Aging and gender are risk factors that are clearly associated with sporadic AD. However, whether or not these risk factors directly affect γ-secretase production of Aβ is not known. Multiple mouse models have been used to recapitulate Aβ pathology and have proved to be useful tools in understanding the pathogenesis of AD 
. We therefore utilized wild type mouse brain to examine the effect of age and gender on the activity and specificity of γ-secretase for Aβ production. First, we found an increased Aβ42
Aβ40 ratio in aging mice indicating that γ-secretase specificity for Aβ40 and Aβ42 processing varies with age. Recent studies suggest that the ratio of Aβ42
Aβ40, rather than the total levels of Aβ, is better correlated with AD pathology and subtle changes to the Aβ42
Aβ40 ratio promotes Aβ aggregation and toxicity 
. Therefore, our data which demonstrates increased Aβ42
Aβ40 ratios in aged mice may account for, in part, the increased incidence of sporadic AD seen during aging (, (a)). In addition, we found that aging also leads to an elevation of BACE1 activity, which is consistent to previous reports showing increased BACE1 activity in both aged and AD brains and further supports a role for aberrant APP processing in sporadic AD (, (b)) 
. Second, we have shown that aged female mouse brain has 65% more in vitro
γ-secretase activity at 24 months compared to male mice. Although female and male mice both have increased Aβ42
Aβ40 ratios at 24 months, the fact that female mice have elevated γ-secretase activity suggests that their overall plaque burden would be increased compared to male mice due to increased APP processing (, (c)). Furthermore, we demonstrated that there were no gender differences in BACE1 activity in aged brain, indicating that the increased in vitro
γ-secretase activity in female mice at 24 months would likely be translated into increased Aβ production in vivo
. These data establish a potential role for elevated APP γ-secretase activity in aged females as an additional causative factor in the increased prevalence of AD in women and increased plaque burden seen in female mouse models of AD.
Model of potential age and gender dependent pathogenic pathways resulting in neurodegeneration.
Photolabeling of PS1 suggests that the reduction in γ-secretase activity seen in aged males is due to either the reduced formation of active complexes or alterations to the active site architecture which reduce APP processing. However, the precise mechanism by which high levels of γ-secretase activity are maintained in aged female mouse brain remains unknown.
The decline in estrogen during menopause has been suggested to be a causative factor in the increased prevalence of AD in women. Early reports of hormone replacement therapy suggested a protective role of estrogen in the pathogenesis of AD 
. However, more recent reports have found that estrogen replacement fails to protect against the development or progression of AD and in some cases worsens disease progression 
. Estrogen has been shown to reduce APP processing in animal and cell model systems via a variety of mechanisms including increased trafficking of APP out of the trans-Golgi network and increased α-secretase mediated APP cleavage which would lead to a reduction in available APP substrate for γ-secretase processing 
. Estrogen deficiencies have been associated with increased Aβ deposition via increased BACE1 activity and decreased neprilysin/insulin-degrading enzyme activity, both of which were detected as gender specific biochemical markers associated with aggressive plaque deposition in mouse models of AD 
. It is known that changes in estrogen levels can influence the lipid and lipoprotein profiles in women 
. Furthermore, it is known that changes to the lipid microenvironment can have a large impact on γ-secretase activity 
. A cellular model of aging in normal human fibroblasts demonstrated a downregulation of γ-secretase activity due to disruptions in the lipid raft microenvironment 
. Therefore, the potential effect of aging and estrogen deficiencies on the lipid environment of brain and γ-secretase activity warrants investigation and may provide insight into the gender specific γ-secretase activity differences in aged brain we observed.
Interestingly, the gender dependant effects on γ-secretase activity appear to be specific for APP processing as both male and female mice exhibited a decline in Notch1 γ-secretase activity during aging and exhibit no gender associated differences at 24 months. Aged brain γ-secretase complexes, therefore, have gender dependent substrate processing abilities with female aged brain strongly favoring APP over Notch1 as a substrate and male brain showing no significant age related changes in its relative substrate preference. These data suggest that discrete γ-secretase complexes exist in brain which exhibit unique activity profiles, substrate processing abilities and regulation.
Notch signaling pathways are critical for cell fate decisions during development and direct proliferative, differentiation, and apoptotic responses 
. In addition to its pivotal role in neurogensis during embryonic development, Notch signaling is involved in many aspects of central nervous system development in the postnatal and adult brain including the maintenance of neuronal stem cells, neuronal proliferation, and dendritic arborization; this Notch signaling is dependent on PS expression and function 
. Importantly, it has been shown that a subset of PS FAD mutations result in the loss of γ-secretase mediated Notch1 cleavage 
. Furthermore, conditional loss of PS in adult mouse brain leads to loss of synaptic plasticity, memory impairments, and age-dependant neurodegeneration through the loss of Notch activation of CRE-dependent gene expression 
. Therefore, the age dependent reduction in Notch1 γ-secretase activity we detected in male and female brain may also contribute to the severity of sporadic AD due to enhanced neurodegeneration (, (d)).
In summary, our data implicate a role for altered γ-secretase activity during aging in the development of sporadic AD and neurodegeneration; aged mice exhibit altered specificity for Aβ40 and Aβ42 production and decreased Notch1 processing, both of which are reminiscent of certain PS FAD mutations that result in the development of early onset AD. Furthermore, our demonstration that female mice have much greater γ-secretase activity in aged brain compared to males offers an additional model to explain the increased prevalence of AD in women and aggressive plaque pathology seen in female mouse models. Increased γ-secretase activity, along with altered Aβ42 and Aβ40 specificity, in aged brain, would potentially exacerbate the plaque load in females. This model would work in concert with previously demonstrated increases in BACE1 activity and reductions in neprilysin activity in female mouse models of AD. Additionally, loss of Notch1 γ-secretase activity in aged mouse brain may also augment the pathological consequences of increased Aβ42
Aβ40 ratios and increased Aβ production by increasing neurodegeneration due to the loss of activated Notch1 mediated CRE-dependent gene expression. Therefore, our data help to clarify the roles of aging and female gender as risk factors for sporadic AD and advance our understanding of aberrant γ-secretase activity as a potential mechanism underlying the pathogenesis of sporadic AD.