We presently investigated the effects of apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease, on the cognitive performance of young targeted replacement apoE4 mice. We revealed that these mice were impaired in the object recognition and Morris water maze tests, both of which are associated with hippocampal learning and memory, relative to that of the apoE3 mice. These results are consistent with previous histological and biochemical findings that hippocampal neurons are specifically affected by apoE4. The suggestion that the behavioral impairments of the apoE4 mice are related to the hippocampal neuropathology of these mice is further supported by the fear conditioning test. This test revealed that the performance of the apoE4 mice in the contextual component, which is hippocampus related, was impaired, whereas their cued test response, which is amygdala driven, was not. The stress levels of the apoE4 and apoE3 mice, as unraveled by the light/dark anxiety test, were similar, suggesting that the observed cognitive impairments of the apoE4 mice are not related to differences in the basal anxiety levels of these mice. In conclusion, the present study shows that young apoE4 targeted replacement mice are impaired in numerous hippocampus-related learning and memory tasks.

Apolipoprotein E4 (ApoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with increased neurodegeneration and vascular impairments. Vascular endothelial growth factor (VEGF), originally described as a key angiogenic factor, has recently been shown to play a crucial role in the nervous system. The objective of this research is to examine the role of VEGF in mediating the apoE4-driven pathologies. We show that hippocampal VEGF levels are lower in apoE4 targeted replacement mice compared to the corresponding apoE3 mice. This effect was accompanied by a specific decrease in both VEGF receptor-2 and HIF1-α. We next set to examine whether upregulation of VEGF can reverse apoE4-driven pathologies, namely the accumulation of hyperphosphorylated tau (AT8) and Aβ42, and reduced levels of the pre-synaptic marker, VGluT1, and of the ApoE receptor, ApoER2. This was first performed utilizing intra-hippocampal injection of VEGF-expressing-lentivirus (LV-VEGF). This revealed that LV-VEGF treatment reversed the apoE4-driven cognitive deficits and synaptic pathologies. The levels of Aβ42 and AT8, however, were increased in apoE3 mice, masking any potential effects of this treatment on the apoE4 mice. Follow-up experiments utilizing VEGF-expressing adeno-associated-virus (AAV-VEGF), which expresses VEGF specifically under the GFAP astrocytic promoter, prevented this effects on apoE3 mice, and reversed the apoE4-related increase in Aβ42 and AT8. Taken together, these results suggest that apoE4-driven pathologies are mediated by a VEGF-dependent pathway, resulting in cognitive impairments and brain pathology. These animal model findings suggest that the VEGF system is a promising target for the treatment of apoE4 carriers in AD.

Apolipoprotein E4 (apoE4), the most prevalent genetic risk factor for Alzheimer's disease (AD), is associated with neuronal and vascular impairments. Recent findings suggest that retina of apoE4 mice have synaptic and functional impairments. We presently investigated the effects of apoE4 on retinal and choroidal vasculature and the possible role of VEGF in these effects. There were no histological differences between the retinal and choroidal vasculatures of naïve apoE3 and apoE4 mice. In contrast, laserdriven choroidal injury induced higher levels of choroidal neovascularization (CNV) in apoE4 than in apoE3 mice. These effects were associated with an inflammatory response and with activation of the Muller cells and asrocytic markers gluthatione synthetase and GFAP, all of which were more pronounced in the apoE4 mice. CNV also induced a transient increase in the levels of the synaptic markers synaptophysin and PSD95 which were however similar in the apoE4 and apoE3 naive mice. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury. Retinal and choroidal VEGF and apoE levels were lower in naïve apoE4 than in corresponding apoE3 mice. In contrast, VEGF and apoE levels rose more pronouncedly following laser injury in the apoE4 than in apoE3 mice. Taken together, these findings suggest that the apoE4-induced retinal impairments, under basal conditions, may be related to reduced VEGF levels in the eyes of these mice. The hyper-neovascularization in the apoE4 mice might be driven by increased inflammation and the associated surge in VEGF following injury.

Apolipoprotein E (APOE) ε4 gene allele and type 2 diabetes mellitus (T2DM) are prime risk factors for Alzheimer’s disease (AD). Despite evidence linking T2DM and apoE4, the mechanism underlying their interaction is yet to be determined. In the present study, we employed a model of APOE-targeted replacement mice and high-fat diet (HFD)-induced insulin resistance to investigate diabetic mechanisms associated with apoE4 pathology and the extent to which they are driven by peripheral and central processes. Results obtained revealed an intriguing pattern, in which under basal conditions, apoE4 mice display impaired glucose and insulin tolerance and decreased insulin secretion, as well as cognitive and sensorimotor characteristics relative to apoE3 mice, while the HFD impairs apoE3 mice without significantly affecting apoE4 mice. Measurements of weight and fasting blood glucose levels increased in a time-dependent manner following the HFD, though no effect of genotype was observed. Interestingly, sciatic electrophysiological and skin intra-epidermal nerve fiber density (IENFD) peripheral measurements were not affected by the APOE genotype or HFD, suggesting that the observed sensorimotor and cognitive phenotypes are related to central nervous system processes. Indeed, measurements of hippocampal insulin receptor and glycogen synthase kinase-3β (GSK-3β) activation revealed a pattern similar to that obtained in the behavioral measurements while Akt activation presented a dominant effect of diet. HFD manipulation induced genotype-independent hyperlipidation of apoE, and reduced levels of brain apoE in apoE3 mice, rendering them similar to apoE4 mice, whose brain apoE levels were not affected by the diet. No such effect was observed in the peripheral plasma levels of apoE, suggesting that the pathological effects of apoE4 under the control diet and apoE3 under HFD conditions are related to the decreased levels of brain apoE. Taken together, our data suggests that diabetic mechanisms play an important role in mediating the pathological effects of apoE4 and that consequently, diabetic-related therapy may be useful in treating apoE4 pathology in AD.

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Background: Alzheimer's Disease (AD) is associated with impairments in key brain Mitogen- Activated Protein Kinase (MAPK) signaling cascades including the p38, c-Jun N-terminal kinase (JNK), ERK and Akt pathways. Apolipoprotein E4 (ApoE4) is the most prevalent genetic risk factor of AD. Objectives: To investigate the extent to which the MAPK signaling pathway plays a role in mediating the pathological effects of apoE4 and can be reversed by experimental manipulations. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Methods: Measurements of total level and activation of MAPK signaling pathway factors, obtained utilizing immunoblot assay of hippocampal tissues from naïve and viral-treated apoE3 and apoE4 targeted replacement mice. Results: ApoE4 mice showed robust activation of the stress related p38 and JNK pathways and a corresponding decrease in Akt activity, which is coupled to activation of GSK3β and tau hyperphosphorylation. There was no effect on the ERK pathway. We have previously shown that the apoE4- related pathology, namely; accumulation of Aβ, hyper-phosphorylated tau, synaptic impairments and decreased VEGF levels can be reversed by up-regulation of VEGF level utilizing a VEGF-expressing adeno-associated virus. Utilizing this approach, we assessed the extent to which the AD-hallmark and synaptic pathologies of apoE4 are related to the corresponding MAPK signaling effects. This revealed that the reversal of the apoE4-driven pathology via VEGF treatment was associated with a reversal of the p38 and Akt related effects. Conclusion: Taken together, these results suggest that the p38 and Akt pathways play a role in mediating the AD-related pathological effects of apoE4 in the hippocampus.

Apolipoprotein E4 ( APOE ε4) is the most prevalent genetic risk factor for Alzheimer's disease (AD). Targeted replacement mice that express either APOE ε4 or its AD benign isoform, APOE ε3, are used extensively in behavioral, biochemical, and physiological studies directed at assessing the phenotypic effects of APOE ε4 and at unraveling the mechanisms underlying them. Such experiments often involve pursuing biochemical and behavioral measurements on the same cohort of mice. In view of the possible cross-talk interactions between brain parameters and cognitive performance, we presently investigated the extent to which the phenotypic expression of APOE ε4 and APOE ε4 in targeted replacement mice is affected by behavioral testing. This was performed using young, naïve APOE ε4 and APOE ε3 mice in which the levels of distinct brain parameters are affected by the APOE genotype (e.g., elevated levels of amyloid beta [Aβ] and hyperphosphorylated tau and reduced levels of vesicular glutamate transporter (VGLUT) in hippocampal neurons of APOE ε4 mice). These mice were exposed to a fear-conditioning paradigm, and the resulting effects on the brain parameters were examined. The results obtained revealed that the levels of Aβ, hyperphosphorylated tau, VGluT, and doublecortin of the APOE ε4 and APOE ε3 mice were markedly affected following the exposure of APOE ε4 and APOE ε3 mice to the fear-conditioning paradigm such that the isoform-specific effects of APOE ε4 on these parameters were greatly diminished. The finding that behavioral testing affects the APOE ε3 and APOE ε4 phenotypes and masks the differences between them has important theoretical and practical implications and suggests that the assessment of brain and behavioral parameters should be performed using different cohorts.

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