Alzheimer’s disease (AD) is an aging-related neurodegenerative disease characterized by an initial memory impairment that progresses to a widespread cerebrocortical failure, culminating in death. Understanding the molecular mechanisms that protect brain function during aging may help reveal novel targets for the development of effective treatments for the memory and cognitive deficits associated with AD. In this study, we analyzed a gene expression dataset generated from the prefrontal cortices of individuals showing no neurological or cognitive abnormalities. The gene expression profiles were used to identify candidate protective genes. We then compared the expression patterns of these genes in aging with their expression patterns in AD, thereby enabling us to pinpoint the genes that potentially contribute to brain resilience that delays or prevents aging-related dementia. We selected seven genes that are potentially protective for aging and AD, and have known homologues in Caenorhabditis elegans (C. elegans). Among these genes, SRPK2, AAK1, EFR3A and MAPK10 were previously implicated in attenuating AD-related cognitive decline. Our experiments demonstrated that all seven genes prioritized by our resilience model significantly extended the lifespan of C. elegans. Given the important relationship between neuronal functional integrity and lifespan (i.e., lifespan vs. brain health span), this work suggests the predicted AD resilience genes could serve as important candidate targets for therapeutic intervention.

Apolipoprotein E (APOE) has been shown to influence amyloid-β (Aβ) clearance from the brain in an isoform-specific manner. Our prior work showed that Aβ transit across the blood-brain-barrier was reduced by apoE4, compared to other apoE isoforms, due to elevated lipoprotein receptor shedding in brain endothelia. Recently, we demonstrated that matrix metallopeptidase 9 (MMP-9) induces lipoprotein receptor proteolysis in an apoE isoform-dependent manner, which impacts Aβ elimination from the brain. The current studies interrogated the relationship between apoE and MMP-9 and found that apoE impacted proMMP-9 cellular secretion from brain endothelia (apoE2 < apoE3 = apoE4). In a cell-free assay, apoE dose-dependently reduced MMP-9 activity, with apoE4 showing a significantly weaker ability to inhibit MMP-9 function than apoE2 or apoE3. Finally, we observed elevated MMP-9 expression and activity in the cerebrovasculature of both human and animal AD brain specimens with an APOE4 genotype. Collectively, these findings suggest a role for apoE in regulating MMP-9 disposition and may describe the effect of apoE4 on Aβ pathology in the AD brain.

A pathological characteristic of repetitive traumatic brain injury (TBI) is the deposition of hyperphosphorylated and aggregated tau species in the brain and increased levels of extracellular monomeric tau are believed to play a role in the pathogenesis of neurodegenerative tauopathies. The pathways by which extracellular tau is eliminated from the brain, however, remains elusive. The purpose of this study was to examine tau uptake by cerebrovascular cells and the effect of TBI on these processes. We found monomeric tau interacts with brain vascular mural cells (pericytes and smooth muscle cells) to a greater extent than other cerebrovascular cells, indicating mural cells may contribute to the elimination of extracellular tau, as previously described for other solutes such as beta-amyloid. Consistent with other neurodegenerative disorders, we observed a progressive decline in cerebrovascular mural cell markers up to 12 months post-injury in a mouse model of repetitive mild TBI (r-mTBI) and human TBI brain specimens, when compared to control. These changes appear to reflect mural cell degeneration and not cellular loss as no difference in the mural cell population was observed between r-mTBI and r-sham animals as determined through flow cytometry. Moreover, freshly isolated r-mTBI cerebrovessels showed reduced tau uptake at 6 and 12 months post-injury compared to r-sham animals, which may be the result of diminished cerebrovascular endocytosis, as caveolin-1 levels were significantly decreased in mouse r-mTBI and human TBI cerebrovessels compared to their respective controls. Further emphasizing the interaction between mural cells and tau, similar reductions in mural cell markers, tau uptake, and caveolin-1 were observed in cerebrovessels from transgenic mural cell-depleted animals. In conclusion, our studies indicate repeated injuries to the brain causes chronic mural cell degeneration, reducing the caveolar-mediated uptake of tau by these cells. Alterations in tau uptake by vascular mural cells may contribute to tau deposition in the brain following head trauma and could represent a novel therapeutic target for TBI or other neurodegenerative disorders.

Though discovered over 100 years ago, the molecular foundation of sporadic Alzheimer’s disease (AD) remains elusive. To elucidate its complex nature, we constructed multiscale causal network models on a large human AD multi-omics dataset, integrating clinical features of AD, DNA variation, and gene and protein expression into probabilistic causal models that enabled detection and prioritization of high-confidence key drivers of AD, including the top predicted key driver VGF. Overexpression of neuropeptide precursor VGF in 5xFAD mice partially rescued beta-amyloid-mediated memory impairment and neuropathology. Molecular validation of network predictions downstream of VGF was achieved, with significant enrichment for homologous genes identified as differentially expressed in 5xFAD brains overexpressing VGF versus controls. Our findings support a causal and/or protective role for VGF in AD pathogenesis and progression. One sentence summary VGF protects against Alzheimer’s disease

Lipoprotein receptor transport across the blood-brain barrier (BBB) mediates beta-amyloid (Aβ) accumulation in the brain and may be a contributing factor in Alzheimer’s disease (AD) pathogenesis. Lipoprotein receptors are susceptible to proteolytic shedding at the cell surface, which precludes the endocytic transport of ligands. A ligand that closely interacts with the lipoprotein receptors is apolipoprotein E (apoE), which exists as three isoforms (apoE2, apoE3, apoE4). Our prior work showed an inverse relationship between lipoprotein receptor shedding and Aβ transport across the BBB, which was apoE-isoform dependent. To interrogate this further, the current studies investigated an enzyme implicated in lipoprotein receptor shedding, matrix metalloproteinase 9 (MMP9). Treatment with MMP9 dose-dependently elevated lipoprotein receptor shedding in brain endothelial cells and freshly isolated mouse cerebrovessels. Furthermore, treatment with a MMP9 inhibitor (SB-3CT) mitigated Aβ-induced lipoprotein receptor shedding in brain endothelial cells and the brains of apoE4 animals. In terms of BBB transit, SB-3CT treatment increased the transport of Aβ across an in vitro model of the BBB. In vivo, administration of SB-3CT to apoE4 animals significantly enhanced Aβ clearance from the brain to the periphery following intracranial administration of Aβ. The current studies show that MMP9 impacts lipoprotein receptor shedding and Aβ transit across the BBB, in an apoE  isoform-specific manner. In total, MMP9 inhibition can facilitate Aβ clearance across the BBB, which could be an effective approach to lowering Aβ levels in the brain and mitigating the AD phenotype, particularly in subjects carrying the apoE4 allele.

Alzheimer's disease (AD) is a neurodegenerative process characterized, in part, by an accumulation of the beta-amyloid (Aβ) protein in the brain and cerebrovasculature. Mounting evidence suggests the excessive accumulation of Aβ in the AD brain is the result of impaired Aβ clearance mechanisms such as dysfunctional Aβ transport at the blood-brain barrier (BBB). The BBB transporter primarily responsible for the brain-to-blood elimination of Aβ is the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1). While LRP1 interacts with an array of ligands, one of the more closely associated is apolipoprotein E (apoE). Previously, we found an inverse relationship between LRP1 proteolysis (i.e. shedding) and Aβ transit across the BBB, one that is apoE isoform-specific. To elucidate the mechanisms driving these observations, we examined the interactions between apoE and a known LRP1 sheddase, matrix metallopeptidase 9 (MMP-9). We examined MMP-9 expression and activity according to apoE genotype in cerebrovascular tissue from human and mouse AD brains. In addition we looked at the effect of apoE genotype on MMP-9 dynamics in vitro. We found that MMP-9 expression and activity is increased in human cerebrovascular tissue in AD subjects and exhibits an apoE genotype difference. In both a cell-free assay and in primary human brain endothelial cells, MMP-9 activity was significantly attenuated by apoE in an isoform-specific manner (apoE2>apoE3>apoE4). In apoE4 animals, treatment with an MMP-9 inhibitor, SB-3CT, resulted in a significant reduction in brain LRP1 shedding and increased Aβ clearance from the brain to the periphery. Our findings indicate apoE4 is less effective in modulating MMP-9 activity and LRP1 shedding than the other apoE isoforms. These studies show that MMP-9 modulation can facilitate Aβ removal from the brain, which may provide a novel approach to the treatment of AD, particularly in individuals with an apoE4 genotype.

The contribution of apolipoprotein E (APOE) e4 to Alzheimer's disease (AD) pathology is characterized by the presence of severe cerebral amyloid angiopathy (CAA), increased blood-brain-barrier (BBB) breakdown and reduced cerebral vascularization in e4 carriers. It has been proposed that the diminished capacity of the apoE4 protein to transport docosahexaenoic acid (DHA), an essential fatty acid that is required for the structural and functional maintenance and vascular integrity of the brain also contributes to AD pathogenesis. However, it remains to be determined if there are changes in brain DHA content of phospholipids (PL) and in DHA transporters in relation to the e4 allele and AD diagnosis and brain cerebrovascular amyloid pathology. We performed liquid chromatography/mass spectrometry based PL analysis of the cerebrovascular and parenchymal fractions from autopsied human brain tissue of pathologically confirmed AD cases and controls. We performed antibody-based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects. In the cerebrovascular and parenchymal fractions, DHA containing PL species were lower in e4 carriers with AD cases compared to control ε4 carriers. We observed an APOE e4 dependent decreases in mfsd2a expression in the brain cerebrovasculature. The mfsd2a expression was lower in e4 carriers compared to non-carriers. Stratification of DHA containing PL by CAA showed that these PL levels were reduced in e4 positive AD patients with severe CAA. These findings demonstrate that brain DHA deficiencies in e4 carriers may be due to reduced mfsd2a expression and partly associated with CAA. Thus, targeting this transport mechanism may improve the bioavailability of DHA to the brain of e4 carriers who are at risk of developing AD.

BACKGROUND:The APOE4 allele is the strongest genetic risk factor for Alzheimer's disease (AD). It has been associated with an accumulation of amyloid-β (Aβ) in the brain, which is produced through the sequential cleavage of the amyloid-β precursor protein (AβPP) by β - and γ-secretases. Alternatively, AβPP is also cleaved by α -secretases such as A Disintegrin and Metalloproteinase Domain-containing Protein 10 (ADAM10).OBJECTIVE:While several studies have investigated the impact of apoE on β- and γ-secretase, interactions between apoE and α-secretases have not been fully examined. We investigated the effect of each apoE isoform on ADAM10 in vitro and in human cortex samples.METHOD:ADAM10 activity and kinetics was assessed in cell-free assays and the biological activity of ADAM10 further investigated in 7WCHO cells over-expressing wild type AβPP through ELISA. Finally, ADAM10 expression and activity was observed in the soluble fraction of both control and Alzheimer's Disease human cortex samples through ELISA.RESULTS:In a cell free assay, ADAM10 activity was found to be significantly lower in apoE4 samples compared to apoE2. 7WCHO cells over expressing wild type AβPP exposed to apoE4 demonstrated reduced formation of sAβPPα compared to other apoE isoforms. We also identified APOE and AD dependent changes in ADAM10 activity and expression in the soluble brain fraction of human brain cortex.CONCLUSION:Overall, our data demonstrates an apoE isoform-dependent effect on ADAM10 function and AβPP processing which may describe the elevated amyloid levels in the brains of AD subjects carrying the APOE4 allele.

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.

The apolipoprotein (APOE) e4 allele is one of the strongest genetic risk factors for Alzheimer’s disease (AD). While the apoE4 protein is well known for its limited capacity to transport lipids, little examination has been performed on blood lipids in the context of the e4 status and their collective contributions to AD. Lipidomic analyses were performed on serum samples from a longitudinal cohort of cognitively normal subjects, a subset of whom converted to mild cognitive impairment (MCI)/AD within 2 to 3 years. To examine lipids in relation to the APOE genotypes and AD pathology in a mouse model, we performed lipidomic profiling of plasma samples from human APOE targeted replacement mice (APOE-TR) and APOE-TR mice crossed with mice harboring 5 AD mutations (EFAD). To gain a mechanistic understanding of the differences in lipid transport in relation to the e4 allele, we assessed arachidonic acid (AA) and docosahexaenoic acid (DHA) transit in an in vitro blood brain barrier (BBB) model in the presence of different apoE isoforms. Serum from cognitively normal e4 carriers who converted to MCI/AD had increased AA/DHA ratios in major phospholipid (PL) classes compared to non-carriers and cognitively normal e4 carriers. Combining these PL species, e4 status, and the Abeta42/40 ratio gave 91% accuracy for detecting preclinical MCI/AD. Both e4 carriers and non-carriers who consumed fish oil/omega-3 had lower AA/DHA ratios within several PL classes compared to non-users. The AA/DHA ratio within PL was increased in plasma from mice expressing human APOE4 relative to E2 or E3 isoforms and in E4FAD mice compared to EFAD animals with other APOE isoforms. In vitro BBB studies showed the apical-to-basolateral transit of both AA and DHA was diminished using human serum from e4/e4 compared to e3/e3 individuals. The elevated AA/DHA ratios within PL observed in serum of e4 carriers with preclinical MCI/AD could be due to impaired lipid transport into the brain. Finally, these studies indicate that determination of AA and DHA containing PL together with APOE genotype could assist in the detection of preclinical MCI/AD.

The cerebrovascular system and the Blood-Brain barrier (BBB) are important factors in the aetiology and pathogenesis of Alzheimer's Disease. The clearance of Aβ species to the periphery through the BBB is a primary route for removing Aβ from the brain. One of the main receptors responsible for the transport and removal of Aβ from the brain to the periphery is the Low density lipoprotein Receptor like Protein 1 (LRP1). LRP1 exists as both a membrane bound receptor, where it is able to facilitate clearance of Aβ through transcytosis, and as a soluble receptor (sLRP) that maintains its binding properties but loses the ability to transport Aβ to the periphery. Therefore, reducing the formation of sLRP would increase the pool of functional LRP1, facilitating removal of Aβ from the brain. Several proteases are capable of inducing the ectodomain shedding of LRP1, including A Desintegrin and Metalloproteinase Domain containing protein 10 (ADAM10). Levels of sLRP1 and clearance of Aβ were assessed in an in vitro BBB model and in an in vivo study utilising ADAM10 knockout (KO) and ADAM10 endothelial-cell KO mice. Additionally, PSAPP mice were treated with an ADAM10 inhibitor and brain Aβ load, Aβ transport to the periphery and production of sLRP1 was assessed. ADAM10 inhibition reduced the production of sLRP1 and increased Aβ transit in an in vitro BBB model. This was confirmed in vivo, as ADAM10 KO showed significantly lower levels of sLRP1 in the brain compared to wild type (WT) mice. Aβ clearance across the BBB in ADAM10 KO and ADAM10 endothelial-cell KO mice was also significantly higher than WT mice. Treatment of PSAPP mice with an ADAM10 specific inhibitor reduced sLRP1 levels and increased Aβ40 levels in the plasma. Levels of soluble and insoluble Aβ in the brain, were consistently lower than vehicle-treated mice, though these effects did not reach statistical significance. Inhibition of enzymes responsible for the shedding of LRP1 may facilitate removal of Aβ from the brain and provide a novel target for the treatment of AD. Our future work will examine the impact of a more chronic treatment paradigm on Aβ levels in the brain.

The contribution of apolipoprotein E (APOE) E4 to the cerebrovascular dysfunction in Alzheimer’s disease (AD) is characterized by the presence of severe cerebral amyloid angiopathy, increased blood-brain-barrier (BBB) breakdown, reduced cerebral vascularization, and basement membrane thinning in E4 carriers compared to non-carriers. It has also been proposed that the diminished capacity of the apoE4 protein to transport essential polyunsaturated fatty acids (PUFAs) that are required for the structural and functional maintenance and vascular integrity of the brain also contribute to AD pathogenesis. However, it remains to be determined if there are changes in the profiles of phospholipids (PL) and in the expression of lipid transporters within the brain vasculature in relation to the E4 allele and AD diagnosis. We performed liquid chromatography/mass spectrometry based lipidomic analysis of the cerebrovascular and parenchymal fractions from autopsied human brain tissue of pathologically confirmed AD cases and controls stratified by APOE genotype. In order to determine if there were changes in the expression of lipid transporters in relation to the APOE E4 allele, we performed antibody based examination of the major facilitator superfamily domain containing 2A (mfsd2a) protein in the cerebrovasculature from these subjects. Total phosphatidylcholine (PC) was significantly lower in the cerebrovascular fractions of AD patients compared to controls. While docosahexaenoic acid (DHA) containing PL species were lower in heterozygous E4 AD patients compared to E4 controls in both the cerebrovascular and parenchymal fractions, an ether PC species containing arachidonic acid (AA) was elevated within the cerebrovasculature of E4 carriers relative to non-carriers and was highest among E4 AD specimens compared to E4 controls. We also observed an APOE E4 dependent difference in mfsd2a expression. Among AD patients, E4 homozygotes had lower expression of mfsd2a than E4 heterozygotes and non-carriers. These findings demonstrate that deficiencies in DHA within the brains of APOE E4 carriers may, in part, be due to lower expression of mfsd2a. Thus, targeting this transport mechanism may improve the bioavailability of DHA to the brain of APOE E4 individuals providing a novel approach to the treatment of AD.

Transport across the blood–brain barrier (BBB) is an important mediator of beta-amyloid (Aβ) accumulation in the brain and a contributing factor in the pathogenesis of Alzheimer’s disease (AD). One of the receptors responsible for the transport of Aβ in the BBB is the low density lipoprotein receptor-related protein 1 (LRP1). LRP1 is susceptible to proteolytic shedding at the cell surface, which prevents endocytic transport of ligands. Previously, we reported a strong inverse correlation between LRP1 shedding in the brain and Aβ transit across the BBB. Several proteases contribute to the ectodomain shedding of LRP1 including the α-secretase, a desintegrin and metalloproteinase domain containing protein 10 (ADAM10).

Recent findings indicate an isoform-specific role for apolipoprotein E (apoE) in the elimination of beta-amyloid (Aβ) from the brain. ApoE is closely associated with various lipoprotein receptors, which contribute to Aβ brain removal via metabolic clearance or transit across the blood–brain barrier (BBB). These receptors are subject to ectodomain shedding at the cell surface, which alters endocytic transport and mitigates Aβ elimination. To further understand the manner in which apoE influences Aβ brain clearance, these studies investigated the effect of apoE on lipoprotein receptor shedding. Consistent with prior reports, we observed an increased shedding of the low-density lipoprotein receptor (LDLR) and the LDLR-related protein 1 (LRP1) following Aβ exposure in human brain endothelial cells. When Aβ was co-treated with each apoE isoform, there was a reduction in Aβ-induced shedding with apoE2 and apoE3, while lipoprotein receptor shedding in the presence of apoE4 remained increased. Likewise, intracranial administration of Aβ to apoE-targeted replacement mice (expressing the human apoE isoforms) resulted in an isoform-dependent effect on lipoprotein receptor shedding in the brain (apoE4 > apoE3 > apoE2). Moreover, these results show a strong inverse correlation with our prior work in apoE transgenic mice in which apoE4 animals showed reduced Aβ clearance across the BBB compared to apoE3 animals. Based on these results, apoE4 appears less efficient than other apoE isoforms in regulating lipoprotein receptor shedding, which may explain the differential effects of these isoforms in removing Aβ from the brain.