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SummaryApolipoprotein E4 (APOE4) is the strongest risk allele associated with the development of late onset Alzheimer’s disease (AD). Across the CNS, astrocytes are the predominant expressor ofAPOEwhile also being critical mediators of neuroinflammation and cerebral metabolism. APOE4 has been consistently linked with dysfunctional inflammation and metabolic processes, yet insights into the molecular constituents driving these responses remain unclear. Utilizing complementary approaches across humanized APOE mice and isogenic human iPSC astrocytes, we demonstrate that ApoE4 alters the astrocyte immunometabolic response to pro-inflammatory stimuli. Our findings show that ApoE4-expressing astrocytes acquire distinct transcriptional repertoires at single-cell and spatially-resolved domains, which are driven in-part by preferential utilization of the cRel transcription factor. Further, inhibiting cRel translocation in ApoE4 astrocytes abrogates inflammatory-induced glycolytic shifts and in tandem mitigates production of multiple pro-inflammatory cytokines. Altogether, our findings elucidate novel cellular underpinnings by which ApoE4 drives maladaptive immunometabolic responses of astrocytes.

Elucidating the actions of genetic polymorphisms associated with the risk of Alzheimer's disease (AD) may provide novel insights into underlying mechanisms. Two polymorphisms have implicated

Background: Genetic variants in TREM2 are strongly associated with Alzheimer’s disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described. Objective: Recognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2. Methods: Human anterior cingulate autopsy tissue from 61 donors was used for end-point and quantitative PCR and western blotting to identify and quantify novel TREM2 isoforms. Results: In addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues with similar frequency (range 5.3 –13.0%). We found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, that both proteins are primarily retained in the Golgi complex, and that D2-TREM2 is expressed in AD and non-AD brain. Conclusion: Since the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and targeting TREM2 splicing may be a novel therapeutic pathway for AD.

BACKGROUND Adherence to a low-sodium diet is essential to self-care of heart failure (HF). Genetic determinants of preference for high-sodium foods may impede adherence but have not been well-studied. OBJECTIVE Our purpose was to examine if TAS2R38 haplotype predicted salt taste sensitivity and dietary sodium intake among patients with HF. METHOD This pilot study used baseline data from a large interventional randomized control trial to support adherence to a low-sodium diet in patients with HF and their family caregivers. Participants were tested for salt taste sensitivity and provided a 24-hour urinary sodium sample and a blood sample for DNA analysis at baseline. Fungiform papillae were counted. χ2 Test and 1-way analysis of variance were used to compare haplotype groups. Linear regression was performed to examine predictors of salt taste sensitivity and 24-hour urinary sodium excretion, controlling for age, gender, ethnicity, smoking status, and fungiform papillae density. RESULTS There were 42 patients with HF and their family caregivers (age, 64.6 ± 13.4 years, 46.5% male, 97.7% white, and 90.7% nonsmoker). Pronine-alanine-valine homozygous haplotype predicted lower urinary sodium excretion (b = -1780.59, t41 = -2.18, P = .036), but genotype was not a significant predictor of salt taste sensitivity. CONCLUSIONS The results of our study partially supported our hypothesis that PAV homozygous haplotype predicts 24-hour urinary sodium excretion. With our small sample size, more research is needed. Understanding genetic influences on taste can lead to development of educational interventions tailored to patients with HF and their family caregivers to better support dietary adherence.

Introduction: A heart-healthy diet might reduce cardiovascular disease (CVD) risk. Genetic variants that affect taste are associated with food choices. This study aims to investigate the associations of the TAS2R38 haplotype with consumption of sodium, sugar, saturated fats, and vegetables. Hypothesis: We hypothesized that, compared to people who are alanine-valine-isoleucine (AVI) homozygous for the TAS2R38 gene, those who are heterozygous or homozygous for the proline-alanine-valine (PAV) haplotype would have (a) a higher intake of sodium, sugar, and saturated fat, and (b) a lower vegetable intake. Methods: DNA from participants at risk for CVD was genotyped, and participants were assigned to groups by haplotype. Intake for sodium, sugar, saturated fat, and vegetables was assessed using the Viocare Food Frequency Questionnaire. Intake was categorized as higher versus lower consumption, divided at the median, and examined by logistic regressions. All models controlled for age, sex, smoking status, body mass index, education level, and financial status. Results: The 175 participants had a mean age of 52 ± 13 years, 72.6% were female, 100% were Caucasian, 89.1% were overweight or obese, and 82.9% were nonsmokers. Participants with one or two PAVs were grouped together, as PAV is the dominant gene, and comprised a majority of the sample (80.6%). Haplotype did not predict intake of sodium, sugar, or saturated fats. Compared to AVI homozygotes, participants with PAV homozygous or heterozygous haplotype had lower odds of being in the higher vegetable intake group (95% CI [0.17, 0.92], p = .032). Conclusions: PAV haplotype predicted lower consumption of vegetables. Variants of taste-related genes appear to play a role in food choices.

BackgroundGenetic variants in TREM2 are strongly associated with Alzheimer’s Disease (AD) risk but alternative splicing in TREM2 transcripts has not been comprehensively described.ObjectiveRecognizing that alternative splice variants can result in reduced gene expression and/or altered function, we sought to fully characterize splice variation in TREM2.MethodsHuman blood and anterior cingulate autopsy tissue from 61 donors were used for end-point and quantitative PCR and Western blotting to identify and quantify novel TREM2 isoforms.ResultsIn addition to previously described transcripts lacking exon 3 or exon 4, or retaining part of intron 3, we identified novel isoforms lacking exon 2, along with isoforms lacking multiple exons. Isoforms lacking exon 2 were predominant at approximately 10% of TREM2 mRNA in the brain. Expression of TREM2 and frequency of exon 2 skipping did not differ between AD samples and non-AD controls (p = 0.1268 and p = 0.4909, respectively). Further, these novel splice isoforms were also observed across multiple tissues with similar frequency (range 5.3 – 13.0%). We found that the exon 2 skipped isoform D2-TREM2 is translated to protein and localizes similarly to full-length TREM2 protein, that both proteins are primarily retained in the Golgi complex, and that D2-TREM2 is expressed in AD and non-AD brain.ConclusionSince the TREM2 ligand binding domain is encoded by exon 2, and skipping this exon retains reading frame while conserving localization, we hypothesize that D2-TREM2 acts as an inhibitor of TREM2 and targeting TREM2 splicing may be a novel therapeutic pathway for AD.

Ethnic discrimination is frequently experienced among U.S. Latinx communities, and is linked to CVD risk factors, such as depression. Genetic variants may influence this relationship.The objectives of this study were to examine associations between experiences of discrimination, rs4680 genotype, and depressive symptoms in Latinx adults.We analyzed data from 124 Latinx adults with two or more CVD risk factors, and conducted hierarchical linear regression, adjusting for sex, age, income, education, and acculturation.Participants were predominately female (74.2%) and aged 40.2 ± 9.3 years. More experiences of discrimination were associated with higher depressive symptoms (p = 0.041). Those with Met-Met-and Val-Met-genotypes had increased depressive symptoms than those with Val-Val-genotype (p = 0.049). Rs4680 was not a moderator.Findings suggest discrimination and rs4680 genotype are associated with depressive symptoms in Latinx adults, which may increase CVD risk. Further research is needed to better understand biological mechanisms of these relationships.

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines, which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

Apolipoprotein E (APOE) alleles impact pathogenesis and risk for multiple human diseases, making them primary targets for disease treatment and prevention. Previously, we and others reported an association between APOE alleles and the gut microbiome. Here, we evaluated effects of APOE heterozygosity and tested whether these overall results extended to mice maintained under ideal conditions for microbiome analyses. To model human APOE alleles, this study used APOE targeted replacement (TR) mice on a C57Bl/6 background. To minimize genetic drift, homozygous APOE3 mice were crossed to homozygous APOE2 or homozygous APOE4 mice prior to the study, and the resulting heterozygous progeny crossed further to generate the study mice. To maximize environmental homogeneity, mice with mixed genotypes were housed together and used bedding from the cages was mixed and added back as a portion of new bedding. Fecal samples were obtained from mice at 3-, 5- and 7-months of age, and microbiota analyzed by 16S ribosomal RNA gene amplicon sequencing. Linear discriminant analysis of effect size (LefSe) identified taxa associated with APOE status, depicted as cladograms to show phylogenetic relatedness. The influence of APOE status was tested on alpha-diversity (Shannon H index) and beta-diversity (principal coordinate analyses and PERMANOVA). Individual taxa associated with APOE status were identified by classical univariate analysis. Whether findings in the APOE mice were replicated in humans was evaluated by using published microbiome genome wide association data. Cladograms revealed robust differences with APOE in male mice and limited differences in female mice. The richness and evenness (alpha-diversity) and microbial community composition (beta-diversity) of the fecal microbiome was robustly associated with APOE status in male but not female mice. Classical univariate analysis revealed individual taxa that were significantly increased or decreased with APOE, illustrating a stepwise APOE2-APOE3–APOE4 pattern of association with heterozygous animals trending as intermediate in the stepwise pattern. The relative abundance of bacteria from the class Clostridia, order Clostridiales, family Ruminococacceae and related genera increased with APOE2 status. The relative abundance of Erysipelotrichia increased with APOE4 status, a finding that extended to humans. In this study, wherein mice were maintained in an ideal fashion for microbiome studies, gut microbiome profiles were strongly and significantly associated with APOE status in male APOE-TR mice. Erysipelotrichia are increased with APOE4 in both mice and humans. APOE allelic effects appeared generally intermediate in heterozygous animals. Further evaluation of these findings in humans, as well as studies evaluating the impact of the APOE-associated microbiota on disease-relevant phenotypes, will be necessary to determine if alterations in the gut microbiome represent a novel mechanism whereby APOE alleles impact disease.

We found evidence of late-onset Alzheimer disease (LOAD)-associated genetic polymorphism within an exon of Mucin 6 (MUC6) and immediately downstream from another gene: Adaptor Related Protein Complex 2 Subunit Alpha 2 (AP2A2). PCR analyses on genomic DNA samples confirmed that the size of the MUC6 variable number tandem repeat (VNTR) region was highly polymorphic. In a cohort of autopsied subjects with quantitative digital pathology data (n = 119), the size of the polymorphic region was associated with the severity of pTau pathology in neocortex. In a separate replication cohort of autopsied subjects (n = 173), more pTau pathology was again observed in subjects with longer VNTR regions (p = 0.031). Unlike MUC6, AP2A2 is highly expressed in human brain. AP2A2 expression was lower in a subset analysis of brain samples from persons with longer versus shorter VNTR regions (p = 0.014 normalizing with AP2B1 expression). Double-label immunofluorescence studies showed that AP2A2 protein often colocalized with neurofibrillary tangles in LOAD but was not colocalized with pTau proteinopathy in progressive supranuclear palsy, or with TDP-43 proteinopathy. In summary, polymorphism in a repeat-rich region near AP2A2 was associated with neocortical pTau proteinopathy (because of the unique repeats, prior genome-wide association studies were probably unable to detect this association), and AP2A2 was often colocalized with neurofibrillary tangles in LOAD.

Genome-wide association studies (GWAS) have identified immune-related genes as risk factors for Alzheimer’s disease (AD), including TREM2 and CD33, frequently passing a stringent false-discovery rate. These genes either encode or signal through immunomodulatory tyrosine-phosphorylated inhibitory motifs (ITIMs) or activation motifs (ITAMs) and govern processes critical to AD pathology, such as inflammation and amyloid phagocytosis. To investigate whether additional ITIM and ITAM-containing family members may contribute to AD risk and be overlooked due to the stringent multiple testing in GWAS, we combined protein quantitative trait loci (pQTL) data from a recent plasma proteomics study with AD associations in a recent GWAS. We found that pQTLs for genes encoding ITIM/ITAM family members were more frequently associated with AD than those for non-ITIM/ITAM genes. Further testing of one family member, SIGLEC14 which encodes an ITAM, uncovered substantial copy number variations, identified an SNP as a proxy for gene deletion, and found that gene expression correlates significantly with gene deletion. We also found that SIGLEC14 deletion increases the expression of SIGLEC5, an ITIM. We conclude that many genes in this ITIM/ITAM family likely impact AD risk, and that complex genetics including copy number variation, opposing function of encoded proteins, and coupled gene expression may mask these AD risk associations at the genome-wide level.

Although gene editing workflows typically consider the possibility of off-target editing, pseudogene-directed homology repair has not, to our knowledge, been reported previously. Here, we employed a CRISPR-Cas9 strategy for targeted excision of exon 2 in CD33 in U937 human monocyte cell line. Candidate clonal cell lines were screened by using a clinically relevant antibody known to label the IgV domain encoded by exon 2 (P67.6, gemtuzumab). In addition to the anticipated deletion of exon 2, we also found unexpected P67.6-negative cell lines which had apparently retained CD33 exon 2. Sequencing revealed that these lines underwent gene conversion from the nearby SIGLEC22P pseudogene during homology repair that resulted in three missense mutations relative to CD33. Ectopic expression studies confirmed that the P67.6 epitope is dependent upon these amino acids. In summation, we report that pseudogene-directed homology repair can lead to aberrant CRISPR gene editing.

Introduction: There is emerging evidence that ethnic discrimination, commonly experienced among U.S. Hispanics, is associated with depressive symptoms, increasing cardiovascular disease (CVD) risk. Genetic variants in dopaminergic pathways may moderate this association. The catechol-O-methyltransferase Val158Met polymorphism is a potential candidate because the Met allele is associated with increased risk for depressive symptoms. The purposes of this study were to examine associations among ethnic discrimination, Val158Met polymorphism, and depressive symptoms, and to explore if Val158Met moderates the association between discrimination and depressive symptoms in Hispanic adults. Methods: Participants were 124 Hispanic adults with 2+ CVD risk factors. Ethnic discrimination was measured with the Experiences of Discrimination instrument, which measures discrimination experiences across 9 settings (e.g. at work). Scores range from 0 to 45; higher scores indicate more experiences of discrimination. Depressive symptoms were measured using the Patient Health Questionnaire-8. Scores range from 0 to 21; higher scores indicate more depressive symptoms. DNA was isolated from saliva and analyzed for Val158Met genotype. A hierarchical linear regression was conducted adjusting for sex, age, income, education, acculturation, and years in the U.S. Discrimination was entered in Step 1 and Val158Met in Step 2 to examine the association with depressive symptoms. To assess moderation, a discrimination*Val158Met interaction term was entered in Step 3. Results: Participants were aged 40.2 ± 9.3 years and mostly female (74.2%). Discrimination was positively associated with depressive symptoms (p = 0.041). Participants with at least one Met allele at the Val158Met polymorphism had more depressive symptoms than those with a Val-Val genotype (p = 0.049). No moderation effect was found. Conclusion: Findings suggest that discrimination and Val158Met genotype independently influence depression in Hispanic adults, which may increase risk for depression and CVD in certain individuals. This highlights the need to explore mechanisms underpinning these associations, and to address the impact of discrimination on depression and CVD disparities.

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.

In 2011, genome-wide association studies implicated a polymorphism near CD33 as a genetic risk factor for Alzheimer's disease. This finding sparked interest in this member of the sialic acid-binding immunoglobulin-type lectin family which is linked to innate immunity. Subsequent studies found that CD33 is expressed in microglia in the brain and then investigated the molecular mechanism underlying the CD33 genetic association with Alzheimer's disease. The allele that protects from Alzheimer's disease acts predominately to increase a CD33 isoform lacking exon 2 at the expense of the prototypic, full-length CD33 that contains exon 2. Since this exon encodes the sialic acid ligand-binding domain, the finding that the loss of exon 2 was associated with decreased Alzheimer's disease risk was interpreted as meaning that a decrease in functional CD33 and its associated immune suppression was protective from Alzheimer's disease. However, this interpretation may need to be reconsidered given current findings that a genetic deletion which abrogates CD33 is not associated with Alzheimer's disease risk. Therefore, integrating currently available findings leads us to propose a model wherein the CD33 isoform lacking the ligand-binding domain represents a gain of function variant that reduces Alzheimer's disease risk.

Although abundant genetic and biochemical evidence strongly links Clusterin (CLU) to Alzheimer disease (AD) pathogenesis, the receptor for CLU within the adult brain is currently unknown. Using unbiased approaches, we identified Plexin A4 (PLXNA4) as a novel, high-affinity receptor for CLU in the adult brain. PLXNA4 protein expression was high in brain with much lower levels in peripheral organs. CLU protein levels were significantly elevated in the cerebrospinal fluid (CSF) of Plxna4-/- mice and, in humans, CSF levels of CLU were also associated with PLXNA4 genotype. Human AD brains had significantly increased the levels of CLU protein but decreased levels of PLXNA4 by ∼50%. To determine whether PLXNA4 levels influenced cognition, we analyzed the behaviour of Plxna4+/+, Plxna4+/-, and Plxna4-/- mice. In comparison to WT controls, both Plxna4+/- and Plxna4-/- mice were hyperactive in the open field assay while Plxna4-/- mice displayed a hyper-exploratory (low-anxiety phenotype) in the elevated plus maze. Importantly, both Plxna4+/- and Plxna4-/- mice displayed prominent deficits in learning and memory in the contextual fear-conditioning paradigm. Thus, even a 50% reduction in the level of PLXNA4 is sufficient to cause memory impairments, raising the possibility that memory problems seen in AD patients could be due to reductions in the level of PLXNA4. Both CLU and PLXNA4 have been genetically associated with AD risk and our data thus provide a direct relationship between two AD risk genes. Our data suggest that increasing the levels of PLXNA4 or targeting CLU-PLXNA4 interactions may have therapeutic value in AD.

The International Genomics of Alzheimer's Project (IGAP) is a consortium for characterizing the genetic landscape of Alzheimer's disease (AD). The identified and/or confirmed 19 single-nucleotide polymorphisms (SNPs) associated with AD are located on non-coding DNA regions, and their functional impacts on AD are as yet poorly understood. We evaluated the roles of the IGAP SNPs by integrating data from many resources, based on whether the IGAP SNP was (1) a proxy for a coding SNP or (2) associated with altered mRNA transcript levels. For (1), we confirmed that 12 AD-associated coding common SNPs and five nonsynonymous rare variants are in linkage disequilibrium with the IGAP SNPs. For (2), the IGAP SNPs in CELF1 and MS4A6A were associated with expression of their neighboring genes, MYBPC3 and MS4A6A, respectively, in blood. The IGAP SNP in DSG2 was an expression quantitative trait loci (eQTL) for DLGAP1 and NETO1 in the human frontal cortex. The IGAP SNPs in ABCA7, CD2AP, and CD33 each acted as eQTL for AD-associated genes in brain. Our approach for identifying proxies and examining eQTL highlighted potentially impactful, novel gene regulatory phenomena pertinent to the AD phenotype.

Individuals with genotypes that code for reduced dopaminergic brain activity often exhibit a predisposition toward aggression. However, it remains largely unknown how dopaminergic genotypes may increase aggression. Lower-functioning dopamine systems motivate individuals to seek reward from external sources such as illicit drugs and other risky experiences. Based on emerging evidence that aggression is a rewarding experience, we predicted that the effect of lower-functioning dopaminergic functioning on aggression would be mediated by tendencies to seek the environment for rewards. Caucasian female and male undergraduates (N = 277) were genotyped for five polymorphisms of the dopamine D2 receptor (DRD2) gene; they reported their previous history of aggression and their dispositional reward-seeking. Lower-functioning DRD2 profiles were associated with greater sensation-seeking, which then predicted greater aggression. Our findings suggest that lower-functioning dopaminergic activity puts individuals at risk for violence because it motivates them to experience aggression's hedonically rewarding qualities.

The CD33 single-nucleotide polymorphism (SNP) rs3865444 has been associated with the risk of Alzheimer's disease (AD). Rs3865444 is in linkage disequilibrium with rs12459419 which has been associated with efficacy of an acute myeloid leukemia (AML) chemotherapeutic agent based on a CD33 antibody. We seek to evaluate the extent to which CD33 genetics in AD and AML can inform one another and advance human disease therapy. We have previously shown that these SNPs are associated with skipping of CD33 exon 2 in brain mRNA. Here, we report that these CD33 SNPs are associated with exon 2 skipping in leukocytes from AML patients and with a novel CD33 splice variant that retains CD33 intron 1. Each copy of the minor rs12459419T allele decreases prototypic full-length CD33 expression by ∼ 25% and decreases the AD odds ratio by ∼ 0.10. These results suggest that CD33 antagonists may be useful in reducing AD risk. CD33 inhibitors may include humanized CD33 antibodies such as lintuzumab which was safe but ineffective in AML clinical trials. Here, we report that lintuzumab downregulates cell-surface CD33 by 80% in phorbol-ester differentiated U937 cells, at concentrations as low as 10 ng/ml. Overall, we propose a model wherein a modest effect on RNA splicing is sufficient to mediate the CD33 association with AD risk and suggest the potential for an anti-CD33 antibody as an AD-relevant pharmacologic agent.

Both common and rare polymorphisms within ABCA7 have been associated with Alzheimer’s disease (AD). In particular, the rare AD associated polymorphism rs200538373 was associated with altered ABCA7 exon 41 splicing and an AD risk odds ratio of ~1.9. To probe the role of this polymorphism in ABCA7 splicing, we used minigene studies and qPCR of human brain RNA. We report aberrant ABCA7 exon 41 splicing in the brain of a carrier of the rs200538373 minor C allele. Moreover, minigene studies show that rs200538373 acts as a robust functional variant in vitro. Lastly, although the ABCA7 isoform with an extended exon 41 is predicted to undergo nonsense mediated RNA decay, this was not supported by qPCR analyses, which showed relatively normal ABCA7 mRNA levels in the carrier of the rs200538373 minor C allele. In summary, rs200538373 is a functional polymorphism that alters ABCA7 exon 41 splicing without grossly altering the level of ABCA7 mRNA.

Background: Previous research indicates that total cholesterol levels increase with age during young adulthood and middle age and decline with age later in life. This is attributed to changes in diet, body composition, medication use, physical activity, and hormone levels. In the current study we utilized data from the Framingham Heart Study Original Cohort to determine if variations in apolipoprotein E (APOE), a gene involved in regulating cholesterol homeostasis, influence trajectories of total cholesterol, HDL cholesterol, and total: HDL cholesterol ratio from midlife through late life. Methods: Cholesterol trajectories from midlife through late life were modeled using generalized additive mixed models and mixed-effects regression models. Results: APOE e2+ subjects had lower total cholesterol levels, higher HDL cholesterol levels, and lower total: HDL cholesterol ratios from midlife to late life compared to APOE e3 and APOE e4+ subjects. Statistically significant differences in life span cholesterol trajectories according to gender and use of cholesterol-lowering medications were also detected. Conclusion: The findings from this research provide evidence that variations in APOE modify trajectories of serum cholesterol from midlife to late life. In order to efficiently modify cholesterol through the life span, it is important to take into account APOE allele status.

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.

Hippocampal sclerosis of aging (HS-Aging) is a high-morbidity brain disease in the elderly but risk factors are largely unknown. We report the first genome-wide association study (GWAS) with HS-Aging pathology as an endophenotype. In collaboration with the Alzheimer's Disease Genetics Consortium, data were analyzed from large autopsy cohorts: (#1) National Alzheimer's Coordinating Center (NACC); (#2) Rush University Religious Orders Study and Memory and Aging Project; (#3) Group Health Research Institute Adult Changes in Thought study; (#4) University of California at Irvine 90+ Study; and (#5) University of Kentucky Alzheimer's Disease Center. Altogether, 363 HS-Aging cases and 2,303 controls, all pathologically confirmed, provided statistical power to test for risk alleles with large effect size. A two-tier study design included GWAS from cohorts #1-3 (Stage I) to identify promising SNP candidates, followed by focused evaluation of particular SNPs in cohorts #4-5 (Stage II). Polymorphism in the ATP-binding cassette, sub-family C member 9 (ABCC9) gene, also known as sulfonylurea receptor 2, was associated with HS-Aging pathology. In the meta-analyzed Stage I GWAS, ABCC9 polymorphisms yielded the lowest p values, and factoring in the Stage II results, the meta-analyzed risk SNP (rs704178:G) attained genome-wide statistical significance (p = 1.4 × 10-9), with odds ratio (OR) of 2.13 (recessive mode of inheritance). For SNPs previously linked to hippocampal sclerosis, meta-analyses of Stage I results show OR = 1.16 for rs5848 (GRN) and OR = 1.22 rs1990622 (TMEM106B), with the risk alleles as previously described. Sulfonylureas, a widely prescribed drug class used to treat diabetes, also modify human ABCC9 protein function. A subsample of patients from the NACC database (n = 624) were identified who were older than age 85 at death with known drug history. Controlling for important confounders such as diabetes itself, exposure to a sulfonylurea drug was associated with risk for HS-Aging pathology (p = 0.03). Thus, we describe a novel and targetable dementia risk factor. © 2014 Springer-Verlag.

Genome-wide association studies (GWAS) have implicated a series of single nucleotide polymorphisms (SNPs) in Alzheimer’s disease (AD) risk. Elucidating the function of these SNPs is critical to identifying the underlying pathways and, potentially, novel therapeutic agents. SNPs within the gene ATP binding cassette A7 (ABCA7) reached significance in these studies, warranting investigation into their actions. Here, we analyzed ABCA7 expression in a set of human brain samples as a function of AD-associated SNPs and AD status. We report that the rs3764650T allele that decreases AD risk is associated with increased ABCA7 expression. However, ABCA7 expression is increased in AD individuals. We interpret our findings as suggesting a model wherein increased ABCA7 expression reduces AD risk and that the increased ABCA7 observed in AD reflects an inadequate compensatory change.

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54–55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39 ± 3 (%, mean ± SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19 ± 2 (%, mean ± SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.

As low-density lipoprotein receptor (LDLR) contributes to cholesterol and amyloid beta homeostasis, insights into LDLR regulation may facilitate our understanding of cardiovascular disease and Alzheimer's disease. Previously, we identified LDLR isoforms that lacked exon 12 or exons 11-12 and that are predicted to encode soluble, dominant negative, LDLR. Moreover, these isoforms were associated with rs688, an exon 12 polymorphism that was associated with LDL-cholesterol and Alzheimer's disease risk. In this study, we present evidence that although the truncated LDLR isoforms are translated in vitro, they represent < 0.1% of CSF proteins. As these LDLR isoforms likely represent a loss of mRNA-encoding functional LDLR, we then focused upon identifying intron-exon boundary and exonic splicing enhancer elements critical to splicing. Exon 12 inclusion is enhanced by altering the 5' splice site in intron 12 towards a consensus splice donor sequence, consistent with its being a weak 5' splice site. Additionally, of the nine evolutionarily conserved putative splicing enhancer regions within exon 12, two regions that flank rs688 were critical to exon 12 inclusion. Overall, these results suggest that LDLR splice variants represent a loss of mRNA encoding functional LDLR and provide insights into the regulatory elements critical for LDLR exon 12 splicing.

Adriamycin (ADR), a potent anti-tumor agent, produces reactive oxygen species (ROS) in cardiac tissue. Treatment with ADR is dose-limited by cardiotoxicity. However, the effect of ADR in the other tissues, including the brain, is unclear because ADR does not pass the blood-brain barrier. Some cancer patients receiving ADR treatment develop a transient memory loss, inability to handle complex tasks etc., often referred to by patients as chemobrain. We previously demonstrated that ADR causes CNS toxicity, in part, via systemic release of cytokines and subsequent generation of reactive oxygen and nitrogen species (RONS) in the brain. Here, we demonstrate that treatment with ADR led to an increased circulating level of tumor necrosis factor-alpha in wild-type mice and in mice deficient in the inducible form of nitric oxide (iNOSKO). However, the decline in mitochondrial respiration and mitochondrial protein nitration after ADR treatment was observed only in wild-type mice, not in the iNOSKO mice. Importantly, the activity of a major mitochondrial antioxidant enzyme, manganese superoxide dismutase (MnSOD), was reduced and the protein was nitrated. Together, these results suggest that NO is an important mediator, coupling the effect of ADR with cytokine production and subsequent activation of iNOS expression. We also identified the mitochondrion as an important target of ADR-induced NO-mediated CNS injury.

Plasmin System, Alzheimer's Disease and Stroke

Dysregulation of cholesterol homeostasis may be associated with the pathogenesis of coronary artery disease (CAD) and Alzheimers disease (AD). Recently, several single nucleotide polymorphisms (SNPs) in cholesteryl ester transfer protein (CETP) were associated with altered plasma CETP concentrations, cholesterol concentrations and CAD. Hence, these CETP SNPs represent excellent candidates for evaluating association with AD. To date, one study has evaluated the association between a single CETP SNP and AD. In this study, we examined three CETP SNPs to evaluate the genetic association of CETP with late onset AD on two study cohorts: the Religious Orders Study (ROS) series, including 85 AD and 70 non-AD individuals, and the University of Kentucky (UKY) series, including 78 AD and 84 non-AD individuals. Significant association between CETP genotypes or haplotypes and late onset AD was not detected in these two study cohorts. Moreover, the CETP genotypes and haplotypes were not significantly associated with AD when the populations were stratified for the presence or absence of apolipoprotein E4 (apoE4). In summary, CETP genetic variants were not associated with AD in two series.

Adriamycin (ADR) is a chemotherapeutic agent useful in treating various cancers. ADR is a quinone-containing anthracycline chemotherapeutic and is known to produce reactive oxygen species (ROS) in heart. Application of this drug can have serious side effects in various tissues, including brain, apart from the known cardiotoxic side effects, which limit the successful use of this drug in treatment of cancer. Neurons treated with ADR demonstrate significant protein oxidation and lipid peroxidation. Patients under treatment with this drug often complain of forgetfulness, lack of concentration, dizziness (collectively called somnolence or sometimes called chemobrain). In this study, we tested the hypothesis that ADR induces oxidative stress in brain. Accordingly, we examined the in vivo levels of brain protein oxidation and lipid peroxidation induced by i.p. injection of ADR. We also measured levels of the multidrug resistance-associated protein (MRP1) in brain isolated from ADR- or saline-injected mice. MRP1 mediates ATP-dependent export of cytotoxic organic anions, glutathione S-conjugates and sulphates. The current results demonstrated a significant increase in levels of protein oxidation and lipid peroxidation and increased expression of MRP1 in brain isolated from mice, 72 h post i.p injection of ADR. These results are discussed with reference to potential use of this redox cycling chemotheraputic agent in the treatement of cancer and its chemobrain side effect in brain.

While caspases have been strongly implicated in delayed neuronal death in a variety of experimental paradigms, other proteases such as calpain can also contribute to neuronal death. To evaluate the relative roles of caspase and calpain, we used a model system wherein UV treatment induced moderate or severe delayed cortical neuronal death, as quantified by propidium iodide and calcein AM. UV treatment led to increases in both caspase and calpain activation. Calpain inhibitor III (MDL-28170) reduced caspase activation, suggesting that caspase activation was mediated by calpain. Calpain contributed to neuronal death, as indicated by strong neuroprotection provided by calpain inhibitor III, calpeptin, or Ca2+-free medium. In contrast, caspase inhibitors were not neuroprotective. These results suggest that UV neurotoxicity is mediated by a loss of Ca2+ homeostasis which leads to a calpain-dependent, caspase-independent cell death. That calpain, but not caspase, may mediate death in instances involving the activation of both proteases may have relevance to other neuronal death models.

Reactive oxygen species are a necessary triggering event for apoptosis of sympathetic neurons after nerve growth factor (NGF) withdrawal. Reactive oxygen species can lead to the generation of 4-hydroxynonenal (HNE), a highly reactive aldehyde that forms adducts with proteins. This covalent modification can activate or inhibit signal transduction pathways involved in the induction of apoptosis. This process may be clinically relevant because HNE-adduct immunoreactivity increases in several disease states. Here we evaluate the role of HNE-adducts in sympathetic neurons undergoing NGF-deprivation-induced apoptosis, a model of developmental programmed cell death. We show that HNE-adduct immunoreactivity is dramatically increased after NGF-withdrawal in an NADPH oxidase-dependent manner. Moreover, HNE-adducts appear to contribute to NGF-deprivation-induced apoptotic signal transduction because microinjected HNE-adduct antiserum protects sympathetic neurons from NGF withdrawal. In conclusion, this report suggests the direct contribution of endogenously generated HNE in the stimulation of apoptotic signal transduction in neurons.

Caveolae are cholesterol-rich, membrane microdomains that appear critical to signaling between extracellular and intracellular macromolecules as well as cholesterol homeostasis. Caveolae formation is modulated by caveolin, a protein family that is the proteinaceous hallmark of caveolae. Very little is known regarding the events that modulate caveolin expression and regulation in neurons. To detect caveolin expression in neurons, primary rat hippocampal neurons were harvested at embryonic day 18, maintained for 7 days in vitro, and then analyzed for caveolin immunofluorescence. Caveolin-1 immunoreactivity was detected in cells that were identified as neurons by morphology and concurrent microtubule-associated protein (MAP2) staining. Changes in caveolin-1 expression were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR) analyses of RNA isolated from hippocampal neurons treated with glutamate receptor agonists. Glutamate induced a concentration-dependent increase in caveolin-1 mRNA. The largest increases in caveolin-1 mRNA were detected after 6 hours of treatment. Kainate and AMPA both mimicked glutamate effects on caveolin-1 mRNA expression. Western blot analyses revealed that caveolin was induced at the protein level as well. Taken together, these data suggest that glutamate can regulate caveolin expression through kainate and AMPA ionotropic glutamate receptors.

Amylin forms large beta-pleated neurotoxic oligomers but shows only 38% sequence similarity to A beta. As patterns of gene expression during neuronal apoptosis appear stimulus and cell type specific, we compared the pattern of amylin-induced gene expression in rat cortical neurons with that shown previously to be induced by A beta in order to evaluate whether these two peptides with different primary but similar secondary structure induce apoptosis similarly. Morphologic and quantitative measures of cell death show widespread apoptotic death after amylin treatment. Amylin treatment results in time- and concentration-dependent inductions of oxidative stress genes, such as cox-2 and IkappaB-alpha. "Apoptotic" genes are also induced in a time- and concentration-dependent manner, including c-jun, junB, c-fos, and fosB, followed temporally by a gene known to be modulated by these transcription factors, i.e., transin. In situ hybridization analyses show that c-fos expression is restricted largely to neurons with condensed chromatin, a hallmark of apoptosis. As these genes are not induced in all models of apoptosis, that amylin-induced neuronal death is genetically similar to that of A beta suggests that these peptides may be neurotoxic through a common mechanism.

Amyloid-beta (Abeta) appears central to Alzheimer's disease (AD), aggregates spontaneously, and is neurotoxic to neurons in vitro. Recently, several groups reported a familial AD locus on chromosome 10. Here, we note that urokinase-type plasminogen activator (uPA) is located within this locus. Previously, we reported that uPA and its functional homolog, tissue-type plasminogen activator, are induced by Abeta treatment of neurons in vitro as well as in a mouse model of Abeta accumulation in vivo. Moreover, the target of plasminogen activators, plasmin, degraded nonaggregated and aggregated Abeta and modulated Abeta toxicity and deposition. Here, we have evaluated the effects of uPA and plasminogen on Abeta fibril formation and neurotoxicity. We report that the combination of uPA and plasminogen, but neither alone, inhibits Abeta toxicity, reduces Abeta deposition in vitro, and inhibits Abeta fibrillogenesis. We interpret these observations as suggesting that uPA represents a possible candidate gene for the chromosome 10 familial AD locus.

Alzheimer’s disease (AD) is already the most common cause of dementia, and with rising life expectancies in the developed world, the number of AD cases can be expected to triple in the first half of this century [10]. The resultant economic and public health considerations of caring for AD patients has greatly intensified the search for novel therapies for this debilitating disease. There is considerable evidence that amyloid(A ) peptides play a role in AD, including in vitro studies demonstrating neurotoxicity derived apparently from the process of A aggregation (reviewed in [16]), genetic studies in which mutations in the gene encoding the A precursor protein are associated with familial AD (reviewed in [32,34]) and animal studies demonstrating that expression of mutated human genes linked with AD cause both A deposits and behavioral abnormalities (reviewed in [12,13]). While the exact link between A production/deposition and AD-associated neurodegeneration and dementia is still unclear and subject to rather energetic debate, one does not have to accept A as a cause of the disease to appreciate the possibility that reducing the A burden in the brains of AD patients could potentially result in behavioral and functional benefits. Taken in this overall context, clinical trials aimed at devising a vaccine to reduce the A burden in the AD brain is both timely and potentially therapeutic. Vaccines aimed at reducing A burden could act to reduce A deposits as well as soluble A . In this regard, it is relevant to note that A is present in several fashions in the AD brain. A deposits as fibrils in dense-cored senile plaques, a neuropathological hallmark of AD, as well as non-fibrillar, amorphous aggregates. Additionally, A is found in soluble forms that include diffusible A oligomers. The latter have been suggested to mediate the deleterious actions of A in

Tissue plasminogen (plgn) activator (tPA) modulates neuronal death in models of stroke, excitotoxicity, and oxidative stress. Amyloid-beta (Abeta) appears central to Alzheimer's disease and is neurotoxic to neurons in vitro. Here, we evaluate tPA effects on Abeta toxicity. We report that tPA alone had no effect on Abeta toxicity. However, in combination with plgn, tPA reduced Abeta toxicity in a robust fashion. Moreover, the combined tPA and plgn treatment markedly inhibited Abeta accumulation. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to a sample of tPA, plgn, and Abeta resulted in a marked reduction of Abeta degradation. We interpret the actions of tPA and plgn within the context of the ability of plasmin to degrade Abeta.