RESUMEN
Apolipoprotein E (ApoE) is a lipid transport protein that is hypothesized to suppress proinflammatory cytokine production, particularly after stimulation with Toll-like receptor (TLR) ligands such as lipopolysaccharide (LPS). Studies using transgenic ApoE human replacement mice (APOE) expressing one of three different allelic variants suggest that there is a hierarchy in terms of responsiveness to proinflammatory stimuli such as APOE4/E4 > APOE3/E3 > APOE2/E2. In this study, we test the hypothesis that APOE genotype can also predict susceptibility to infection with the facultative intracellular gram-positive bacterium Listeria monocytogenes. We found that bone-marrow-derived macrophages isolated from aged APOE4/E4 mice expressed elevated levels of nitric oxide synthase 2 and were highly resistant to in vitro infection with L. monocytogenes compared to APOE3/E3 and APOE2/E2 mice. However, we did not find statistically significant differences in cytokine or chemokine output from either macrophages or whole splenocytes isolated from APOE2/E2, APOE3/E3, or APOE4/E4 mice following L. monocytogenes infection. In vivo, overall susceptibility to foodborne listeriosis also did not differ by APOE genotype in either young (2 mo old) or aged (15 mo old) C57BL/6 mice. However, we observed a sex-dependent susceptibility to infection in aged APOE2/E2 male mice and a sex-dependent resistance to infection in aged APOE4/E4 male mice that was not present in female mice. Thus, these results suggest that APOE genotype does not play an important role in innate resistance to infection with L. monocytogenes but may be linked to sex-dependent changes that occur during immune senescence.
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Listeria monocytogenes , Listeriosis , Animales , Femenino , Humanos , Masculino , Ratones , Apolipoproteína E2 , Apolipoproteína E3 , Apolipoproteína E4 , Apolipoproteínas E/genética , Citocinas , Genotipo , Ratones Endogámicos C57BL , Ratones TransgénicosRESUMEN
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.
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Cuidadores , Insuficiencia Cardíaca , Receptores Acoplados a Proteínas G/genética , Sodio/orina , Anciano , Dieta Hiposódica , Femenino , Haplotipos , Insuficiencia Cardíaca/genética , Humanos , Masculino , Persona de Mediana Edad , Proyectos PilotoRESUMEN
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.
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Enfermedad de Alzheimer/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/fisiología , Secuencias de Aminoácidos , Secuencia de Consenso , Dimerización , Exones/genética , Mutación con Ganancia de Función , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Metaanálisis como Asunto , Microglía/fisiología , Familia de Multigenes , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Eliminación de Secuencia , Lectina 3 Similar a Ig de Unión al Ácido Siálico/química , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/genética , Lectinas Similares a la Inmunoglobulina de Unión a Ácido Siálico/metabolismoRESUMEN
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 â¼50%. 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.
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Enfermedad de Alzheimer/genética , Clusterina/genética , Mapas de Interacción de Proteínas/genética , Receptores de Superficie Celular/genética , Enfermedad de Alzheimer/fisiopatología , Animales , Clusterina/biosíntesis , Cognición/fisiología , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Memoria/fisiología , Ratones , Ratones Noqueados , Polimorfismo de Nucleótido Simple , Receptores de Superficie Celular/biosíntesis , Factores de RiesgoRESUMEN
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 â¼ 25% and decreases the AD odds ratio by â¼ 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.
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Enfermedad de Alzheimer/genética , Estudios de Asociación Genética , Leucemia Mieloide Aguda/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Anciano , Anciano de 80 o más Años , Alelos , Empalme Alternativo , Enfermedad de Alzheimer/metabolismo , Anticuerpos Monoclonales Humanizados/farmacología , Línea Celular , Exones , Femenino , Expresión Génica , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Intrones , Leucemia Mieloide Aguda/metabolismo , Masculino , Polimorfismo de Nucleótido Simple , Estabilidad del ARN , ARN Mensajero/genética , Lectina 3 Similar a Ig de Unión al Ácido Siálico/antagonistas & inhibidores , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismoRESUMEN
Genome-wide association studies are identifying novel Alzheimer's disease (AD) risk factors. Elucidating the mechanism underlying these polymorphisms is critical to the validation process and, by identifying rate-limiting steps in AD risk, may yield novel therapeutic targets. Here, we elucidate the mechanism of action of the AD-associated polymorphism rs3865444 in the promoter of CD33, a member of the sialic acid-binding Ig-superfamily of lectins (SIGLECs). Immunostaining established that CD33 is expressed in microglia in human brain. Consistent with this finding, CD33 mRNA expression correlated well with expression of the microglial genes CD11b and AIF-1 and was modestly increased with AD status and the rs3865444C AD-risk allele. Analysis of CD33 isoforms identified a common isoform lacking exon 2 (D2-CD33). The proportion of CD33 expressed as D2-CD33 correlated robustly with rs3865444 genotype. Because rs3865444 is in the CD33 promoter region, we sought the functional polymorphism by sequencing CD33 from the promoter through exon 4. We identified a single polymorphism that is coinherited with rs3865444, i.e., rs12459419 in exon 2. Minigene RNA splicing studies in BV2 microglial cells established that rs12459419 is a functional single nucleotide polymorphism (SNP) that modulates exon 2 splicing efficiency. Thus, our primary findings are that CD33 is a microglial mRNA and that rs3865444 is a proxy SNP for rs12459419 that modulates CD33 exon 2 splicing. Exon 2 encodes the CD33 IgV domain that typically mediates sialic acid binding in SIGLEC family members. In summary, these results suggest a novel model wherein SNP-modulated RNA splicing modulates CD33 function and, thereby, AD risk.
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Empalme Alternativo/genética , Enfermedad de Alzheimer/genética , Exones/genética , Predisposición Genética a la Enfermedad/genética , Polimorfismo de Nucleótido Simple , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Genotipo , Humanos , Inmunohistoquímica , Regiones Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de RiesgoRESUMEN
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.
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Envejecimiento/genética , Envejecimiento/patología , Hipocampo/patología , Polimorfismo de Nucleótido Simple , Receptores de Sulfonilureas/genética , Anciano de 80 o más Años , Envejecimiento/efectos de los fármacos , Estudios de Cohortes , Bases de Datos como Asunto , Endofenotipos , Estudio de Asociación del Genoma Completo , Hipocampo/efectos de los fármacos , Humanos , Esclerosis/genética , Esclerosis/patología , Compuestos de Sulfonilurea/efectos adversos , Compuestos de Sulfonilurea/uso terapéuticoRESUMEN
Traumatic brain injury (TBI) increases gastrointestinal morbidity and associated mortality. Clinical and preclinical studies implicate gut dysbiosis as a consequence of TBI and an amplifier of brain damage. However, little is known about the association of gut dysbiosis with structural and functional changes of the gastrointestinal tract after an isolated TBI. To assess gastrointestinal dysfunction, mice received a controlled cortical impact or sham brain injury and intestinal permeability was assessed at 4 h, 8 h, 1 d, and 3 d after injury by oral administration of 4 kDa FITC Dextran prior to euthanasia. Quantification of serum fluorescence revealed an acute, short-lived increase in permeability 4 h after TBI. Despite transient intestinal dysfunction, no overt morphological changes were evident in the ileum or colon across timepoints from 4 h to 4 wks post-injury. To elucidate the timeline of microbiome changes after TBI, 16 s gene sequencing was performed on DNA extracted from fecal samples collected prior to and over the first month after TBI. Differential abundance analysis revealed that the phylum Verrucomicrobiota was increased at 1, 2, and 3 d after TBI. The Verrucomicrobiota species was identified by qPCR as Akkermansia muciniphila, an obligate anaerobe that resides in the intestinal mucus bilayer and produces short chain fatty acids (e.g. butyrate) utilized by intestinal epithelial cells. We postulated that TBI promotes intestinal changes favorable for the bloom of A. muciniphila. Consistent with this premise, the relative area of mucus-producing goblet cells in the medial colon was significantly increased at 1 d after injury, while colon hypoxia was significantly increased at 3 d. Our findings reveal acute gastrointestinal functional changes coupled with an increase of beneficial bacteria suggesting a potential compensatory response to systemic stress after TBI.
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Lesiones Traumáticas del Encéfalo , Lesiones Encefálicas , Enfermedades Gastrointestinales , Ratones , Animales , Disbiosis/complicaciones , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Encefálicas/complicaciones , Verrucomicrobia , Íleon , Enfermedades Gastrointestinales/complicaciones , Permeabilidad , AkkermansiaRESUMEN
ß-Secretase, the rate-limiting enzymatic activity in the production of the amyloid-ß (Aß) peptide, is a major target of Alzheimer's disease (AD) therapeutics. There are two forms of the enzyme: ß-site Aß precursor protein cleaving enzyme (BACE) 1 and BACE2. Although BACE1 increases in late-stage AD, little is known about BACE2. We conducted a detailed examination of BACE2 in patients with preclinical to late-stage AD, including amnestic mild cognitive impairment, and age-matched controls, cases of frontotemporal dementia, and Down's syndrome. BACE2 protein and enzymatic activity increased as early as preclinical AD and were found in neurons and astrocytes. Although the levels of total BACE2 mRNA were unchanged, the mRNA for BACE2 splice form C (missing exon 7) increased in parallel with BACE2 protein and activity. BACE1 and BACE2 were strongly correlated with each other at all levels, suggesting that their regulatory mechanisms may be largely shared. BACE2 was also elevated in frontotemporal dementia but not in Down's syndrome, even in patients with substantial Aß deposition. Thus, expression of both forms of ß-secretase are linked and may play a combined role in human neurologic disease. A better understanding of the normal functions of BACE1 and BACE2, and how these change in different disease states, is essential for the future development of AD therapeutics.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Química Encefálica , Femenino , Humanos , Masculino , Neprilisina/metabolismo , Neuronas/metabolismo , ARN Mensajero/metabolismoRESUMEN
The single nucleotide polymorphisms rs35349669 and rs10933431 within Inositol Polyphosphate-5-Phosphatase D (INPP5D) are strongly associated with Alzheimer's Disease risk. To better understand INPP5D expression in the brain, we investigated INPP5D isoform expression as a function of rs35349669 and rs10933431, as well as Alzheimer's disease neuropathology, by qPCR and isoform-specific primers. In addition, INPP5D allelic expression imbalance was evaluated relative to rs1141328 within exon 1. Expression of INPP5D isoforms associated with transcription start sites in exon 1 and intron 14 was increased in individuals with high Alzheimer's disease neuropathology. In addition, a novel variant with 47bp lacking from exon 12 increased expression in Alzheimer's Disease brains, accounting for 13% of total INPP5D expression, and was found to undergo nonsense-mediated decay. Although inter-individual variation obscured a possible polymorphism effect on INPP5D isoform expression as measured by qPCR, rs35349669 was associated with rs1141328 allelic expression imbalance, suggesting that rs35349669 is significantly associated with full-length INPP5D isoform expression. In summary, expression of INPP5D isoforms with start sites in exon 1 and intron 14 are increased in brains with high Alzheimer's Disease neuropathology, a novel isoform lacking the phosphatase domain was significantly increased with the disease, and the polymorphism rs35349669 correlates with allele-specific full-length INPP5D expression.
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Enfermedad de Alzheimer , Encéfalo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Humanos , Alelos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Encéfalo/metabolismo , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/genética , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas/metabolismo , Polimorfismo de Nucleótido Simple , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Background: Understanding the mechanisms whereby genetic variants influence the risk of Alzheimer's disease (AD) may provide insights into treatments that could reduce AD risk. Objective: Here, we sought to test the hypothesis that a single nucleotide polymorphism (SNP) associated with AD risk, rs2070902, influences splicing of FCER1G exon 2. Methods: AD and non-AD brain samples were analyzed for FCER1G expression by genotyping, immunohistochemistry, immunofluorescence, and qPCR. Results: The protein encoded by FCER1G, FcRγ, is robustly expressed in microglia in both AD and non-AD brain. The FCER1G isoform lacking exon 2 (D2-FCER1G) was readily detectable. Moreover, the proportion of FCER1G expressed as this isoform was increased in brains with high AD neuropathology. However, the proportion of FCER1G expressed as the D2-FCER1G isoform was not associated with rs2070902 genotype. Conclusions: In summary, the proportion of FCER1G expressed as the D2-FCER1G isoform is increased with AD neuropathology but is not associated with rs2070902.
RESUMEN
Apolipoprotein 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 of APOE while 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.
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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 ABI3 as a modulator of AD risk. Here, we sought to identify ABI3 isoforms expressed in human AD and non-AD brain, quantify the more abundant isoforms as a function of AD genetics and neuropathology, and provide an initial in vitro characterization of the proteins produced by these novel isoforms. We report that ABI3 expression is increased with AD neuropathology but not associated with AD genetics. Single-cell RNAseq of APP/PS1 mice showed that Abi3 is primarily expressed by microglia, including disease-associated microglia. In human brain, several novel ABI3 isoforms were identified, including isoforms with partial or complete loss of exon 6. Expression of these isoforms correlated tightly with total ABI3 expression but were not influenced by AD genetics. Lastly, we performed an initial characterization of these isoforms in transfected cells and found that, while full-length ABI3 was expressed in a dispersed punctate fashion within the cytosol, isoforms lacking most or all of exon six tended to form extensive protein aggregates. In summary, ABI3 expression is restricted to microglia, is increased with Alzheimer's neuropathology, and includes several isoforms that display a variable tendency to aggregate when expressed in vitro.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Enfermedad de Alzheimer , Proteínas Adaptadoras Transductoras de Señales/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Animales , Encéfalo/metabolismo , Humanos , Ratones , Microglía/metabolismo , Enfermedades del Sistema Nervioso/metabolismo , Agregado de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
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.
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Apolipoproteína E3/genética , Apolipoproteína E4/genética , Apolipoproteínas E/genética , Bacterias/crecimiento & desarrollo , Microbioma Gastrointestinal , Tracto Gastrointestinal/microbiología , Animales , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Apolipoproteínas E/metabolismo , Bacterias/genética , Disbiosis , Heces/microbiología , Femenino , Genes Dominantes , Genes Recesivos , Heterocigoto , Homocigoto , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados para ApoE , Fenotipo , Ribotipificación , Factores SexualesRESUMEN
BACKGROUND: 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. OBJECTIVES: The objectives of this study were to examine associations between experiences of discrimination, rs4680 genotype, and depressive symptoms in Latinx adults. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
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Enfermedades Cardiovasculares , Catecol O-Metiltransferasa , Adulto , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/genética , Catecol O-Metiltransferasa/genética , Depresión/genética , Femenino , Genotipo , Hispánicos o Latinos , Humanos , Masculino , Polimorfismo de Nucleótido SimpleRESUMEN
Elucidating the impact of the gut microbiome on Alzheimer's Disease (AD) is an area of intense interest. Short chain fatty acids (SCFAs) are major microbiota metabolites that have been implicated as a mediator of gut microbiome effects in the brain. Here, we tested the effects of SCFA-treated water vs. saline-treated water on APPswe/PSEN1dE9 mice maintained under standard laboratory conditions. Mice were treated with SCFAs from five months of age until ten months of age, when they were evaluated for microbiome profile, impaired spatial memory as evaluated with the radial arm water maze, astrocyte activation as measured by Gfap expression and amyloid burden as assessed by histochemistry and MSD ELISA. We report that SCFA treatment increased alpha-diversity and impacted the gut microbiome profile by increasing, in part, the relative abundance of several bacteria that typically produce SCFAs. However, SCFA treatment did not significantly affect behavior. Similarly, SCFAs did not affect cortical or hippocampal astrocyte activation observed in the APP/PS1 mice. Lastly, although robust levels of soluble and insoluble amyloid were present in the APP/PS1 mice, SCFA treatment had no effect on these indices. Overall, our findings are that SCFA treatment modifies the microbiome in a fashion that may increase further SCFA production. However, SCFA treatment did not alter behavior, astrocyte activation, nor amyloid neuropathology in APP/PS1 mice maintained with a conventional microbiome.
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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 (pâ=â0.1268 and pâ=â0.4909, 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.
Asunto(s)
Empalme Alternativo , Encéfalo , Glicoproteínas de Membrana , Receptores Inmunológicos , Enfermedad de Alzheimer , Encéfalo/metabolismo , Exones/genética , Humanos , Ligandos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismoRESUMEN
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 and 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.
Asunto(s)
Síndrome de Marfan/genética , Síndrome de Marfan/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Empalme del ARN , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Aminoácidos , Encéfalo/metabolismo , Exones , Fibrilina-1 , Fibrilinas , Humanos , Riñón/metabolismo , Hígado/metabolismo , Pulmón/metabolismo , Persona de Mediana Edad , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Adulto JovenRESUMEN
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.
Asunto(s)
Sistemas CRISPR-Cas , Conversión Génica , Edición Génica/métodos , Seudogenes , Lectina 3 Similar a Ig de Unión al Ácido Siálico/genética , Línea Celular Tumoral , Células HEK293 , Humanos , Reparación del ADN por RecombinaciónRESUMEN
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.