Deposition of extracellular amyloid plaques and intracellular neurofibrillary tangles in the central nervous system are hallmarks of Alzheimer's disease (AD). Other components of the disease are inflammation, neuronal loss, and progressive cognitive decline. Furthermore, vascular abnormalities and changes in blood flow are also a feature of AD (de la Torre 1997; Faraci 2011). Indeed, it has been demonstrated that endothelial dysfunction is an early event in AD transgenic mouse models and this precedes overt plaque development (Iadecola et al. 1999; Niwa et al. 2002). The cause of sporadic AD, which accounts for approximately 95% of AD cases, is unknown. Several risk factors have been identified. Of these risk factors, aging is the single greatest risk factor for the development of AD (Kukull et al. 2002). Cardiovascular risk factors are also associated with an increased incidence of AD. Importantly, one commonality between aging and cardiovascular risk factors is the decreased bioavailability of endothelial nitric oxide (NO) (Dudzinski et al. 2006).
Amyloid beta (Aβ) is the primary component of the extracellular plaques. Aβ is generated by the sequential cleavages of amyloid precursor protein (APP) by β-site APP cleaving enzyme (BACE) 1 and γ-secretase. We recently demonstrated that loss of endothelial NO led to increased expression of APP and BACE1 protein and Aβ levels in brain tissue suggesting that endothelial NO may play a role in the modulation of neuronal APP expression and amyloidogenic processing (Austin et al. 2010, 2012). We also demonstrated that supplementation of NO in endothelial nitric oxide synthase deficient (eNOS−/−) mice via nitroglycerin treatment was able to attenuate the up-regulation of APP and BACE1 protein levels in the cerebral microvasculature (Austin et al. 2012). Furthermore, Pak and colleagues reported that NO down-regulated BACE1 in human neuroblastoma cells (Pak et al. 2005). Lastly, Kwak et al. demonstrated that treatment of cultured neurons with low concentrations of NO suppressed BACE1 levels (Kwak et al. 2011). These data support the hypothesis that endothelial NO modulates Aβ levels in the brain.
In this study, we sought to determine the effect of chronic loss of endothelial NO on several AD related pathologies using late middle aged (LMA) (14–15 month old) eNOS−/− mice. Our studies provide evidence that chronic loss of endothelial NO in LMA animals leads to increased APP expression and amyloidogenic processing, increased microglial activation, and impaired performance in a radial arm maze spatial memory test.
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- Materials and methods
- Supporting Information
Our results support the hypothesis that endothelial dysfunction, and specifically loss of endothelial NO, may be an important contributor to the pathogenesis of sporadic AD. We previously demonstrated increased amyloidogenic processing of APP in young eNOS−/− mice as compared to age matched wild-type mice (Austin et al. 2010, 2012). Here, we confirmed that APP and BACE1 expression and production of Aβ were significantly higher in LMA eNOS−/− brains as compared with LMA wild-type animals. However, we also report several important and novel observations in LMA eNOS−/− mice. First, we believe this is the first report of inflammatory changes found in the brains of LMA eNOS−/− animals under basal conditions. Several microglial markers (CD68, Iba-1, and MHC II) were significantly higher in the brains of LMA eNOS−/− mice and this was accompanied by higher levels of the cytokines: GM-CSF, IL-1α, and MIP-1β, as compared with LMA wild-type mice. Second, we report significantly increased APP and Aβ1-40 levels in the hippocampus, a region critically involved in the pathogenesis of AD. Third, we report behavioral alterations in LMA eNOS−/− mice as compared with LMA wild-type mice.
Several studies have suggested an association between NO and the development of AD; however, the literature is controversial. Kummer et al. demonstrated that treating APP/PS1 mice, a transgenic model of AD, with an iNOS inhibitor decreased Aβ deposition and cognitive dysfunction by limiting tyrosine nitrosylation of Aβ (Kummer et al. 2011). Furthermore, several groups examined the role of iNOS in the pathogenesis of AD by utilizing AD mouse models which also lacked iNOS (Nathan et al. 2005; Colton et al. 2008; Kummer et al. 2011). Nathan et al. and Kummer et al. reported that genetic inactivation of iNOS protected AD mice from plaque formation, Aβ accumulation and microgliosis (Nathan et al. 2005) as well as memory deficits (Kummer et al. 2011). In contrast, Colton and colleagues reported that loss of iNOS activity worsened all symptoms in their AD mouse model (Colton et al. 2008). Similarly, several studies also report on nNOS and AD with conflicting findings (Simic et al. 2000; Lahiri et al. 2003; Martin et al. 2006). Simic et al. demonstrated increased nNOS in the hippocampus and entorhinal cortex in areas around plaques and overt cell loss in AD patients as compared to age-matched controls (Simic et al. 2000). In addition, Martin et al. reported increased nNOS activity in Tg2576 mice, a murine model of AD (Martin et al. 2006). Conversely, Lahiri et al. reported no statistical difference in nNOS activity in APP/PS1 transgenic mice as compared with control mice (Lahiri et al. 2003). While numerous studies have examined the role of iNOS and nNOS in relation to AD a systematic examination of the role of eNOS in the pathogenesis of AD is lacking. In this study the rationale for focusing on eNOS was based on the established differences among the three isoforms of NOS in terms of biochemical and functional properties.
In our studies, we have utilized a murine model deficient in eNOS to help determine the effects of a chronic loss of endothelial NO in AD-related pathologies, including APP expression and processing, Aβ generation, inflammation, and cognitive function. Notably, this is not a traditional mouse model for Alzheimer's disease, in that eNOS−/− mice express unaltered wild-type murine APP and secretase enzymes; however, it allows for the measurements of physiological alterations of APP and Aβ in response to a loss of endothelial NO. It is also important to note that, murine Aβ behaves much differently than human Aβ. Most importantly, murine Aβ does not form fibrils and therefore an elevated concentration of murine Aβ does not result in the formation of plaques (Dyrks et al. 1993). Future studies will include examination of AD mouse models that lack eNOS to determine the role of endothelial NO in the progression of AD.
Because of their close proximity, endothelial NO appears to be an important signaling molecule, between the vascular wall and neurons (Garthwaite et al. 2006; Hopper and Garthwaite 2006). Consistent with our previous findings in young eNOS−/− mice, we demonstrated that loss of endothelial NO led to increased expression of APP and BACE1 protein in brain tissue of LMA eNOS−/− mice (Austin et al. 2010, 2012). In line with our observations, Gutsaeva and colleagues detected changes in the expression of mitochondrial proteins within neurons in eNOS−/− mice demonstrating an important role of endothelial NO in control of protein expression in neuronal tissue (Gutsaeva et al. 2008). However, we have yet to determine the mechanism by which NO is able to suppress APP and BACE1 expression. Of note, the APP promoter contains a stimulating protein (Sp)1 site and the BACE1 promoter and 5′ untranslated region contains binding sites for cAMP response element binding protein (CREB), nuclear factor κB (NFκB), Sp1, and yin yang (YY)1 (Hoffman and Chernak 1995; Sambamurti et al. 2004) which can be modified by nitric oxide (Peng et al. 1995; Lu et al. 1999; Berendji-Grun et al. 2001; Hongo et al. 2005).
As previously reported, we detected significant changes in blood pressure, lipid profile, and glucose levels in LMA eNOS−/− mice as compared with LMA wild-type controls (Huang 2009). These metabolic changes could contribute to the phenotype of the LMA eNOS−/− mice we report here; however, it is important to note that we previously reported that inhibition or loss of eNOS derived NO led to increased APP, BACE1, and Aβ levels in vitro in the absence of hemodynamic forces or alterations in metabolic factors (Austin et al. 2010). Furthermore, we reported increased brain APP, BACE1, and Aβ in young eNOS−/− which did not display significant differences in their metabolic profile as compared with young wild-type mice (Austin et al. 2010, 2012).
To the best of our knowledge, this is the first report of an altered inflammatory response, specifically an increase in the microglial markers CD68, Iba-1, and MHC II in the brains of LMA eNOS−/− mice. As the immunocompetent cell of the central nervous system, microglia are constantly contacting neurons, other glia and the cells of the vasculature to survey and sample the environment (Nimmerjahn et al. 2005). Thus, it appears that microglia could detect alterations in the production of endothelial NO although it is unclear whether the increased microglial activation reported here is because of loss of NO per se. We observed increased GM-CSF, IL-1α, and MIP-1β, in the brains of LMA eNOS−/− mice as compared with wild-type mice. As of yet, we do not know the source or the consequence of the increased GM-CSF, IL-1α, and MIP-1β we observed in the brains of eNOS−/− mice and this will require further investigation.
In the hippocampus, we observed an increase in Aβ1-40 levels while Aβ1-42 levels were unchanged. The mechanism or consequence of this preferential generation of Aβ1-40 remains to be determined. Numerous studies have demonstrated that administration of either Aβ1-40 or Aβ1-42 in rodents leads to a number of deleterious effects, such as: LTP dysfunction, neuronal loss, cellular stress, inflammation, and cognitive deficits [reviewed in (Chambon et al. 2011)]. Furthermore, it has been demonstrated that in vitro treatment of hippocampal neuronal cultures with Aβ1-40 and Aβ1-42 is highly neurotoxic; however, Aβ1-42 treatment exhibited significantly higher levels of toxicity (Dore et al. 1997). Other studies suggest that Aβ1-40 may be neuroprotective or at least require seeding of Aβ1-42 to produce toxicity (Zou et al. 2002, 2003; Vasilevko et al. 2010; Abramowski et al. 2012; Viet and Li 2012). Lastly, numerous studies suggest that both the concentration and conformation of Aβ peptides determines the neurotrophic or neurotoxic effects of the Aβ peptide (Yankner et al. 1990; Busciglio et al. 1992; Hoshi et al. 2003).
The hippocampal region is one of the earliest and most greatly altered regions of the brain affected by AD (Hyman et al. 1984). The hippocampus in humans plays a prominent role in the formation of new memories or newly learned information and thus early symptoms of AD include difficulty remembering recent events or newly acquired information (Hyman et al. 1984). In rodents, the hippocampal region is responsible for spatial learning and memory (O'Keefe and Dostrovsky 1971). Therefore, we used the 8-arm radial arm maze to assess the spatial memory and learning abilities of LMA eNOS−/− and wild type mice (Dubreuil et al. 2003). We report a spatial memory task deficit in LMA eNOS−/− animals as these mice made more incorrect runs as compared with LMA wild-type mice in the radial arm maze. Differences were also observed in the number of revisiting errors. At first, LMA eNOS−/− mice appeared to perform better than the LMA wild-type mice as eNOS−/− mice made less revisiting errors on the first day of the test. However, by day 5, LMA eNOS−/− mice were actually committing more revisiting errors than their wild-type counterparts. These data illustrate that the LMA eNOS−/− mice have a deficit in spatial learning. It is unclear exactly why LMA eNOS−/− mice initially made less revisiting errors than wild-type mice in the radial arm maze experiment; however, previous behavioral studies on young wild-type and eNOS−/− mice have reported conflicting findings. Dere et al. reported no change in performance of young eNOS−/− and wild type in the radial arm maze while Frisch and colleagues reported superior performance by eNOS−/− mice in a water maze as compared to wild type (Frisch et al. 2000; Dere et al. 2001). One possible explanation is that while both the water maze and the radial arm maze are testing spatial memory, the water maze is a negative reinforcement test while the radial arm maze is a positive reinforcement test.
We did not observe any differences in locomotor activity between LMA eNOS−/− and LMA wild-type control mice; however, some groups have reported decreased activity in the open field test by eNOS−/− mice as compared with wild-type control animals at both a young and old age (Frisch et al. 2000; Dere et al. 2002). We only examined LMA eNOS−/− and LMA wild-type mice in this study. While we do not have an exact explanation for the difference there are several considerations. Dere et al. examined locomotor activity of eNOS−/− and wild-type mice that were 18–22 months old whereas our studies were performed on mice 14–15 months old (Dere et al. 2002). Importantly, it has been published that age-dependent cardiac abnormalities, including: perturbations in left ventricular function, decreased ejection fraction, and septum thickening, occur in 18 month and older eNOS−/− mice (Li et al. 2004). These changes in cardiac phenotype could lead to altered locomotor activity. We also wish to point out that the eNOS−/− mice used in the above mentioned studies were generated by disrupting the NADPH binding site (exons 24 and 25) whereas the eNOS−/− mice we used were generated by disrupting the calmodulin binding domain (exon 12) (Godecke et al. 1998; Frisch et al. 2000; Dere et al. 2001, 2002).
The exact cause of the spatial memory deficit observed in our study remains to be elucidated; however, several reports demonstrate the importance of the NO pathway in the formation of LTP and hippocampal-derived cognitive function (Arancio et al. 1996; Lu et al. 1999). Indeed, several groups have suggested modulation of the NO/cGMP to be used as a therapeutic target for the treatment of cognitive dysfunction in AD (Thatcher et al. 2004; Puzzo et al. 2009; Reneerkens et al. 2009; Domek-Lopacinska and Strosznajder 2010). Further studies will be needed to determine the exact changes in the brain causing the decreased performance by the LMA eNOS−/− mice in this particular maze.
In summary, our studies demonstrate that NO produced by the vasculature modulates APP expression and processing in brain tissue of LMA mice. The present studies also report several novel observations regarding the chronic loss of endothelial derived NO and microglia and cognitive performance. Taken together, our studies suggest that preservation of endothelial NO production and biological signaling are important potential therapeutic targets in the treatment of MCI and AD.