Androgen cell signaling pathways involved in neuroprotective actions

As a normal consequence of aging in men, testosterone levels significantly decline in both serum and brain. Age-related testosterone depletion results in increased risk of dysfunction and disease in androgen-responsive tissues, including brain. Recent evidence indicates that one deleterious effect of age-related testosterone loss in men is increased risk for Alzheimer's disease (AD). We discuss recent findings from our laboratory and others that identify androgen actions implicated in protecting the brain against neurodegenerative diseases and begin to define androgen cell signaling pathways that underlie these protective effects. Specifically, we focus on the roles of androgens as (1) endogenous negative regulators of beta-amyloid accumulation, a key event in AD pathogenesis, and (2) neuroprotective factors that utilize rapid non-genomic signaling to inhibit neuronal apoptosis. Continued elucidation of cell signaling pathways that contribute to protective actions of androgens should facilitate the development of targeted therapeutic strategies to combat AD and other age-related neurodegenerative diseases.     

Aging, gender and APOE isotype modulate metabolism of Alzheimer's Abeta peptides and F-isoprostanes in the absence of detectable amyloid deposits

Aging and apolipoprotein E (APOE) isoform are among the most consistent risks for the development of Alzheimer's disease (AD). Metabolic factors that modulate risk have been elusive, though oxidative reactions and their by-products have been implicated in human AD and in transgenic mice with overt histological amyloidosis. We investigated the relationship between the levels of endogenous murine amyloid beta (Abeta) peptides and the levels of a marker of oxidation in mice that never develop histological amyloidosis [i.e. APOE knockout (KO) mice with or without transgenic human APOEepsilon3 or human APOEepsilon4 alleles]. Aging-, gender-, and APOE-genotype-dependent changes were observed for endogenous mouse brain Abeta40 and Abeta42 peptides. Levels of the oxidized lipid F2-isoprostane (F2-isoPs) in the brains of the same animals as those used for the Abeta analyses revealed aging- and gender-dependent changes in APOE KO and in human APOEepsilon4 transgenic KO mice. Human APOEepsilon3 transgenic KO mice did not exhibit aging- or gender-dependent increases in F2-isoPs. In general, the changes in the levels of brain F2-isoPs in mice according to age, gender, and APOE genotype mirrored the changes in brain Abeta levels, which, in turn, paralleled known trends in the risk for human AD. These data indicate that there exists an aging-dependent, APOE-genotype-sensitive rise in murine brain Abeta levels despite the apparent inability of the peptide to form histologically detectable amyloid. Human APOEepsilon3, but not human APOEepsilon4, can apparently prevent the aging-dependent rise in murine brain Abeta levels, consistent with the relative risk for AD associated with these genotypes. The fidelity of the brain Abeta/F2-isoP relationship across multiple relevant variables supports the hypothesis that oxidized lipids play a role in AD pathogenesis, as has been suggested by recent evidence that F2-isoPs can stimulate Abeta generation and aggregation.     

Development of Fetal Testicular Cells in Androgen Receptor Deficient Mice

During mammalian development, androgens produced by the fetal testis are the most important hormones controlling the masculinization of the reproductive tract and the genitalia. New findings show that the male germ line is the most sensitive to anti-androgenic endocrine disruptors during the embryonic period. In a recent study, we reported that endogenous androgens physiologically control germ cell growth in the male mouse fetus during early fetal life. In the present study, we extended this result by showing the presence of a functional androgen receptor in the gonocytes in the latter part of the fetal life. We also studied the effect of androgens on the development of the somatic testicular cells using the Tfm mice which carry a naturally inactivating mutation of the androgen receptor. Fetal Leydig cell are largely independent of endogenous androgens during fetal development whereas fetal Sertoli cell number is decreased following a default of peritubular myoid cells differenciation. They also point to the gonocyte as a special target for androgens during the embryonic period and indicate a novel mechanism of androgen action on gonocytes. Elucidation of this new pathway in the fetal testis will clarify not only fetal testis physiology but also the effects of environmental anti-androgens that act during fetal life and open new perspectives for future investigations into the sensitivity of fetal germ cell to androgens.     

Age-dependent high-density clustering of GM1 ganglioside at presynaptic neuritic terminals promotes amyloid beta-protein fibrillogenesis

The deposition of amyloid beta-protein (Abeta) is an invariable feature of Alzheimer's disease (AD); however, the biological mechanism underlying Abeta assembly into fibrils in the brain remains unclear. Here, we show that a high-density cluster of GM1 ganglioside (GM1), which was detected by the specific binding of a novel peptide (p3), appeared selectively on synaptosomes prepared from aged mouse brains. Notably, the synaptosomes bearing the high-density GM1 cluster showed extraordinary potency to induce Abeta assembly, which was suppressed by an antibody specific to GM1-bound Abeta, an endogenous seed for AD amyloid. Together with evidence that Abeta deposition starts at presynaptic terminals in the AD brain and that GM1 levels significantly increase in amyloid-positive synaptosomes prepared from the AD brain, our results suggest that the age-dependent high-density GM1 clustering at presynaptic neuritic terminals is a critical step for Abeta deposition in AD.     

AR, apoE, and cognitive function

Reduced androgen levels in aged men and women might be risk factors for age-related cognitive decline and Alzheimer's disease (AD). Ongoing clinical trials are designed to evaluate the potential benefit of estrogen in women and of testosterone in men. In this review, we discuss the potential beneficial effects of androgens and androgen receptors (ARs) in males and females. In addition, we discuss the hypothesis that AR interacts with apolipoprotein (apoE)4, encoded by epsilon4 and a risk factor for age-related cognitive decline and AD, and the potential consequences of this interaction.     

Sex Steroid Receptors in the Perinatal Rat Brain

Many sex differences in the regulation of reproductive functionin rats are the result of a differentiation of the brain whichoccurs neonatally. Although injections of either androgens orestrogens are capable during the neonatal period of alteringhypothalamic systems involved in reproductive behavior and gonadotropinregulation, the physiological role of each type of hormone hasnot been clearly established. In both sexes, circulating estrogensare normally kept from interacting with estrogen receptors inthe limbic brain by the high levels of alpha-fetoprotein inthe blood. The local aromatization of androgens in the braincould circumvent alpha-fetoprotein, since androgens do not bindto this serum protein. The "paromatization hypothesis" statesthat testosterone, which is abundant in neonatal male circulationbut absent in females, is locally converted to estradiol inthe limbic brain. There it binds to estrogen receptor proteinsto produce tissue differentiation. The ontogeny of estradiolbinding proteins in limbic areas is consistent with the aromatizationhypothesis, with a rapid increase in receptor levels occurringshortly after birth. Also, the presence of endogenous estradiol-receptorcomplexes has been demonstrated in the cell nuclei of male neonatesbut not female neonales. Furthermore, the presence of estradiolbinding proteins in other regions of the neonatal male and femalebrain suggests an additional role of estradiol. unknown as ofyet. Several studies with agents which block the aromatizationof androgens to estrogens or the binding of estrogens to theirreceptors are consistent with the aromatization hypothesis,since these agents prevent the differentiation of intact neonatalmales. However, specific androgen binding proteins are alsopresent in neonatal brains, and androgen-receptor complexescan be found in cell nuclei of neonates after an injection oftritiated androgen. The possible involvement of these receptorsin sexual differentiation of the brain is suggested by the findingthat an antiandrogen inhibits both the binding of androgens(but not estrogens) and the differentiation of males.     

Oxidized neprilysin in aging and Alzheimer's disease brains

Deposition of amyloid in the brain is important in the pathogenesis of Alzheimer's disease (AD), but it remains to be determined if deposition is due to increased production or decreased clearance of fibrillogenic forms of beta-amyloid (Abeta). Except for rare genetic forms of AD, there is little evidence for increased production of Abeta, but decreases in enzymes involved in the clearance of Abeta are increasingly being investigated. Neprilysin (NEP) is a major enzyme for degradation of Abeta and changes in amount or activity of NEP may play a role in Abeta deposition in AD. Since oxidative damage to proteins, including formation of adducts such as 4-hydroxynonenal (HNE), has been reported in AD, it was of interest to determine if NEP might be susceptible to oxidative modification. To address this question, monoclonal antibody immunoprecipitates of NEP were probed with polyclonal antibodies to NEP and HNE. The results showed decreased NEP in AD compared to normal controls. NEP in both AD and controls had HNE-modification and the ratio of oxidized to total NEP was greater in AD than in controls. These findings suggest that decreased NEP may contribute to Abeta deposition in AD and that age-related oxidative damage to NEP may play a role in age-related cerebral amyloidosis that is exacerbated in AD.     

Amyloid beta-peptide induces cholinergic dysfunction and cognitive deficits: a minireview

Amyloid beta-peptide (Abeta) plays a critical role in the development of Alzheimer's disease (AD). Much progress has been made in understanding this age-related neurodegenerative disorder, thus an insight into the cellular actions of Abeta and resulting functional consequences may contribute to preventive and therapeutic approaches for AD. In this review, recent evidence of Abeta-induced brain dysfunction, particularly of cholinergic impairment and memory deficits is summarized. Moreover, proposed mechanisms for Abeta-induced neurotoxicity such as oxidative stress, ion-channel formation, and Abeta-receptor interaction are discussed.     

Proteomic changes in rat spermatogenesis in response to in vivo androgen manipulation; impact on meiotic cells

The production of mature sperm is reliant on androgen action within the testis, and it is well established that androgens act on receptors within the somatic Sertoli cells to stimulate male germ cell development. Mice lacking Sertoli cell androgen receptors (AR) show late meiotic germ cell arrest, suggesting Sertoli cells transduce the androgenic stimulus co-ordinating this essential step in spermatogenesis. This study aimed to identify germ cell proteins responsive to changes in testicular androgen levels and thereby elucidate mechanisms by which androgens regulate meiosis. Testicular androgen levels were suppressed for 9 weeks using testosterone and estradiol-filled silastic implants, followed by a short period of either further androgen suppression (via an AR antagonist) or the restoration of intratesticular testosterone levels. Comparative proteomics were performed on protein extracts from enriched meiotic cell preparations from adult rats undergoing androgen deprivation and replacement in vivo. Loss of androgenic stimulus caused changes in proteins with known roles in meiosis (including Nasp and Hsp70-2), apoptosis (including Diablo), cell signalling (including 14-3-3 isoforms), oxidative stress, DNA repair, and RNA processing. Immunostaining for oxidised DNA adducts confirmed spermatocytes undergo oxidative stress-induced DNA damage during androgen suppression. An increase in PCNA and an associated ubiquitin-conjugating enzyme (Ubc13) suggested a role for PCNA-mediated regulation of DNA repair pathways in spermatocytes. Changes in cytoplasmic SUMO1 localisation in spermatocytes were paralleled by changes in the levels of free SUMO1 and of a subunit of its activating complex, suggesting sumoylation in spermatocytes is modified by androgen action on Sertoli cells. We conclude that Sertoli cells, in response to androgens, modulate protein translation and post-translational events in spermatocytes that impact on their metabolism, survival, and completion of meiosis.     

Cysteine protease inhibitors effectively reduce in vivo levels of brain beta-amyloid related to Alzheimer's disease

Abnormal accumulation of neurotoxic beta-amyloid peptides (Abeta) in brain represents a key factor in the progression of Alzheimer's disease (AD). Identification of small molecules that effectively reduce brain levels of Abeta is important for development of Abeta-lowering agents for AD. In this study, we demonstrate that in vivo Abeta levels in brain are significantly reduced by the cysteine protease inhibitor E64d and the related CA074Me inhibitor, which inhibits cathepsin B. Direct infusion of these inhibitors into brains of guinea pigs resulted in reduced levels of Abeta by 50-70% after 30 days of treatment. Substantial decreases in Abeta also occurred after only 7 days of inhibitor infusion, with a reduction in both Abeta40 and Abeta42 peptide forms. A prominent decrease in Abeta peptides was observed in brain synaptosomal nerve terminal preparations after CA074Me treatment. Analyses of APP-derived proteolytic fragments showed that CA074Me reduced brain levels of the CTFbeta fragment, and increased amounts of the sAPPalpha fragment. These results suggest that CA074Me inhibits Abeta production by modulating APP processing. Animals appeared healthy after treatment with these inhibitors. These results, showing highly effective in vivo decreases in brain Abeta levels by these cysteine protease inhibitors, indicate the feasibility of using related compounds for lowering Abeta in AD.     

Alzheimer's disease beta-amyloid peptide is increased in mice deficient in endothelin-converting enzyme

The abnormal accumulation of beta-amyloid (Abeta) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Abeta. As with any peptide, however, the degree of Abeta accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models we have previously characterized endothelin-converting enzyme-1 (ECE-1) as an Abeta-degrading enzyme that appears to act intracellularly, thus limiting the amount of Abeta available for secretion. To determine the physiological significance of this activity, we analyzed Abeta levels in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Abeta40 and Abeta42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Abeta levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Abeta accumulation in the brain and also provides further insight into the factors involved in Abeta clearance in vivo.     

Neuroendocrine consequences of androgen excess in female rodents

Androgens exert significant organizational and activational effects on the nervous system and behavior. Despite the fact that female mammals generally produce low levels of androgens, relative to the male of the same species, increasing evidence suggests that androgens can exert profound effects on the normal physiology and behavior of females during fetal, neonatal, and adult stages of life. This review examines the effects of exposure to androgens at three stages of development--as an adult, during early postnatal life and as a fetus, on reproductive hormone secretions in female rats. We examine the effects of androgen exposure both as a model of neuroendocrine sexual differentiation and with respect to the role androgens play in the normal female. We then discuss the hypothesis that androgens may cause epigenetic modification of estrogen target genes in the brain. Finally we consider the clinical consequences of excess androgen exposure in women.     

The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease

Amyloid beta-peptide (Abeta) accumulation in specific brain regions is a pathological hallmark of Alzheimer's disease (AD). We have previously reported that a well-characterized acyl-coenzyme A: cholesterol acyltransferase (ACAT) inhibitor, CP-113,818, inhibits Abeta production in cell-based experiments. Here, we assessed the efficacy of CP-113,818 in reducing AD-like pathology in the brains of transgenic mice expressing human APP(751) containing the London (V717I) and Swedish (K670M/N671L) mutations. Two months of treatment with CP-113,818 reduced the accumulation of amyloid plaques by 88%-99% and membrane/insoluble Abeta levels by 83%-96%, while also decreasing brain cholesteryl-esters by 86%. Additionally, soluble Abeta(42) was reduced by 34% in brain homogenates. Spatial learning was slightly improved and correlated with decreased Abeta levels. In nontransgenic littermates, CP-113,818 also reduced ectodomain shedding of endogenous APP in the brain. Our results suggest that ACAT inhibition may be effective in the prevention and treatment of AD by inhibiting generation of the Abeta peptide.     

The molecular chaperone alphaB-crystallin enhances amyloid beta neurotoxicity.

Amyloid beta (Abeta) is a 40- to 42-residue peptide that is implicated in the pathogenesis of Alzheimer's Disease (AD). As a result of conformational changes, Abeta assembles into neurotoxic fibrils deposited as 'plaques' in the diseased brain. In AD brains, the small heat shock proteins (sHsps) alphaB-crystallin and Hsp27 occur at increased levels and colocalize with these plaques. In vitro, sHsps act as molecular chaperones that recognize unfolding peptides and prevent their aggregation. The presence of sHsps in AD brains may thus reflect an attempt to prevent amyloid fibril formation and toxicity. Here we report that alphaB-crystallin does indeed prevent in vitro fibril formation of Abeta(1-40). However, rather than protecting cultured neurons against Abeta(1-40) toxicity, alphaB-crystallin actually increases the toxic effect. This indicates that the interaction of alphaB-crystallin with conformationally altering Abeta(1-40) may keep the latter in a nonfibrillar, yet highly toxic form.     

Suppression of Abeta deposition in brain by peripheral administration of Fab fragments of anti-seed antibody

Assembly and deposition of amyloid beta-protein (Abeta) in the brain is a fundamental process of Alzheimer's disease (AD). We previously hypothesized that GM1 ganglioside-bound Abeta (GAbeta) is an endogenous seed for Abeta assembly in brain. Recently, we have succeeded in generation of a monoclonal antibody specific to GAbeta. Notably, this antibody, 4396C, per se substantially inhibits Abeta assembly in vitro. Here we report that the peripheral administration of Fab fragments of 4396C into transgenic mice expressing a mutant amyloid precursor protein gene, following the conjugation of the protein transduction domain of the Tat protein, markedly suppressed Abeta deposition in the brain. This result further supports our previous hypothesis and also provides a new insight into develop AD therapy through targeting seed Abeta in the brain, which selectively inhibits the initial step of the pathological process of AD.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain.

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad-intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad-intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen-responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor-immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28+/-0.3 units) than in males (2.21+/-0.2 units; p < .05). These sex differences in the number and staining intensity of androgen-responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens.     

The effects of ABCA1 on cholesterol efflux and Abeta levels in vitro and in vivo

ABCA1 promotes cholesterol efflux from cells and is required for maintaining plasma cholesterol levels. Cholesterol homeostasis is important in the production of beta-amyloid (Abeta), a peptide that is overproduced in Alzheimer's disease (AD). Overexpression of ABCA1 can be achieved by stimulating Liver X Receptors (LXR), and changes in Abeta have been reported after LXR stimulation in vitro. To determine whether ABCA1 could alter endogenous Abeta levels, we used two different in vivo systems. We first examined the effects of an LXR agonist (TO-901317) on wild-type mice and found an increase in brain ABCA1 and apoE levels, which caused an increase in plasma cholesterol. This was accompanied by a decrease in brain Abeta levels. We then examined endogenous Abeta levels in ABCA1 knockout mice and found that, despite having no ABCA1, lowered brain apoE levels, and lowered plasma cholesterol, there was no change in Abeta levels. To assess these in vivo models in an in vitro system, we designed a model in which cholesterol transport via ABCA1 (or related transporters) was prevented. Switching off cholesterol efflux, even in the presence of TO-901317, caused no change in Abeta levels. However, when efflux capability was restored, TO-901317 reduced Abeta levels. These data show that promoting cholesterol efflux is a viable target for Abeta reducing strategies; however, knockout of cholesterol transporters is not sufficient to alter Abeta in vitro or in vivo.     

Acetyl-l-Carnitine Protects Against Amyloid-Beta Neurotoxicity: Roles of Oxidative Buffering and ATP Levels

Acetyl-l-carnitine (ALCAR), normally produced in mitochondria, is a precursor of acetyl-CoA in the tricarboxylic (TCA) cycle. Since mitochondrial compromise and ATP depletion have been considered to play a role in neuronal degeneration in Alzheimer's disease (AD), we examined whether ALCAR attenuated oxidative stress and/or ATP depletion after exposure of cells to beta-amyloid (Abeta), a neurotoxic peptide that accumulates in AD brain. Differentiated SH-SY-5Y human neuroblastoma cells were exposed for 2–24 h to 20 M Abeta in the presence and absence of 50 M ALCAR. ALCAR attenuated oxidative stress and cell death induced by Abeta neurotoxicity. Abeta depleted ATP levels, suggesting Abeta may induce neurotoxicity in part by compromising neuronal energy. ALCAR prevented ATP depletion; therefore, ALCAR may mediate its protective effect by buffering oxidative stress and maintaining ATP levels.     

Oxidative damage in brain from human mutant APP/PS-1 double knock-in mice as a function of age

Oxidative stress is strongly implicated in the progressive decline of cognition associated with aging and neurodegenerative disorders. In the brain, free radical-mediated oxidative stress plays a critical role in the age-related decline of cellular function as a result of the oxidation of proteins, lipids, and nucleic acids. A number of studies indicate that an increase in protein oxidation and lipid peroxidation is associated with age-related neurodegenerative diseases and cellular dysfunction observed in aging brains. Oxidative stress is one of the important factors contributing to Alzheimer's disease (AD), one of whose major hallmarks includes brain depositions of amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP). Mutation in APP and PS-1 genes, which increases production of the highly amyloidogenic amyloid beta-peptide (Abeta42), is the major cause of familial AD. In the present study, protein oxidation and lipid peroxidation in the brain from knock-in mice expressing human mutant APP and PS-1 were compared with brain from wild type, as a function of age. The results suggest that there is an increased oxidative stress in the brain of wild-type mice as a function of age. In APP/PS-1 mouse brain, there is a basal increase (at 1 month) in oxidative stress compared to the wild type (1 month), as measured by protein oxidation and lipid peroxidation. In addition, age-related elevation of oxidative damage was observed in APP/PS-1 mice brain compared to that of wild-type mice brain. These results are discussed with reference to the importance of Abeta42-associated oxidative stress in the pathogenesis of AD.     

Anti-amyloid activity of neprilysin in plaque-bearing mouse models of Alzheimer's disease

Abnormally high concentrations of beta-amyloid peptide (Abeta) and amyloid plaque formation in Alzheimer's disease (AD) may be caused either by increased generation or by decreased degradation of Abeta. Therefore, activation of mechanisms that lower brain Abeta levels is considered valuable for AD therapy. Neuronal upregulation of neprilysin (NEP) in young transgenic mice expressing the AD-causing amyloid precursor protein mutations (SwAPP) led to reduction of brain Abeta levels and delayed Abeta plaque deposition. In contrast, a comparable increase of brain NEP levels in aged SwAPP mice with pre-existing plaque pathology did not result in a significant reduction of plaque pathology. Therefore, we suggest that the potential of NEP for AD therapy is age-dependent and most effective early in the course of AD pathophysiology.