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Progressive hippocampal degeneration is a key component of Alzheimer’s disease (AD) progression. Therefore, identifying how hippocampal neuronal function is modulated early in AD is an important approach to eventually prevent degeneration. AD-risk factors and signaling molecules likely modulate neuronal function, including APOE genotype and angiotensin II. Compared to APOE3, APOE4 increases AD risk up to 12-fold, and high levels of angiotensin II are hypothesized to disrupt neuronal function in AD. However, the extent that APOE and angiotensin II modulates the hippocampal neuronal phenotype in AD-relevant models is unknown. To address this issue, we used electrophysiological techniques to assess the impact of APOE genotype and angiotensin II on basal synaptic transmission, presynaptic and post-synaptic activity in mice that express human APOE3 (E3FAD) or APOE4 (E4FAD) and overproduce Aβ. We found that compared to E3FAD mice, E4FAD mice had lower basal synaptic activity, but higher levels of paired pulse facilitation (PPF) and Long-Term Potentiation (LTP) in the Schaffer Collateral Commissural Pathway (SCCP) of the hippocampus. We also found that exogenous angiotensin II has a profound inhibitory effect on hippocampal LTP in both E3FAD and E4FAD mice. Collectively, our data suggests that APOE4 and Aβ are associated with a hippocampal phenotype comprised of lower basal activity and higher responses to high frequency stimulation, the latter of which is suppressed by angiotensin II. These novel data suggest a potential mechanistic link between hippocampal activity, APOE4 genotype and angiotensin II in AD.

CXCL10 is an interferon-inducible chemokine that can recruit CXCR3+leukocytes to the central nervous system, leading to neuroinflammation, demyelination, and neuronal losses. How CXCL10 promotes leukocyte extravasation and diapedesis across the blood-brain barrier – formed by brain endothelial cells – is poorly understood. Here, we report that CXCL10 mediates CD4+ T cell migration through the brain endothelial cell cytoplasm (transcellular), but not cell-cell junctions (paracellular), via the vesicular trafficking protein Caveolin-1. Caveolin-1 promotes CXCL10 aggregation into cytoplasmic stores in brain endothelial cellsin vitroto provide the local, high concentration necessary for recruitment of CXCR3+ leukocytes. This process also requires LFA-1 activity. In the absence of Caveolin-1, endothelial CXCL10 is secreted, and the local signaling cues are lost. Consistent with ourin vitrodata, genetic ablation of Caveolin-1 in endothelial cells reduces the severity of active experimental autoimmune encephalomyelitis (EAE), a murine model for multiple sclerosis, by decreasing the infiltration of CXCR3+ T cells into the CNS. Moreover, loss of Caveolin-1 protects against the adoptive transfer of autoreactive T cells. Our findings establish a novel mechanism by which brain endothelial cells utilize Caveolin-1 dependent CXCL10 intracellular stores to license T cells for transcellular migration across the blood-brain barrier.

Neonatal AAV9-gene therapy of the lysosomal enzyme galactosylceramidase (GALC) significantly ameliorates central and peripheral neuropathology, prolongs survival, and largely normalizes motor deficits in Twitcher mice. Despite these therapeutic milestones, new observations identified the presence of multiple small focal demyelinating areas in the brain after 6-8 months. These lesions are in stark contrast to the diffuse, global demyelination that affects the brain of naive Twitcher mice. Late-onset lesions exhibited lysosomal alterations with reduced expression of GALC and increased psychosine levels. Furthermore, we found that lesions were closely associated with the extravasation of plasma fibrinogen and activation of the fibrinogen-BMP-SMAD-GFAP gliotic response. Extravasation of fibrinogen correlated with tight junction disruptions of the vasculature within the lesioned areas. The lesions were surrounded by normal appearing white matter. Our study shows that the dysregulation of therapeutic GALC was likely driven by the exhaustion of therapeutic AAV episomal DNA within the lesions, paralleling the presence of proliferating oligodendrocyte progenitors and glia. We believe that this is the first demonstration of diminishing expression in vivo from an AAV gene therapy vector with detrimental effects in the brain of a lysosomal storage disease animal model. The development of this phenotype linking localized loss of GALC activity with relapsing neuropathology in the adult brain of neonatally AAV-gene therapy-treated Twitcher mice identifies and alerts to possible late-onset reductions of AAV efficacy, with implications to other genetic leukodystrophies.

[Image: see text] The calpain–cathepsin hypothesis posits a key role for elevated calpain-1 and cathepsin-B activity in the neurodegeneration underlying neurotrauma and multiple disorders including Alzheimer’s disease (AD). AD clinical trials were recently halted on alicapistat, a selective calpain-1 inhibitor, because of insufficient exposure of neurons to the drug. In contrast to neuroprotection, the ability of calpain-1 and cathepsin-B inhibitors to protect the blood–brain barrier (BBB), is understudied. Since cerebrovascular dysfunction underlies vascular dementia, is caused by ischemic stroke, and is emerging as an early feature in the progression of AD, we studied protection of brain endothelial cells (BECs) by selective and nonselective calpain-1 and cathepsin-B inhibitors. We show these inhibitors protect both neurons and murine BECs from ischemia–reperfusion injury. Cultures of primary BECs from ALDH2(–/–) mice that manifest enhanced oxidative stress were sensitive to ischemia, leading to reduced cell viability and loss of tight junction proteins; this damage was rescued by calpain-1 and cathepsin-B inhibitors. In ALDH2(–/–) mice 24 h after mild traumatic brain injury (mTBI), BBB damage was reflected by significantly increased fluorescein extravasation and perturbation of tight junction proteins, eNOS, MMP-9, and GFAP. Both calpain and cathepsin-B inhibitors alleviated BBB dysfunction caused by mTBI. No clear advantage was shown by selective versus nonselective calpain inhibitors in these studies. The lack of recognition of the ability of calpain inhibitors to protect the BBB may have led to the premature abandonment of this therapeutic approach in AD clinical trials and requires further mechanistic studies of cerebrovascular protection by calpain-1 inhibitors.

Persistent cognitive impairment and neuropsychiatric disorders are prevalent sequelae of SARS-CoV-2-induced COVID-19 in middle-aged adults. To model age-related neurological vulnerability to COVID-19, we induced respiratory SARS-CoV-2 MA10 infections by nasal inoculation in young (2 months) and middle-aged (12 months) mice. We hypothesized that aging and SARS-CoV-2 synergistically damage the blood-brain barrier (BBB). Indeed, the combined action of aging and SARS-CoV-2 infection caused more fibrinogen leakage, T cell infiltration, and neuroinflammation in middle-aged SARS-CoV-2-infected mice than in similarly inoculated young adults. Mechanistically, SARS-CoV-2 exacerbated age-related increases in Caveolin-1 BBB transcellular permeability and loss of Wnt/β-catenin ligands, with no apparent changes in tight junction proteins. Finally, SARS-CoV-2 infection induced age-dependent neuropsychiatric abnormalities including bradykinesia and obsessive-compulsive-like behavior. These observations indicate that cerebrovascular aging, including loss of Wnt suppression of Caveolin-1, heightens vulnerability to SARS-CoV-2-induced neuroinflammation and neuropsychiatric sequalae. Our work suggests that modulation of Wnt signaling or its downstream effectors at the BBB could be potential interventional strategies for Long COVID.HighlightsTo our knowledge, we have for the first time used a small animal model to experimentally test the impact of age on SARS-CoV-2 neuropathology.Aged mice were uniquely vulnerable to neuropsychiatric signs after SARS-CoV-2 infectionMiddle-age increased gliosis, cerebrovascular inflammation, BBB permeability, and T cell infiltration in SARS-CoV-2 infected miceBBB permeability was related to loss of Wnt7a suppression of Caveolin-1

Arterial spin labeling (ASL) MRI, based on endogenous contrast from blood water, is used in research and diagnosis of cerebral vascular conditions. However, artifacts due to imperfect imaging conditions such as B0-inhomogeneity (ΔB0) could lead to variations in the quantification of relative cerebral blood flow (CBF). In this study, we evaluate a new approach using tagging distance dependent Z-spectrum (TADDZ) data, similar to the ΔB0 corrections in the chemical exchange saturation transfer (CEST) experiments, to remove the imaging plane B0 inhomogeneity induced CBF artifacts in ASL MRI. Our results indicate that imaging-plane B0-inhomogeneity can lead to variations and errors in the relative CBF maps especially under small tagging distances. Along with an acquired B0 map, TADDZ data helps to eliminate B0-inhomogeneity induced artifacts in the resulting relative CBF maps. We demonstrated the effective use of TADDZ data to reduce variation while subjected to systematic changes in ΔB0. In addition, TADDZ corrected ASL MRI, with improved consistency, was shown to outperform conventional ASL MRI by differentiating the subtle CBF difference in Alzheimer's disease (AD) mice brains with different APOE genotypes.

Although the cause of Alzheimer’s disease (AD) is unknown, glial-induced neuroinflammation is an early symptom. Familial AD is caused by increases in amyloid-beta (Aβ) peptide, particularly soluble oligomeric (oAβ), considered a proximal neurotoxin and neuroinflammatory stimuli. APOE4, a naturally occurring genotype of APOE, is the greatest genetic risk factor for AD; increasing risk up to 12-fold compared to APOE3 and APOE2. oAβ-induced neuroinflammation is greater with APOE4 compared to APOE3 and APOE2. As sinapates and flavonoids have anti-inflammatory properties, a protocol was developed for optimizing polyphenol production in seedlings of Arabidopsis thaliana (A. thaliana). Three mutants (cop1, prn1, xpf3) were identified and the extracts treated with liver microsomes to mimic physiological metabolism, with HPLC and MS performed on the resulting metabolites for peak identification. These extracts were used to treat primary glial cells isolated from human APOE-targeted-replacement (APOE-TR) and APOE-knock-out (KO) mice, with neuroinflammation induced by lipopolysaccharide (LPS) or oAβ. The dose-response data for TNFα secretion demonstrate the followed the order: APOE-KO > APOE4 > APOE3 > APOE2, with xpf3 the most effective anti-neuroinflammatory across APOE genotypes. Thus, the plant-based approach described herein may be particularly valuable in treating the APOE4-induced neuroinflammatory component of AD risk.

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.

Seizures are emerging as a common symptom in Alzheimer’s disease (AD) patients, often attributed to high levels of amyloid β (Aβ). 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 Aβ. The goal of the present study was to evaluate the role of APOE genotype in modulating seizures in the absence and presence of high Aβ 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 Aβ 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.

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, Aβ 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 Aβ 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 Aβ 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.

Over the last several decades, a number of mouse models have been generated for mechanistic and preclinical therapeutic research on Alzheimer's disease (AD)-like behavioral impairments and pathology. Acceptance or rejection of these models by the scientific community is playing a prominent role in how research findings are viewed and whether grants get funded and manuscripts published. The question of whether models are useful has become an exceptionally contentious issue. Much time and effort have gone into investigators debating comments such as “there are no mouse models of AD,” “…nice work but needs to be tested in another mouse model,” or “only data from humans is valid.” This leads to extensive written justifications for the choice of a model in grant applications, to the point of almost apologizing for the use of models. These debates also lead to initiatives to create new, better models of AD without consideration of what “better” may mean in this context. On the “other side,” an argument supporting the use of mouse models is one cannot dissect a biological mechanism in postmortem human tissue. In this chapter, we examine issues that we believe must be addressed if in vivo AD research is to progress. We opine that it is not the models that are the issue, but rather a lack of understanding the aspects of AD-like pathology the models were designed to mimic. The goal here is to improve the utilization of models to address critical issues, not to offer a critique of existing models or make endorsements.

APOE4 is a major genetic risk factor for Alzheimer's disease and high amyloid-β (Aβ) levels in the brain are a pathological hallmark of the disease. However, the contribution of specific APOE-modulated Aβ-dependent and Aβ-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 Aβ 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 Aβ-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., Biochemistry; Cell biology; Neuroscience; Pathology; Pathophysiology; APOE4, EGF, Cerebrovascular function

Scope Currently available omega-3 fatty acid supplements do not enrich the docosahexaenoic acid (DHA) of the adult brain because they are absorbed as triacylglycerol, whereas the transporter at the blood brain barrier requires lysophosphatidylcholine (LPC)-DHA. The hypothesis that treatment of krill oil (KO), which contains DHA/eicosapentaenoic acid (EPA) at the SN2 position of phosphatidylcholine, with SN1-specific lipase will generate LPC-DHA/EPA and which can be absorbed intact and transported into the brain, is tested. Methods KO and fish oil (FO) are treated with Mucor meihei lipase, incorporated into AIN 93G diet, and fed to 2-month-old mice for 30 days. Fatty acid composition is analyzed by gas chromatography/mass spectroscopy. Brain derived neurotrophic factor (BDNF) is measured by ELISA. Results Lipase-treated (LT) KO increases brain DHA and EPA, respectively, 5-and 70-fold better than untreated (UT) KO. FO, whether lipase-treated or not, has no effect on brain DHA/EPA. LTKO is also more efficient in enriching liver DHA/EPA, but less efficient than UTKO and FO in enriching adipose tissue and heart. Brain BDNF is significantly increased by LTKO, but only marginally by other preparations. Conclusions Pretreatment of dietary KO with lipase enables it to efficiently increase brain DHA/EPA because of the generation of LPC-DHA/EPA.

This work describes a role for endothelial N-cadherin in the regulation of endothelial permeability in the brain and lung. N-cadherin adhesions formed between endothelial cells and pericytes increase the abundance of VE-cadherin at adherens junctions through the RhoGEF Trio-dependent activation of RhoA and Rac1., Vascular endothelial (VE)–cadherin forms homotypic adherens junctions (AJs) in the endothelium, whereas N-cadherin forms heterotypic adhesion between endothelial cells and surrounding vascular smooth muscle cells and pericytes. Here we addressed the question whether both cadherin adhesion complexes communicate through intracellular signaling and contribute to the integrity of the endothelial barrier. We demonstrated that deletion of N-cadherin (Cdh2) in either endothelial cells or pericytes increases junctional endothelial permeability in lung and brain secondary to reduced accumulation of VE-cadherin at AJs. N-cadherin functions by increasing the rate of VE-cadherin recruitment to AJs and induces the assembly of VE-cadherin junctions. We identified the dual Rac1/RhoA Rho guanine nucleotide exchange factor (GEF) Trio as a critical component of the N-cadherin adhesion complex, which activates both Rac1 and RhoA signaling pathways at AJs. Trio GEF1-mediated Rac1 activation induces the recruitment of VE-cadherin to AJs, whereas Trio GEF2-mediated RhoA activation increases intracellular tension and reinforces Rac1 activation to promote assembly of VE-cadherin junctions and thereby establish the characteristic restrictive endothelial barrier.

Identified in 1993, APOE4 is the greatest genetic risk factor for sporadic Alzheimerʼns disease (AD), increasing risk up to 15-fold compared with APOE3, with APOE2 decreasing AD risk. However, the functional effects of APOE4 on AD pathology remain unclear and, in some cases, controversial. In vivo progress to understand how the human (h)-APOE genotypes affect AD pathology has been limited by the lack of a tractable familial AD-transgenic (FAD-Tg) mouse model expressing h-APOE rather than mouse (m)-APOE. The disparity between m- and h-apoE is relevant for virtually every AD-relevant pathway, including amyloid-β (Aβ) deposition and clearance, neuroinflammation, tau pathology, neural plasticity and cerebrovascular deficits. EFAD mice were designed as a temporally useful preclinical FAD-Tg-mouse model expressing the h-APOE genotypes for identifying mechanisms underlying APOE-modulated symptoms of AD pathology. From their first description in 2012, EFAD mice have enabled critical basic and therapeutic research. Here we review insights gleaned from the EFAD mice and summarize future directions.

This study was designed to explore the influence of apolipoprotein E (APOE) on blood phospholipids (PL) in predicting preclinical Alzheimer's disease (AD). Lipidomic analyses were also performed on blood from an AD mouse model expressing human APOE isoforms (EFAD) and five AD mutations and from 195 cognitively normal participants, 23 of who converted to mild cognitive impairment (MCI)/AD within 3 years. APOE ε4-carriers converting to MCI/AD had high arachidonic acid (AA)/docosahexaenoic acid (DHA) ratios in PL compared to cognitively normal ε4 and non-ε4 carriers. Arachidonic acid and DHA containing PL species, ε4-status and Aβ42/Aβ40 ratios provided 91% accuracy in detecting MCI/AD. Fish oil/omega-3 fatty acid consumption was associated with lower AA/DHA ratios even among ε4 carriers. High plasma AA/DHA ratios were observed in E4FAD compared to EFAD mice with other isoforms. In particular, alterations in plasma AA and DHA containing PL species were also observed in the brains of E4FAD mice compared to E3FAD mice. Despite the small sample size and a short follow-up, these results suggest that blood PL could potentially serve as biomarkers of preclinical MCI/AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques, composed of amyloid-beta peptide (Aβ) and neurofibrillary tangles, composed of aberrantly phosphorylated tau. APOE4 is the greatest genetic risk factor for AD, increasing risk up to 12- fold with a double allele compared to APOE3. In contrast, APOE2 reduces AD risk ~2-fold per allele. Accumulating evidence demonstrates that apolipoprotein E4 (apoE4) plays a multifactorial role in AD pathogenesis, although the exact mechanisms remain unclear. Further data support roles for apoE4 as a toxic gain of function or loss of positive function in AD, a discrepancy that has significant implications for the future of apoE-directed therapeutics. However, recent evidence repurposing retinoid X receptor (RXR) agonists, or rexinoids, for the treatment of AD demonstrates conflicting, though potentially beneficial effects in familial AD-transgenic (FAD-Tg) mouse models. Of particular note is bexarotene (Targretin®), a selective rexinoid previously utilized in cancer treatment emerging as a viable candidate for AD clinical trials. However, the mechanism of action of bexarotene and similar rexinoids remains controversial, particularly in the context of human APOE. In addition, rexinoids demonstrate distinct adverse event profiles in humans that may have greater detrimental effects in an elderly AD population. Therefore, this special issue review discusses the implications for rexinoiddirected therapeutic strategies in AD, the potential mechanistic targets, and future directions for the improvement of rexinoid-based therapies in AD.

Type 2 diabetes mellitus (T2DM) is a risk factor for the development of late-onset Alzheimer's disease (AD). However, the mechanism underlying the development of late-onset AD is largely unknown. Here we show that levels of the endothelial-enriched protein caveolin-1 (Cav-1) are reduced in the brains of T2DM patients compared with healthy aging, and inversely correlated with levels of β-amyloid (Aβ). Depletion of Cav-1 is recapitulated in the brains ofdb/db(Leprdb) diabetic mice and corresponds with recognition memory deficits as well as the upregulation of amyloid precursor protein (APP), BACE-1, a trending increase in β-amyloid Aβ42/40ratio and hyperphosphorylated tau (p-tau) species. Importantly, we show that restoration of Cav-1 levels in the brains of maledb/dbmice using adenovirus overexpressing Cav-1 (AAV-Cav-1) rescues learning and memory deficits and reduces pathology (i.e., APP, BACE-1 and p-tau levels). Knocking down Cav-1 using shRNA in HEK cells expressing the familial AD-linked APPswe mutant variant upregulates APP, APP carboxyl terminal fragments, and Aβ levels. In turn, rescue of Cav-1 levels restores APP metabolism. Together, these results suggest that Cav-1 regulates APP metabolism, and that depletion of Cav-1 in T2DM promotes the amyloidogenic processing of APP and hyperphosphorylation of tau. This may suggest that depletion of Cav-1 in T2DM underlies, at least in part, the development of AD and imply that restoration of Cav-1 may be a therapeutic target for diabetic-associated sporadic AD.SIGNIFICANCE STATEMENTMore than 95% of the Alzheimer's patients have the sporadic late-onset form (LOAD). The cause for late-onset Alzheimer's disease is unknown. Patients with Type 2 diabetes mellitus have considerably higher incidence of cognitive decline and AD compared with the general population, suggesting a common mechanism. Here we show that the expression of caveolin-1 (Cav-1) is reduced in the brain in Type 2 diabetes mellitus. In turn, reduced Cav-1 levels induce AD-associated neuropathology and learning and memory deficits. Restoration of Cav-1 levels rescues these deficits. This study unravels signals underlying LOAD and suggests that restoration of Cav-1 may be an effective therapeutic target.

Docosahexaenoic acid (DHA) is highly concentrated in the brain and its deficiency is associated with several neurological disorders including Alzheimer’s disease. However, the currently used supplements do not appreciably enrich brain DHA, although they enrich most other tissues. We tested the hypothesis that the ability of the dietary carrier to augment brain DHA depends upon the generation of DHA-lysophosphatidylcholine (LPC), the preferred carrier of DHA across the blood brain barrier. We compared the efficacy of DHA-triacylglycerol (TAG), di-DHA phosphatidylcholine (PC) and DHA-LPC to enrich brain DHA following their gavage to normal rats for 30 days, all at a dose of 10 mg DHA/day. The results show that DHA from TAG, which is released as free DHA or monoacylglycerol during digestion, and is absorbed as TAG in chylomicrons, was incorporated preferentially into adipose tissue and heart, but not into brain. In contrast, LPC-DHA increased brain DHA by up to 100%, but had no effect on adipose tissue. Di-DHA PC, which generates both free DHA and LPC-DHA during the digestion, enriched DHA in brain, as well as in heart and liver. Brain-derived neurotrophic factor was increased by di-DHA PC and DHA-LPC, but not by TAG-DHA, showing that enrichment of brain DHA correlated with its functional effect. We conclude that dietary DHA from TAG or from natural PC (sn-2 position) is not suitable for brain enrichment, whereas DHA from LPC (at either sn-1 or sn-2 position) or from sn-1 position of PC efficiently enriches the brain, and is functionally effective.

Background: Since APOE alleles represent the most impactful genetic risk factors for Alzheimer's disease (AD), their differential mechanism(s) of action are under intense scrutiny. APOE4 is robustly associated with increased AD risk compared to the neutral APOE3 and protective APOE2. APOE alleles have also been associated with differential inflammation and gastrointestinal recovery after insult in human and murine studies, leading us to hypothesize that APOE alleles impact the gut microbiome. Methods: To assess this hypothesis, we compared 16S ribosomal RNA gene amplicon-based microbiome profiles in a cohort of mice that were homozygous for APOE2, APOE3, or APOE4, and included both males and females as well as carriers and non-carriers of five familial AD (5xFAD) mutations. Fecal samples were analyzed from mice at 4 and 6 months of age. APOE genotype, as well as sex and 5xFAD status, was then tested for influence on alpha diversity (Shannon H index) and beta diversity (principal coordinate analyses and PERMANOVA). A Random Forest analysis was used to identify features that predicted APOE, sex and 5xFAD status. Results: The richness and evenness (alpha diversity) of the fecal microbiome was not robustly associated with APOE genotype, 5xFAD status or sex. In contrast, microbial community composition (beta-diversity) was consistently and strongly associated with APOE genotype. The association between beta-diversity and sex or 5xFAD status was less consistent and more modest. Comparison of the differences underlying APOE effects showed that the relative abundance of multiple bacterial taxa was significantly different as a function of APOE genotype. Conclusions: The structure of the gut microbiome was strongly and significantly associated with APOE alleles in this murine model. Further evaluation of these findings in humans, as well as studies evaluating the impact of the APOE-associated microbiota on AD-relevant phenotypes in murine models, will be necessary to determine if alterations in the gut microbiome represent a novel mechanism whereby APOE genotype impacts AD.

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.

Three months of exercise training (ET) decreases soluble A&beta, 40 and A&beta, 42 levels in an intensity dependent manner early in life in Tg2576 mice (Moore et al., 2016). Here, we examined the effects of 12 months of low- and high- intensity exercise training on cognitive function and amyloid plaque load in the cortex and hippocampus of 15-month-old Tg2576 mice. Low- (LOW) and high- (HI) intensity ET animals ran at speeds of 15 m/min on a level treadmill and 32 m/min at a 10% grade, respectively, for 60 min/day, five days/week, from 3 to 15 months of age. Sedentary mice (SED) were placed on a level, non-moving, treadmill for the same duration. ET mice demonstrated a significantly lower amyloid plaque load in the cortex and hippocampus that was intensity dependent. Improvement in cognitive function, assessed by Morris Water Maze and Novel Object Recognition tests, was greater in the HI group compared to the LOW and SED groups. LOW mice performed better in the initial latency to the platform location during the probe trial of the Morris Water Maze (MWM) test than SED, but not in any other aspect of MWM or the Novel Object Recognition test. The results of this study indicate that exercise training decreases amyloid plaque load in an intensity dependent manner and that high-intensity exercise training improves cognitive function relative to SED mice, but the intensity of the LOW group was below the threshold to demonstrate robust improvement in cognitive function in Tg2576 mice.

Docosahexaenoic acid (DHA) is uniquely concentrated in the brain, and is essential for its function, but must be mostly acquired from diet. Most of the current supplements of DHA, including fish oil and krill oil, do not significantly increase brain DHA, because they are hydrolyzed to free DHA and are absorbed as triacylglycerol, whereas the transporter at blood brain barrier is specific for phospholipid form of DHA. Here we show that oral administration of DHA to normal adult mice as lysophosphatidylcholine (LPC) (40 mg DHA/kg) for 30 days increased DHA content of the brain by >2-fold. In contrast, the same amount of free DHA did not increase brain DHA, but increased the DHA in adipose tissue and heart. Moreover, LPC-DHA treatment markedly improved the spatial learning and memory, as measured by Morris water maze test, whereas free DHA had no effect. The brain derived neurotrophic factor increased in all brain regions with LPC-DHA, but not with free DHA. These studies show that dietary LPC-DHA efficiently increases brain DHA content and improves brain function in adult mammals, thus providing a novel nutraceutical approach for the prevention and treatment of neurological diseases associated with DHA deficiency, such as Alzheimer’s disease.

Blood-brain barrier (BBB) coverage plays a central role in the homeostasis of the central nervous system (CNS). The BBB is dynamically maintained by astrocytes, pericytes and brain endothelial cells (BECs). Here, we detail methods to assess BBB coverage using single cultures of immortalized human BECs, single cultures of primary mouse BECs, and a humanized triple culture model (BECs, astrocytes and pericytes) of the BBB. To highlight the applicability of the assays to disease states, we describe the effect of oligomeric amyloid-β (oAβ), which is an important contributor to Alzheimer's disease (AD) progression, on BBB coverage. Further, we utilize the epidermal growth factor (EGF) to illuminate the drug screening potential of the techniques. Our results show that single and triple cultured BECs form meshwork-like structures under basal conditions, and that oAβ disrupts this cell meshwork formation and degenerates the preformed mesh structures, but EGF blocks this disruption. Thus, the techniques described are important for dissecting fundamental and disease-relevant processes that modulate BBB coverage.

We explored whether white matter (WM) integrity in cognitively normal (CN) older adults is associated with cerebrospinal fluid (CSF) markers of Alzheimer's disease pathology. Twenty CN older adults underwent lumbar puncture and magnetic resonance imaging within a few days of each other. Analysis of diffusion tensor imaging data involved a priori region of interest and voxelwise approaches. The region of interest results revealed a positive correlation between CSF measures of amyloid-beta (Aβ 42 and Aβ 42 /p-Tau 181 ) and WM integrity in the fornix, a relationship which persisted after controlling for hippocampal volume and fornix volume. Lower WM integrity in the same portion of the fornix was also associated with reduced performance on the Digit Symbol test. Subsequent exploratory voxelwise analyses indicated a positive correlation between CSF Aβ 42 /p-Tau 181 and WM integrity in bilateral portions of the fornix, superior longitudinal fasciculus, inferior fronto-occipital fasciculus, and in the corpus callosum and left inferior longitudinal fasciculus. Our results link lower WM microstructural integrity in CN older adults with CSF biomarkers of Alzheimer's disease and suggest that this association in the fornix may be independent of volumetric measures.

Previous data demonstrate that bexarotene (Bex), retinoid X receptor (RXR) agonist, reduces soluble and insoluble amyloid-β (Aβ) in Alzheimer disease (AD)-transgenic mice either by increasing the levels of mouse apolipoprotein E (apoE) or increasing ABCA1/ABCG1-induced apoE lipoprotein association/lipidation. However, although the mechanism of action of RXR agonists remains unclear, a major concern for their use is human (h)-APOE4, the greatest AD genetic risk factor. If APOE4 imparts a toxic gain-of-function, then increasing apoE4 may increase soluble Aβ, likely the proximal AD neurotoxin. If the APOE4 loss-of-function is lipidation of apoE4, then induction of ABCA1/ABCG1 may be beneficial. In novel EFAD-Tg mice (overexpressing h-Aβ42 with h-APOE), levels of soluble Aβ (Aβ42 and oligomeric Aβ) are highest in E4FAD hippocampus (HP) > E3FAD-HP > E4FAD cortex (CX) > E3FAD-CX, whereas levels of lipoprotein-associated/lipidated apoE have the opposite pattern (6 months). In E4FAD-HP, short-term RXR agonist treatment (Bex or LG100268; 5.75–6 months) increased ABCA1, apoE4 lipoprotein-association/lipidation, and apoE4/Aβ complex, decreased soluble Aβ, and increased PSD95. In addition, hydrogel delivery, which mimics low sustained release, was equally effective as gavage for Bex and LG100268. RXR agonists induced no beneficial effects in the E4FAD-HP in a prevention protocol (5–6 months) and actually increased soluble Aβ levels in E3FAD-CX and E4FAD-CX with the short-term protocol, possibly the result of systemic hepatomegaly. Thus, RXR agonists address the loss-of-function associated with APOE4 and exacerbated by Aβ pathology, i.e. low levels of apoE4 lipoprotein association/lipidation. Further studies are vital to address whether RXR agonists are an APOE4-specific AD therapeutic and the systemic side effects that limit translational application.

Human apolipoprotein E (apoE) isoforms may differentially modulate amyloid-β (Aβ) levels. Evidence suggests physical interactions between apoE and Aβ are partially responsible for these functional effects. However, the apoE/Aβ complex is not a single static structure; rather, it is defined by detection methods. Thus, literature results are inconsistent and difficult to interpret. An ELISA was developed to measure soluble apoE/Aβ in a single, quantitative method and was used to address the hypothesis that reduced levels of soluble apoE/Aβ and an increase in soluble Aβ, specifically oligomeric Aβ (oAβ), are associated with APOE4 and AD. Previously, soluble Aβ42 and oAβ levels were greater with APOE4 compared with APOE2/APOE3 in hippocampal homogenates from EFAD transgenic mice (expressing five familial AD mutations and human apoE isoforms). In this study, soluble apoE/Aβ levels were lower in E4FAD mice compared with E2FAD and E3FAD mice, thus providing evidence that apoE/Aβ levels isoform-specifically modulate soluble oAβ clearance. Similar results were observed in soluble preparations of human cortical synaptosomes; apoE/Aβ levels were lower in AD patients compared with controls and lower with APOE4 in the AD cohort. In human CSF, apoE/Aβ levels were also lower in AD patients and with APOE4 in the AD cohort. Importantly, although total Aβ42 levels decreased in AD patients compared with controls, oAβ levels increased and were greater with APOE4 in the AD cohort. Overall, apoE isoform-specific formation of soluble apoE/Aβ modulates oAβ levels, suggesting a basis for APOE4-induced AD risk and a mechanistic approach to AD biomarkers.

Clinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer's disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD.NMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aβ in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function.The multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aβ pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2 (-/-) ) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aβ and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.

Clinical failures singularly targeting amyloid-β pathology indicate a critical need for alternative Alzheimer’s disease (AD) therapeutic strategies. The mixed pathology reported in a large population of AD patients demands a multifunctional drug approach. Since activation of cAMP response element binding protein (CREB) plays a crucial role in synaptic strengthening and memory formation, we retooled a clinical drug with known neuroprotective and anti-inflammatory activity to activate CREB, and validated this novel multifunctional drug, NMZ, in 4 different mouse models of AD. NMZ was tested in three mouse models of familial AD and one model of sporadic AD. In 3 × Tg hippocampal slices, NMZ restored LTP. In vivo, memory was improved with NMZ in all animal models with robust cognitive deficits. NMZ treatment lowered neurotoxic forms of Aβ in both APP/PS1 and 3 × Tg transgenic mice while also restoring neuronal plasticity biomarkers in the 3 × Tg mice. In EFAD mice, incorporation of the major genetic AD risk factor, hAPOE4, did not mute the beneficial drug effects. In a novel sporadic mouse model that manifests AD-like pathology caused by accelerated oxidative stress in the absence of any familial AD mutation, oral administration of NMZ attenuated hallmark AD pathology and restored biomarkers of synaptic and neuronal function. The multifunctional approach, embodied by NMZ, was successful in mouse models of AD incorporating Aβ pathology (APP/PS1), tau pathology (3xTg), and APOE4, the major human genetic risk factor for AD (EFAD). The efficacy observed in a novel model of sporadic AD (Aldh2 −/− ) demonstrates that the therapeutic approach is not limited to rare, familial AD genetic mutations. The multifunctional drug, NMZ, was not designed directly to target Aβ and tau pathology; however, the attenuation of this hallmark pathology suggests the approach to be a highly promising, disease-modifying strategy for AD and mixed pathology dementia.

Cerebrovascular dysfunction is a critical component of Alzheimer’s disease (AD) pathogenesis. Oligomeric amyloid-β42 (oAβ42) is considered a major contributor to AD progression. However, data are limited on the role of oAβ42 in brain endothelial cell vessel degeneration/angiogenesis, including the interaction with angiogenic mediators. Thus, the current study determined the effect of oAβ42 on angiogenesis in vitro, utilizing single brain endothelial cell cultures and triple cultures mimicking the microvascular unit (MVU: brain endothelial cells, astrocytes, and pericytes). oAβ42 dose-dependently reduced angiogenesis and induced vessel disruption. Critically, epidermal growth factor prevented oAβ42-induced deficits, implicating angiogenic pathways as potential therapeutics for AD.