.
If somatostatin plays an important role in the aging process, it is possible that somatostatin receptor (sst) expression also changes as a function of age. ssts exist as six different subtypes encoded by five genes (262). Of these, subtype-2 (sst2) and subtype-5 (sst5) are primarily involved in the regulation of GH release, and both sst2 and sst5 mRNA expression in the pituitary gland decline during aging (262, 263, 264, 265, 266, 267, 268). sst2 is also abundantly expressed in the CNS (269, 270). In stress situations, compared with wild-type mice, sst2–/– mice release more ACTH, show increased anxiety, and exhibit reduced locomotor and exploratory behavior (264, 270). Hence, sst2 is apparently involved in regulation of locomotor, exploratory, and emotional reactivity (270). sst2 is also expressed in the retina, and treating a mouse model of diabetic retinopathy with the sst2 selective agonist
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Abstract
I. Introduction
