Recent studies have suggested that testosterone reduction might be a risk factor for AD in aged males (Hogervorst et al., 2001
; Moffat et al., 2004
; Rosario et al., 2004
; Rosario and Pike, 2008
). Consistent with this hypothesis, research data has shown that testosterone treatment can reduce AD neuropathology both in clinical observation (Moffat et al., 2006
) and in cell culture research (Gouras et al., 2000
). Because testosterone can be converted into estrogen by aromatase, some researchers have suggested that the effects of testosterone on preventing AD may be a secondary effect of estrogen (estrogen-dependent) and/or may be estrogen-independent (Li and Shen, 2005
). To differentiate between the estrogen-dependent and -independent mechanisms of testosterone’s association with AD neuropathology, previous studies have used nonaromatized androgen dihydrotestosterone (DHT) as estrogen-independent agent and have found that DHT treatment caused a reduction in amyloid levels in the brains of male mice that was similar to the effect of testosterone treatment (Ramsden et al., 2003
), suggesting that estrogen-independent mechanisms may be involved. However, recent studies showed that the DHT metabolite 5α
-diol can stimulate estrogen receptor subtype β
) (Lund et al., 2006
). Therefore, there is a need for a more specific model to investigate the possible estrogen-independent pathways of testosterone’s action on amyloid deposition.
Here, we used genetic knock-out of aromatase in APP transgenic mice as a model to study the estrogen-independent effect of testosterone on AD-like neuropathology and on cognition. This model was advantageous because it allowed us to study the effects of increased testosterone levels in males while avoiding a corresponding increase in estrogen generated from the aromatization of testosterone. As expected, the APP23/Ar+/− male mice showed elevated levels of endogenous testosterone and reduced levels of endogenous estrogen compared with age-matched APP23 male mice due to the depletion of aromatase. Therefore, the improvements in AD-like neuropathology and cognitive function that were exhibited in the male APP23/Ar+/− mice compared with the APP23 mice are most likely due to the estrogen-independent action of testosterone rather than due to the aromatization of testosterone into estrogen.
We predicted that if testosterone plays a role in preventing amyloid deposition independently from estrogen, we should see a decrease in amyloid levels in the APP23/Ar+/− male mice. Indeed, we found reductions not only in amyloid plaque formation but also in the Aβ42/Aβ40 ratio and in neuronal loss in the male APP23/Ar+/− mice compared with age-matched APP23 control mice, suggesting that increased endogenous testosterone limited amyloid deposition via an estrogen-independent mechanism.
One of the major enzymes responsible for Aβ production is BACE1. To examine whether testosterone plays a role in the regulation of BACE1, we detected levels of BACE1 protein from the brains of male APP23/Ar+/− and APP23 mice. Our data showed that BACE1 protein levels are reduced in APP23/Ar+/− compared with APP23 mice at the age of 18 months (), suggesting that the inhibitory action of testosterone on Aβ production involves the reduction of BACE1 protein expression. To confirm the effect of testosterone on BACE1 activity, in addition to examine the protein levels of C99 and APP c-terminal fragments (both of which result from the cleavage of APP by BACE1), we also measured BACE1 enzyme activity. We found a reduction of BACE1 enzyme activity as well as lower levels of C99 and intracellular c-terminal fragments in APP23/Ar+/− male mice compared with APP23 mice (), suggesting that BACE1 activity was reduced in the APP23/Ar+/− mice. This is the first time, to our knowledge, that testosterone has been shown to regulate Aβ levels by reducing BACE1 activity in the brain.
To further understand molecular mechanisms by which testosterone regulates BACE1 expression in APP23/Ar+/− mice, we performed RT-PCR and found that the level of BACE1 mRNA is significantly reduced in APP23/Ar+/− male mice at 18 months of age compared with that in age-matched APP23 mice (), suggesting the testosterone-induced reduction of BACE1 activity and protein expression may be mediated at the transcriptional level. To confirm the action of testosterone on BACE1 mRNA, we also transfected BACE1 cDNA into 293 cells and found that testosterone treatment alone caused a decrease in BACE1 mRNA levels (). To study the stability of mRNA, we also treated the cells with testosterone and actinomycin D and found no further reduction of BACE1 mRNA compared with cells receiving testosterone alone while actinomycin D did block retinoic acid-induced BACE1 transcription. Because we found that testosterone reduces the activity, protein expression, and mRNA abundance of BACE1, we conclude that the effect of testosterone on BACE1 regulation is more likely mediated at the transcriptional level through estrogen-independent mechanism.
To examine whether increased endogenous testosterone promotes Aβ
clearance, we examined protein levels and enzyme activities of IDE and NEP in the brains of APP23/Ar+/−
, APP23, and WT mice. Our results showed that NEP protein levels and activities were increased in APP23/Ar+/−
mice compared with APP23 control mice (), which is in line with a recent study which showed that androgen (DHT) treatment upregulates NEP protein expression in APP transfected cells (Yao et al., 2008
). The effect of testosterone on NEP protein expression might be mediated by promoting NEP transcription via stimulation of the androgen response element located on the NEP promoter (Shen et al., 2000
; Zheng et al., 2006
). Although IDE protein expression was increased in male APP23/Ar+/−
mice compared with age-matched APP23 mice (), the differences in IDE enzyme activity failed to reach significance (). Further investigation of the connection between androgen and IDE-mediated Aβ
degradation is needed.
The effect of testosterone on learning and memory has been controversial. Many studies have found that androgens improve spatial learning and memory (Flood and Roberts, 1988
; Alexander et al., 1994
; Janowsky et al., 1994
; Ishunina et al., 2002
; Raber et al., 2002
). However, other studies have reported that androgens have the opposite effect (Goudsmit et al., 1990
; Gouchie and Kimura, 1991
; Galea et al., 1995
; Hampson, 1995
; Naghdi et al., 2001
). One possible explanation for this discrepancy is that some studies only looked at serum levels of testosterone rather than the levels of testosterone in the brain. Because our APP23/Ar+/−
male mice exhibited increased testosterone levels in the brain as well as in the serum (), the effect of systemic testosterone levels versus brain-localized testosterone levels is not an issue.
To investigate the estrogen-independent effect of endogenous testosterone on cognitive function in aged male mice, we examined cognitive function in male APP23/Ar+/−
, APP23, and WT mice at various ages by hole-board testing. The hole-board test is a measurement of spatial learning and memory, both of which have been attributed to hippocampal function (Kesner et al., 1993
; Dodart et al., 2002
). At the age of 6 months, there were no differences in performance among the three genotypes (), suggesting that the increase of endogenous testosterone at young ages does not alter cognitive function in males. At the age of 18 months, however, male APP23/Ar+/−
mice showed a great improvement in spatial memory compared with APP23 and WT mice (). The 12- and 18-month old APP23/Ar+/−
mice not only found the target hole with greater accuracy (), but they also found the target hole in less time (). In contrast, APP23 mice, at 18 months old, showed a great reduction of memory performance and made more mistakes than did WT or APP23/Ar+/−
mice (). Together, these data suggest that increased endogenous testosterone, independent of estrogen, not only prevents Aβ
-induced impairment of spatial memory and cognitive function, but also may prevent deficits due to normal aging.
In summary, the genetic knock-out of aromatase in male APP23 mice leads to increased endogenous testosterone levels in the serum and brain without a corresponding increase in estrogen () and prevents the development of AD-like neuropathology as measured by the Aβ42/Aβ40 ratio, the level of amyloid plaque formation, and the extent of neuronal loss in the hippocampus (). Furthermore, the estrogen-independent mechanisms by which testosterone reduces plaque formation in the brain involves the reduction of Aβ production through attenuation of BACE1 enzyme activity via downregulation of BACE1 protein expression at transcription level () as well as the promotion of Aβ clearance through the enhancement of NEP protein expression and enzyme activity (). The APP23/Ar+/− mice also showed improved spatial memory and cognitive function compared with APP23 and WT mice (), suggesting that endogenous testosterone in males is essential in preventing age- and AD-related cognitive declines.