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, 53 (3), 1015-31

Human ApoE ɛ2 Promotes Regulatory Mechanisms of Bioenergetic and Synaptic Function in Female Brain: A Focus on V-type H+-ATPase

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Human ApoE ɛ2 Promotes Regulatory Mechanisms of Bioenergetic and Synaptic Function in Female Brain: A Focus on V-type H+-ATPase

Sarah K Woody et al. J Alzheimers Dis.

Abstract

Humans possess three major isoforms of the apolipoprotein E (ApoE) gene encoded by three alleles: ApoE ɛ2 (ApoE2), ApoE ɛ3 (ApoE3), and ApoE ɛ4 (ApoE4). It is established that the three ApoE isoforms confer differential susceptibility to Alzheimer's disease (AD); however, an in-depth molecular understanding of the underlying mechanisms is currently unavailable. In this study, we examined the cortical proteome differences among the three ApoE isoforms using 6-month-old female, human ApoE2, ApoE3, and ApoE4 gene-targeted replacement mice and two-dimensional proteomic analyses. The results reveal that the three ApoE brains differ primarily in two areas: cellular bioenergetics and synaptic transmission. Of particular significance, we show for the first time that the three ApoE brains differentially express a key component of the catalytic domain of the V-type H+-ATPase (Atp6v), a proton pump that mediates the concentration of neurotransmitters into synaptic vesicles and thus is crucial in synaptic transmission. Specifically, our data demonstrate that ApoE2 brain exhibits significantly higher levels of the B subunit of Atp6v (Atp6v1B2) when compared to both ApoE3 and ApoE4 brains, with ApoE4 brain exhibiting the lowest expression. Our additional analyses show that Atp6v1B2 is significantly impacted by aging and AD pathology and the data suggest that Atp6v1B2 deficiency could be involved in the progressive loss of synaptic integrity during early development of AD. Collectively, our findings indicate that human ApoE isoforms differentially modulate regulatory mechanisms of bioenergetic and synaptic function in female brain. A more efficient and robust status in both areas-in which Atp6v may play a role-could serve as a potential mechanism contributing to the neuroprotective and cognition-favoring properties associated with the ApoE2 genotype.

Keywords: Alzheimer’s disease; ApoE2; ApoE3; ApoE4; V-type H+-ATPase; cellular bioenergetics; synaptic transmission.

Figures

Figure 1
Figure 1. ApoE2 brain expresses significantly higher levels of Atp6v1B2
(A) A representative 2D gel image of protein spots derived from 6-month-old female hApoE2-TR mouse cortex. Selected proteins are circled on the gel image and protein identification number corresponding to Table 1 is indicated adjacent to protein spot. (B) Proteomic analysis of 150 μg of cortical protein demonstrates that the expression of Atp6v1B2 is significantly higher in ApoE2 brain when compared to both ApoE3 and ApoE4 brains. The data are shown as a vertical point plot with the mean value indicated by a horizontal line; n=3. (C) Immunoblot analysis of cortical tissues harvested from 6-month-old female hApoE2-TR, hApoE3-TR, and hApoE4-TR mice confirmed the proteomic results. The data are shown as a box plot with the bottom bar representing the lowest data point, the middle bar representing the median, and the upper bar representing the highest data point; n=5. *p<0.05 when compared to ApoE2 brain.
Figure 2
Figure 2. Aging and AD pathology alter the expression of Atp6v1B2 in female brain
Immunoblot analysis was performed on cortical protein samples derived from female (A) 129/C57BL/6 and (B) 3xTg-AD mice at ages of 6, 9, 12, and 15 months. n=4–5; *p<0.05. (C) Immunoblot analysis of Atp6v1B2 expression in cortical protein samples derived from age-matched female 129/C57BL/6 and 3xTg-AD mice. n=4–5; *p<0.05. (D) Campbell-Switzer staining of mouse brain hemispherical sections harvested from 9- and 12-month-old 3xTg-AD mice. Campbell-Switzer staining detects the presence of Aβ plaques shown in black.
Figure 3
Figure 3. Atp6v1B2 is localized to the vesicular membrane of synaptic vesicles
(A–C) Primary hippocampal neurons were fixed on day 15 and stained for tau (green), synaptophysin (blue) and Atp6v1B2 (red) immunoreactivity. Images were visualized using 60× oil immersion confocal microscopy and acquired in three-dimensional stacks with 0.10 μm planes. (D–F) A schematic representation of Atp6v on the membrane of synaptic vesicles in presynaptic (axonal) nerve terminals. The 8-subunit V1 complex generates energy via ATP hydrolysis which allows the 5-subunit V0 complex to transport protons across the vesicular membrane. Simultaneously, the neurotransmitter transporters utilize this electrochemical gradient to concentrate neurotransmitters into the synaptic vesicles.
Figure 3
Figure 3. Atp6v1B2 is localized to the vesicular membrane of synaptic vesicles
(A–C) Primary hippocampal neurons were fixed on day 15 and stained for tau (green), synaptophysin (blue) and Atp6v1B2 (red) immunoreactivity. Images were visualized using 60× oil immersion confocal microscopy and acquired in three-dimensional stacks with 0.10 μm planes. (D–F) A schematic representation of Atp6v on the membrane of synaptic vesicles in presynaptic (axonal) nerve terminals. The 8-subunit V1 complex generates energy via ATP hydrolysis which allows the 5-subunit V0 complex to transport protons across the vesicular membrane. Simultaneously, the neurotransmitter transporters utilize this electrochemical gradient to concentrate neurotransmitters into the synaptic vesicles.
Figure 4
Figure 4. A representative schematic of our proposed hypothesis
In order for synaptic transmission to occur, neurotransmitters must first be synthesized, concentrated in synaptic vesicles, and released across the synaptic cleft. V-ATPase (Atp6v), a multi-subunit proton pump, is the primary mediator of ATP-dependent concentration of neurotransmitters into synaptic vesicles and is thus vital for healthy synaptic transmission. Our data indicate that human ApoE isoforms differentially modulate the expression of Atp6v1B2, a key component of the catalytic core of Atp6v, with ApoE2 brain exhibiting the highest expression and ApoE4 brain exhibiting the lowest. Moreover, several studies have indicated that the glycolytic pathway of glucose metabolism is a direct regulator of Atp6v function. Based on these findings, we hypothesize that ApoE2 upregulates Atp6v in the brain and that the increased glucose uptake and metabolism previously observed in ApoE2 brain provides a direct source of ATP and protons for Atp6v thus facilitating increased Atp6v activity. Moreover, we hypothesize that the increased activity of Atp6v results in significantly increased neurotransmitter concentration and thus enhanced synaptic function.

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