Endothelial Cell APOE3 Regulates Neurovascular, Neuronal, and Behavioral Function.

BACKGROUND: Specialized brain endothelial cells and human APOE3 are independently important for neurovascular function, yet whether APOE3 expression by endothelial cells contributes to brain function is currently unknown. In the present study, we determined whether the loss of endothelial cell APOE3 impacts brain vascular and neural function. METHODS: We developed APOE3fl/fl/Cdh5(PAC)-CreERT2+/- (APOE3Cre+/-) and APOE3fl/fl/Cdh5(PAC)-CreERT2-/- (APOE3Cre-/-, control) mice and induced endothelial cell APOE3 knockdown with tamoxifen at ≈4 to 5 weeks of age. Neurovascular and neuronal function were evaluated by biochemistry, immunohistochemistry, behavioral testing, and electrophysiology at 9 months of age. RESULTS: We found that the loss of endothelial APOE3 expression was sufficient to cause neurovascular dysfunction including higher permeability and lower vessel coverage in tandem with deficits in spatial memory and fear memory extinction and a disruption of cortical excitatory/inhibitory balance. CONCLUSIONS: Our data collectively support the novel concept that endothelial APOE3 plays a critical role in the regulation of the neurovasculature, neural circuit function, and behavior.

Dysfunction of platelet-derived growth factor receptor α (PDGFRα) represses the production of oligodendrocytes from arylsulfatase A-deficient multipotential neural precursor cells.

The membrane-bound receptor for platelet-derived growth factor A (PDGFRα) is crucial for controlling the production of oligodendrocytes (OLs) for myelination, but regulation of its activity during OL differentiation is largely unknown. We have examined the effect of increased sulfated content of galactosylceramides (sulfatides) on the regulation of PDGFRα in multipotential neural precursors (NPs) that are deficient in arylsulfatase A (ASA) activity. This enzyme is responsible for the lysosomal hydrolysis of sulfatides. We show that sulfatide accumulation significantly impacts the formation of OLs via deregulation of PDGFRα function. PDGFRα is less associated with detergent-resistant membranes in ASA-deficient cells and showed a significant decrease in AKT phosphorylation. Rescue experiments with ASA showed a normalization of the ratio of long versus short sulfatides, restored PDGFRα levels, corrected its localization to detergent-resistant membranes, increased AKT phosphorylation, and normalized the production of OLs in ASA-deficient NPs. Moreover, our studies identified a novel mechanism that regulates the secretion of PDGFRα in NPs, in glial cells, and in the brain cortex via exosomal shedding. Our study provides a first step in understanding the role of sulfatides in regulating PDGFRα levels in OLs and its impact in myelination.

The role of APOE in cerebrovascular dysfunction.

The ε4 allele of the apolipoprotein E gene (APOE4) is associated with cognitive decline during aging, is the greatest genetic risk factor for Alzheimer's disease and has links to other neurodegenerative conditions that affect cognition. Increasing evidence indicates that APOE genotypes differentially modulate the function of the cerebrovasculature (CV), with apoE and its receptors expressed by different cell types at the CV interface (astrocytes, pericytes, smooth muscle cells, brain endothelial cells). However, research on the role of apoE in CV dysfunction has not advanced as quickly as other apoE-modulated pathways. This review will assess what aspects of the CV are modulated by APOE genotypes during aging and under disease states, discuss potential mechanisms, and summarize the therapeutic significance of the topic. We propose that APOE4 induces CV dysfunction through direct signaling at the CV, and indirectly via modulation of peripheral and central pathways. Further, that APOE4 predisposes the CV to damage by, and exacerbates the effects of, additional risk factors (such as sex, hypertension, and diabetes). ApoE4-induced detrimental CV changes include reduced cerebral blood flow (CBF), modified neuron-CBF coupling, increased blood-brain barrier leakiness, cerebral amyloid angiopathy, hemorrhages and disrupted transport of nutrients and toxins. The apoE4-induced detrimental changes may be linked to pericyte migration/activation, astrocyte activation, smooth muscle cell damage, basement membrane degradation and alterations in brain endothelial cells.

APOE4 Promotes Tonic-Clonic Seizures, an Effect Modified by Familial Alzheimer's Disease Mutations.

Seizures are emerging as a common symptom in Alzheimer's disease (AD) patients, often attributed to high levels of amyloid ß (Aß). However, the extent that AD disease risk factors modulate seizure activity in aging and AD-relevant contexts is unclear. APOE4 is the greatest genetic risk factor for AD and has been linked to seizures independent of AD and Aß. The goal of the present study was to evaluate the role of APOE genotype in modulating seizures in the absence and presence of high Aß levels in vivo. To achieve this goal, we utilized EFAD mice, which express human APOE3 or APOE4 in the absence (EFAD-) or presence (EFAD+) of familial AD mutations that result in Aß overproduction. When quantified during cage change day, we found that unlike APOE3, APOE4 is associated with tonic-clonic seizures. Interestingly, there were lower tonic-clonic seizures in E4FAD+ mice compared to E4FAD- mice. Restraint handing and auditory stimuli failed to recapitulate the tonic-clonic phenotype in EFAD mice that express APOE4. However, after chemical-induction with pentylenetetrazole, there was a higher incidence of tonic-clonic seizures with APOE4 compared to APOE3. Interestingly, the distribution of seizures to the tonic-clonic phenotype was higher with FAD mutations. These data support that APOE4 is associated with higher tonic-clonic seizures in vivo, and that FAD mutations impact tonic-clonic seizures in a paradigm dependent manner.

Systemic Candesartan Treatment Modulates Behavior, Synaptic Protein Levels, and Neuroinflammation in Female Mice That Express Human APOE4.

Evidence suggests that angiotensin receptor blockers (ARBs) could be beneficial for Alzheimer's disease (AD) patients independent of any effects on hypertension. However, studies in rodent models directly testing the activity of ARB treatment on behavior and AD-relevent pathology including neuroinflammation, Aß levels, and cerebrovascular function, have produced mixed results. APOE4 is a major genetic risk factor for AD and has been linked to many of the same functions as those purported to be modulated by ARB treatment. Therefore, evaluating the effects of ARB treatment on behavior and AD-relevant pathology in mice that express human APOE4 could provide important information on whether to further develop ARBs for AD therapy. In this study, we treated female and male mice that express the human APOE4 gene in the absence (E4FAD-) or presence (E4FAD+) of high Aß levels with the ARB prodrug candesartan cilexetil for a duration of 4 months. Compared to vehicle, candesartan treatment resulted in greater memory-relevant behavior and higher hippocampal presynaptic protein levels in female, but not male, E4FAD- and E4FAD+ mice. The beneficial effects of candesartan in female E4FAD- and E4FAD+ mice occurred in tandem with lower GFAP and Iba1 levels in the hippocampus, whereas there were no effects on markers of cerebrovascular function and Aß levels. Collectively, these data imply that the effects of ARBs on AD-relevant pathology may be modulated in part by the interaction between APOE genotype and biological sex. Thus, the further development of ARBs could provide therapeutic options for targeting neuroinflammation in female APOE4 carriers.

LPC-DHA/EPA-Enriched Diets Increase Brain DHA and Modulate Behavior in Mice That Express Human APOE4.

Compared with APOE3, APOE4 is associated with greater age-related cognitive decline and higher risk of neurodegenerative disorders. Therefore, development of supplements that target APOE genotype-modulated processes could provide a great benefit for the aging population. Evidence suggests a link between APOE genotype and docosahexaenoic acid (DHA); however, clinical studies with current DHA supplements have produced negative results in dementia. The lack of beneficial effects with current DHA supplements may be related to limited bioavailability, as the optimal form of DHA for brain uptake is lysophosphatidylcholine (LPC)-DHA. We previously developed a method to enrich the LPC-DHA content of krill oil through lipase treatment (LT-krill oil), which resulted in fivefold higher enrichment in brain DHA levels in wild-type mice compared with untreated krill oil. Here, we evaluated the effect of a control diet, diet containing krill oil, or a diet containing LT-krill oil in APOE3- and APOE4-targeted replacement mice (APOE-TR mice; treated from 4 to 12 months of age). We found that DHA levels in the plasma and hippocampus are lower in APOE4-TR mice and that LT-krill oil increased DHA levels in the plasma and hippocampus of both APOE3- and APOE4-TR mice. In APOE4-TR mice, LT-krill oil treatment resulted in higher levels of the synaptic vesicle protein SV2A and improved performance on the novel object recognition test. In conclusion, our data demonstrate that LPC-DHA/EPA-enriched krill oil can increase brain DHA and improve memory-relevant behavior in mice that express APOE4. Therefore, long-term use of LT-krill oil supplements may on some level protect against age-related neurodegeneration.

Autocrine Effects of Brain Endothelial Cell-Produced Human Apolipoprotein E on Metabolism and Inflammation in vitro.

Reports of APOE4-associated neurovascular dysfunction during aging and in neurodegenerative disorders has led to ongoing research to identify underlying mechanisms. In this study, we focused on whether the APOE genotype of brain endothelial cells modulates their own phenotype. We utilized a modified primary mouse brain endothelial cell isolation protocol that enabled us to perform experiments without subculture. Through initial characterization we found, that compared to APOE3, APOE4 brain endothelial cells produce less apolipoprotein E (apoE) and have altered metabolic and inflammatory gene expression profiles. Further analysis revealed APOE4 brain endothelial cultures have higher preference for oxidative phosphorylation over glycolysis and, accordingly, higher markers of mitochondrial activity. Mitochondrial activity generates reactive oxygen species, and, with APOE4, there were higher mitochondrial superoxide levels, lower levels of antioxidants related to heme and glutathione and higher markers/outcomes of oxidative damage to proteins and lipids. In parallel, or resulting from reactive oxygen species, there was greater inflammation in APOE4 brain endothelial cells including higher chemokine levels and immune cell adhesion under basal conditions and after low-dose lipopolysaccharide (LPS) treatment. In addition, paracellular permeability was higher in APOE4 brain endothelial cells in basal conditions and after high-dose LPS treatment. Finally, we found that a nuclear receptor Rev-Erb agonist, SR9009, improved functional metabolic markers, lowered inflammation and modulated paracellular permeability at baseline and following LPS treatment in APOE4 brain endothelial cells. Together, our data suggest that autocrine signaling of apoE in brain endothelial cells represents a novel cellular mechanism for how APOE regulates neurovascular function.

Epidermal growth factor treatment of female mice that express APOE4 at an age of advanced pathology mitigates behavioral and cerebrovascular dysfunction.

APOE4 is a major genetic risk factor for Alzheimer's disease and high amyloid-ß (Aß) levels in the brain are a pathological hallmark of the disease. However, the contribution of specific APOE-modulated Aß-dependent and Aß-independent functions to cognitive decline remain unclear. Increasing evidence supports a role of APOE in modulating cerebrovascular function, however whether ameliorating this dysfunction can improve behavioral function is still under debate. We have previously demonstrated that systemic epidermal growth factor (EGF) treatment, which is important for vascular function, at early stages of pathology (treatment from 6 to 8 months) is beneficial for recognition and spatial memory and cerebrovascular function in female mice that express APOE4. These data raise the important question of whether EGF can improve APOE4-associated cerebrovascular and behavioral dysfunction when treatment is initiated at an age of advanced pathology. Positive findings would support the development of therapies that target cerebrovascular dysfunction associated with APOE4 in aging and AD in individuals with advanced cognitive impairment. Therefore, in this study female mice that express APOE4 in the absence (E4FAD- mice) or presence (E4FAD+ mice) of Aß overproduction were treated from 8 to 10 months of age systemically with EGF. EGF treatment mitigated behavioral dysfunction in recognition memory and spatial learning and improved hippocampal neuronal function in both E4FAD+ and E4FAD- mice, suggesting that EGF treatment improves Aß-independent APOE4-associated deficits. The beneficial effects of EGF treatment on behavior occurred in tandem with improved markers of cerebrovascular function, including lower levels of fibrinogen, lower permeability when assessed by MRI and higher percent area coverage of laminin and CD31 in the hippocampus. These data suggest a mechanistic link among EGF signaling, cerebrovascular function and APOE4-associated behavioral deficits in mice with advanced AD-relevant pathology.

EGF Treatment Improves Motor Behavior and Cortical GABAergic Function in the R6/2 Mouse Model of Huntington's Disease.

Recent evidence indicates that disruption of epidermal growth factor (EGF) signaling by mutant huntingtin (polyQ-htt) may contribute to the onset of behavioral deficits observed in Huntington's disease (HD) through a variety of mechanisms, including cerebrovascular dysfunction. Yet, whether EGF signaling modulates the development of HD pathology and the associated behavioral impairments remain unclear. To gain insight on this issue, we used the R6/2 mouse model of HD to assess the impact of chronic EGF treatment on behavior, and cerebrovascular and cortical neuronal functions. We found that bi-weekly treatment with a low dose of EGF (300 µg/kg, i.p.) for 6 weeks was sufficient to effectively improve motor behavior in R6/2 mice and diminish mortality, compared to vehicle-treated littermates. These beneficial effects of EGF treatment were dissociated from changes in cerebrovascular leakiness, a result that was surprising given that EGF ameliorates this deficit in other neurodegenerative diseases. Rather, the beneficial effect of EGF on R6/2 mice behavior was concomitant with a marked amelioration of cortical GABAergic function. As GABAergic transmission in cortical circuits is disrupted in HD, these novel data suggest a potential mechanistic link between deficits in EGF signaling and GABAergic dysfunction in the progression of HD.

Peripheral Inflammation, Apolipoprotein E4, and Amyloid-ß Interact to Induce Cognitive and Cerebrovascular Dysfunction.

Cerebrovascular dysfunction is rapidly reemerging as a major process of Alzheimer's disease (AD). It is, therefore, crucial to delineate the roles of AD risk factors in cerebrovascular dysfunction. While apolipoprotein E4 ( APOE4), Amyloid-ß (Aß), and peripheral inflammation independently induce cerebrovascular damage, their collective effects remain to be elucidated. The goal of this study was to determine the interactive effect of APOE4, Aß, and chronic repeated peripheral inflammation on cerebrovascular and cognitive dysfunction in vivo. EFAD mice are a well-characterized mouse model that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce human Aß42 via expression of 5 Familial Alzheimer's disease (5xFAD) mutations. Here, we utilized EFAD carriers [5xFAD+/-/ APOE+/+ (EFAD+)] and noncarriers [5xFAD-/-/ APOE+/+ (EFAD-)] to compare the effects of peripheral inflammation in the presence or absence of human Aß overproduction. Low-level, chronic repeated peripheral inflammation was induced in EFAD mice via systemic administration of lipopolysaccharide (LPS; 0.5 mg/kg/wk i.p.) from 4 to 6 months of age. In E4FAD+ mice, peripheral inflammation caused cognitive deficits and lowered post-synaptic protein levels. Importantly, cerebrovascular deficits were observed in LPS-challenged E4FAD+ mice, including cerebrovascular leakiness, lower vessel coverage, and cerebral amyloid angiopathy-like Aß deposition. Thus, APOE4, Aß, and peripheral inflammation interact to induce cerebrovascular damage and cognitive deficits.

Epidermal growth factor prevents APOE4 and amyloid-beta-induced cognitive and cerebrovascular deficits in female mice.

Cerebrovascular (CV) dysfunction is emerging as a critical component of Alzheimer's disease (AD), including altered CV coverage. Angiogenic growth factors (AGFs) are key for controlling CV coverage, especially during disease pathology. Therefore, evaluating the effects of AGFs in vivo can provide important information on the role of CV coverage in AD. We recently demonstrated that epidermal growth factor (EGF) prevents amyloid-beta (Aß)-induced damage to brain endothelial cells in vitro. Here, our goal was to assess the protective effects of EGF on cognition, CV coverage and Aß levels using an AD-Tg model that incorporates CV relevant AD risk factors. APOE4 is the greatest genetic risk factor for sporadic AD especially in women and is associated with CV dysfunction. EFAD mice express human APOE3 (E3FAD) or APOE4 (E4FAD), overproduce human Aß42 and are a well characterized model of APOE pathology. Thus, initially the role of APOE and sex in cognitive and CV dysfunction was assessed in EFAD mice in order to identify a group for EGF treatment. At 8 months E4FAD female mice were cognitively impaired, had low CV coverage, high microbleeds and low plasma EGF levels. Therefore, E4FAD female mice were selected for an EGF prevention paradigm (300 µg/kg/wk, 6 to 8.5 months). EGF prevented cognitive decline and was associated with lower microbleeds and higher CV coverage, but not changes in Aß levels. Collectively, these data suggest that EGF can prevent Aß-induced damage to the CV. Developing therapeutic strategies based on AGFs may be particularly efficacious for APOE4-induced AD risk.