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Apolipoprotein E (APOE) genotype affects outcomes of Alzheimer's disease and other conditions of brain damage. Using APOE knock-in mice, we have previously shown that APOE-ε4 Targeted Replacement (TR) mice have fewer dendritic spines and reduced branching in cortical neurons. As dendritic spines are post-synaptic sites of excitatory neurotransmission, we used APOE TR mice to examine whether APOE genotype affected the various elements of the glutamate–glutamine cycle. We found that levels of glutamine synthetase and glutamate uptake transporters were unchanged among the APOE genotypes. However, compared with APOE-ε3 TR mice, APOE-ε4 TR mice had decreased glutaminase levels (18%, p < 0.05), suggesting decreased conversion of glutamine to glutamate. APOE-ε4 TR mice also had increased levels of the vesicular glutamate transporter 1 (20%, p < 0.05), suggesting that APOE genotype affects pre-synaptic terminal composition. To address whether these changes affected normal neurotransmission, we examined the production and metabolism of glutamate and glutamine at 4–5 months and 1 year. Using high-frequency 13C/1H nuclear magnetic resonance spectroscopy, we found that APOE-ε4 TR mice have decreased production of glutamate and increased levels of glutamine. These factors may contribute to the increased risk of neurodegeneration associated with APOE-ε4, and also act as surrogate markers for Alzheimer's disease risk.
Apolipoprotein E (APOE) is the strongest genetic risk factor for late-onset Alzheimer's disease (AD), and there are several hypotheses about how APOE affects AD pathogenesis [for review see (Bales et al. 2002)]. APOE also affects normal brain function independent of AD pathology, as evidenced by studies of young, non-demented humans (Reiman et al. 2004; Scarmeas et al. 2005; Filippini et al. 2009). The ε4 allele of APOE is associated with deficits in glucose metabolism in cognitively unimpaired individuals in their 50s–60s (Small et al. 1995; Reiman et al. 1996), and with alterations in cerebral activation (measured by PET or fMRI) in young adults (Reiman et al. 2004; Scarmeas et al. 2005; Filippini et al. 2009). These findings suggest that APOE's impact on brain function earlier in life could contribute to the susceptibility to damages later in life. Consistent with this idea, APOE genotype affects outcomes of other conditions of brain damage, including traumatic brain injury (Zhou et al. 2008), HIV dementia (Burt et al. 2008), and stroke (Savva and Stephan 2010). These observations support the development of a new class of therapeutics to compensate for APOE-ε4-related changes prior to overt signs of disease. However, before one could evaluate such therapies, it would be necessary to identify biomarkers that can be used in assessing therapeutic outcome.
To address this need, we and others have utilized APOE Targeted Replacement (TR) mice. These animals express the human APOE alleles (APOE-ε2, APOE-ε3, or APOE-ε4) under the mouse APOE promoter, and do not develop the plaques and tangles diagnostic of AD (Sullivan et al. 1997). Despite the lack of neuropathological changes, APOE-ε4 TR mice have simpler neuronal structures in the amygdala (Wang et al. 2005) and cortex (Dumanis et al. 2009), abnormalities in hippocampal long-term potentiation (LTP), (Trommer et al. 2005; Korwek et al. 2009), and alterations in hippocampal structure (Andrews-Zwilling et al. 2010). The APOE-ε4 animals have behavioral deficits in some Morris Water Maze tasks such as the probe test, but not the learning rates while training (Grootendorst et al. 2005; Bour et al. 2008; Andrews-Zwilling et al. 2010). Moreover, aged female APOE-ε4 TR mice display increased errors in avoidance-conditioning tasks that are not observed in younger cohorts (Grootendorst et al. 2005; Bour et al. 2008). Interestingly, a recent study has reported an emergence of a seizure phenotype in aged APOE-ε4 TR mice and abnormal cortical EEG activity (Hunter et al. 2012). These studies support the human studies described above, which suggest that APOE genotype affects brain structure and activity in the absence of overt signs of AD.
On the basis of these findings, we asked whether APOE genotype affected normal excitatory neurotransmission at the synapse by examining the elements of the glutamate–glutamine cycle in the brain. APOE-ε4 TR mice had lower levels of glutaminase and higher levels of the vesicular glutamate transporter 1 (VGLUT1) transporter. Moreover, we examined the production and metabolism of glutamate (GLU) and glutamine (GLN). Using high-frequency 13C/1H nuclear magnetic resonance (NMR), we found that incorporation of 13C label from glucose into C4 and/or C3 isotopomers of glutamate was decreased in APOE-ε4 TR mice. However, APOE-ε4 TR mice had higher levels of brain glutamine. Taken together, these data suggest that APOE genotype affects pre-synaptic terminal composition and impacts the normal GLU–GLN cycle.
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Various findings in humans and APOE knock-in mice indicate that APOE genotype affects normal brain function independent of the accumulation of AD-associated neuropathological changes. We have been able to address this hypothesis by analyzing whether there are effects of APOE genotype in knock-in mice on the metabolism of GLU, the major excitatory amino acid in the brain. We used immunoblots to measure proteins important to GLU metabolic pathways and 1H-decoupled 13C NMR to measure various GLU metabolites. We found several indications that GLU metabolism was altered in APOE-ε4 TR mouse brains, including decreased levels of glutaminase and increased levels of VGLUT1. We also found that 13C incorporation into GLU is lower in APOE-ε4 TR mice compared with APOE-ε3 and APOE-ε2 TR mice at 4–5 months and at 1 year of age. Human APOE-ε4 carriers have reduced glucose utilization (Small et al. 1995; Reiman et al. 2001; Scarmeas et al. 2005) and the APOE-ε4 knock-in mice have a more permeable blood–brain barrier (Nishitsuji et al. 2011; Bell et al. 2012), suggesting that reduced GLU levels observed via 13C NMR may be related to reduced uptake of glucose by astrocytic endfeet. However, APOE-ε4 TR mice did not have a lower 13C incorporation into succinate C2/C3 or GLN C4 (Figs 6, 5c and f). If decreased glucose uptake in APOE-ε4 TR mice were responsible for the reduced 13C incorporation into GLU C4 observed, then all measured glucose metabolites should have a reduced rate of production.
We also found that total GLN levels were over twofold higher in the APOE-ε4 TR mice compared with APOE-ε3 and APOE-ε2 TR mice; this result was consistent in two independent cohorts of animals (Fig. 4). We examined the various transporters and enzymes involved in the GLU–GLN cycle. The lower glutaminase levels in APOE-ε4 TR brains (Fig. 1) would result in reduced recycling of GLN back into GLU and thus explain the higher levels of total available GLN. The GLU–GLN cycle is a major pathway for GLU repletion in the brain (Laake et al. 1995). These findings have suggested that pre-synaptic mechanisms among the APOE genotypes may account for the observed differences in GLN levels (See model, Fig. 7). We hypothesize that apoE maybe inhibiting vesicle release either by altering pre-synaptic membrane lipid composition or altering pre-synaptic ApoE receptor signaling. Future studies will begin to address this hypothesis.
Figure 7. The effects of APOE4 on pre-synaptic markers. A model depicting the alterations in APOE-ε4 TR mice compared to APOE-ε3 TR mice. APOE-ε4 TR mice have increased total GLN (in green) and increased VGLUT1 (in green), but decreased glutaminase (red) compared with APOE-ε3 TR mice. All other aspects of the GLU–GLN cycle are unaffected (such as Glutamine Synthetase and the other Glutamate transporters). We hypothesize that apoE maybe inhibiting vesicle release either by altering synaptic lipid membrane composition or affecting signaling via a pre-synaptic ApoE receptor.
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Although the total levels of GLU in APOE-ε4 TR mice were unchanged at 1 year compared to the APOE-ε3 TR mice (Fig. 4e), the metabolic flux of 13C into GLU C4 was lower (Fig. 5), suggesting that APOE-ε4 TR mice may compensate by having a larger pool of readily available GLU. VGLUTs are transporters which use a chloride and proton gradient to shuttle cytoplasmic GLU into pre-synaptic vesicles. The total level of VGLUTs correlates with the amount of GLU stored and released (Takamori et al. 2000; Wojcik et al. 2004; Wilson et al. 2005; Daniels et al. 2006) at the pre-synaptic terminal. A recent report showed that APOE-ε4 TR mice on a control diet have higher total VGLUT1 levels compared with APOE-ε3 TR mice (Kariv-Inbal et al. 2012), consistent with our findings (Fig. 3), suggesting increased vesicular GLU in APOE-ε4 TR mice. The higher VGLUT1 in APOE-ε4 TR mice would thus compensate for the reduced GLU turnover by increasing the quantel content of GLU in synaptic vesicles (See model, Fig. 6). Although VGLUT1 is predominately expressed on synaptic vesicles, when the vesicles fuse to the plasma membrane and release GLU, VGLUT1 is also present on the plasma membrane. Plasma membrane VGLUT1 is involved in phosphate transport (Takamori 2006). High concentrations of phosphate are needed to activate glutaminase, and VGLUT1 on the plasma membrane correlates with an increase in glutaminase activity (Takamori 2006). Thus, in APOE-ε4 TR mice, higher VGLUT1 levels at the plasma membrane may be a compensatory mechanism for the observed reduction in glutaminase levels.
In contrast to the membrane fractions, we observed decreases in VGLUT1 levels in the cytoplasmic fractions of APOE-ε4 TR mice (Fig. 3e). The presence of full-length VGLUT1 in cytoplasmic extracts may reflect turnover of membrane-bound VGLUT1, although little is known about VGLUT1 metabolism (Lobo et al. 2011). Recently, it has been reported that VGLUT1 levels on synaptic vesicles can be impacted by the diurnal cycle (Yelamanchili et al. 2006; Darna et al. 2009); however, the precise mechanism regulating VGLUT1 levels are unknown. The observation of lower levels of VGLUT1 immunoreactive bands in APOE-ε4 mice may indicate that they have reduced rates of VGLUT1 turnover. The apoE4 protein has been observed to reduce recycling of endocytic vesicles (Heeren et al. 2004; Chen et al. 2010), and it may similarly impact VGLUT1 recycling in synaptic vesicles.
Here, we focused on elements of the GLU–GLN cycle, but GLU can also be derived from other sources in the brain. For example, leucine, which is transported across the blood–brain barrier, can be transaminated into GLU (Kanamori et al. 1998). It is possible that the leaky blood–brain barrier of APOE-ε4 knock-in mice may impact leucine trafficking into the brain, which subsequently could impact GLU metabolism.
Our observed alterations in GLU metabolism support earlier studies showing synaptic alterations in APOE-ε4 TR mice (Trommer et al. 2004; Wang et al. 2005; Dumanis et al. 2009; Korwek et al. 2009). They may also relate to a recent study that found seizure activity as well as abnormal EEG cortical activity in APOE-ε4 TR mice (Hunter et al. 2012). Alterations in the precise balance of excitatory and inhibitory neurotransmission could underlie the susceptibility of APOE-ε4 TR mice to seizures. This recent finding in mice correlates with another recent study, which has found that healthy human APOE-ε4 carriers have abnormal EEG activity following hyperventilation (Ponomareva et al. 2008).
Understanding the biochemical variations that correlate with APOE genotype in these mouse models could allow advancement for clinical approaches in APOE-ε4 carriers. There are reports of aberrant expression of glutaminase as well as glutamine synthetase and glutamate dehydrogenase (which converts α-ketoglutarate to glutamate) in AD brains compared with controls (Robinson 2001; Burbaeva et al. 2005). Unfortunately, in these studies, it is impossible to delineate whether these changes were present prior to onset of AD. These APOE TR mice allow us to examine such changes independent of the AD pathology.
Because of the limitations of NMR sensitivity, we were using whole brain extracts enriched with 13C label to achieve our signal, therefore the effects that we observe have been averaged across brain regions. Future studies will begin to elucidate the brain-specific region effects of APOE-ε4 TR compared to the other genotypes. Such studies would be interesting, especially with the conflicting reports of LTP abnormalities in APOE-ε4 TR mice: LTP is enhanced in the CA1 region of the hippocampus (Korwek et al. 2009) but reduced in the dentate gyrus (Trommer et al. 2004).
Here, we have defined several pre-synaptic effects of APOE genotypes. However, we and others have found that APOE genotype also affects post-synaptic measures. APOE-ε4 correlates with decreased dendritic spines in both mice (Wang et al. 2005; Dumanis et al. 2009) and humans (Ji et al. 2003). Primary neurons treated with recombinant apoE4 recombinant protein have reduced surface-level expression of glutamate receptors (AMPA and NMDA subunits) compared with control and apoE3-treated neurons (Chen et al. 2010). Future in vitro studies will address whether apoE receptors are involved in modulating the pre-synaptic (glutamate synthesis, VGLUT1 levels, glutaminase levels) and post-synaptic (GLU trafficking, dendritic spines) effects of APOE4, to begin elucidating the mechanism of these isoform-specific effects. The various in vivo measures have thus allowed us to focus future in vitro analyses on specific neurotransmission pathways and test whether the pre- and post-synaptic changes in APOE-ε4 TR mice are independent or related events.
A potential approach to manipulating apoE isoform effects is through diet. APOE-ε4 TR mice fed on a diet high in DHA content resulted in lower levels of VGLUT1, abolishing the effects of APOE-ε4 (Kariv-Inbal et al. 2012). These results indicate that not only does diet interact with APOE genotype to affect the lifecycle of GLU, but also that preventative measures may allow reduction of the differences in APOE-ε4 and APOE-ε3 TR genotypes. The various measures of proteins and metabolites from this study could act as biomarkers of AD risk. ApoE is a cholesterol transporter and essential to maintaining lipid and cholesterol homeostasis in the membrane. Although total membrane composition of cholesterol and lipids is not strikingly different in these animal models (Kariv-Inbal et al. 2012), it is quite possible that the membrane composition at the synaptic vesicle is altered. Thus, AD-preventative treatments of normal mice could be monitored using these new in vivo markers of APOE genotype.
Interestingly, APOE-ε2 TR mice are markedly different from the APOE-ε3 and APOE-ε4 TR mice, potentially relating to the decreased risk of AD associated with APOE-ε2 (Sullivan et al. 1998). We observed some opposite effects of APOE-ε2 compared with APOE-ε4 in these mice, such as higher 13C label incorporation into GLU in the APOE-ε2 TR animals (Fig. 5a, Table 1), as well as higher levels of brain GLU (Fig. 4). As in humans, APOE-ε2 is associated with type III hyperlipoproteinemia in these mice (Sullivan et al. 1998), resulting in atherosclerotic plaques and over double the cholesterol and triglyceride levels of the other APOE genotypes. The observed differences in brain GLU flux or level of 13C incorporation (E2 > E3 > E4) could be related to differences in lipid metabolism or could reflect differences in risk of neurodegeneration by APOE genotype.
In this work, using a variety of measures, we have shown that APOE genotype modulates glutamate metabolism in a mouse model displaying normal expression of human APOE alleles. In the APOE-ε4 TR mice, glutamate level was low whereas glutamine levels were high; in addition, glutaminase levels were low and VGLUT1 levels were high. These various factors may contribute to the increased risk of neurodegeneration associated with APOE-ε4, and could thus act as surrogate markers for AD risk.