New insights into the genetic etiology of Alzheimer's disease and related dementias.

Characterization of the genetic landscape of Alzheimer's disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/'proxy' AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele.

Common variants in Alzheimer's disease and risk stratification by polygenic risk scores.

Genetic discoveries of Alzheimer's disease are the drivers of our understanding, and together with polygenetic risk stratification can contribute towards planning of feasible and efficient preventive and curative clinical trials. We first perform a large genetic association study by merging all available case-control datasets and by-proxy study results (discovery n = 409,435 and validation size n = 58,190). Here, we add six variants associated with Alzheimer's disease risk (near APP, CHRNE, PRKD3/NDUFAF7, PLCG2 and two exonic variants in the SHARPIN gene). Assessment of the polygenic risk score and stratifying by APOE reveal a 4 to 5.5 years difference in median age at onset of Alzheimer's disease patients in APOE ɛ4 carriers. Because of this study, the underlying mechanisms of APP can be studied to refine the amyloid cascade and the polygenic risk score provides a tool to select individuals at high risk of Alzheimer's disease.

Long runs of homozygosity are associated with Alzheimer's disease.

Long runs of homozygosity (ROH) are contiguous stretches of homozygous genotypes, which are a footprint of inbreeding and recessive inheritance. The presence of recessive loci is suggested for Alzheimer's disease (AD); however, their search has been poorly assessed to date. To investigate homozygosity in AD, here we performed a fine-scale ROH analysis using 10 independent cohorts of European ancestry (11,919 AD cases and 9181 controls.) We detected an increase of homozygosity in AD cases compared to controls [ßAVROH (CI 95%) = 0.070 (0.037-0.104); P = 3.91 × 10-5; ßFROH (CI95%) = 0.043 (0.009-0.076); P = 0.013]. ROHs increasing the risk of AD (OR > 1) were significantly overrepresented compared to ROHs increasing protection (p < 2.20 × 10-16). A significant ROH association with AD risk was detected upstream the HS3ST1 locus (chr4:11,189,482‒11,305,456), (ß (CI 95%) = 1.09 (0.48 ‒ 1.48), p value = 9.03 × 10-4), previously related to AD. Next, to search for recessive candidate variants in ROHs, we constructed a homozygosity map of inbred AD cases extracted from an outbred population and explored ROH regions in whole-exome sequencing data (N = 1449). We detected a candidate marker, rs117458494, mapped in the SPON1 locus, which has been previously associated with amyloid metabolism. Here, we provide a research framework to look for recessive variants in AD using outbred populations. Our results showed that AD cases have enriched homozygosity, suggesting that recessive effects may explain a proportion of AD heritability.

Decreased Blood Level of MFSD2a as a Potential Biomarker of Alzheimer's Disease.

The protein Major Facilitator Superfamily Domain containing 2A (MFSD2a) was recently described as the primary carrier for docosahexaenoic acid (DHA) into the brain. Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by lower DHA levels in blood lipids. The aim of this study was to investigate the expression of MFSD2a in the whole blood and brain as a potential biomarker of AD. Three groups were established: 38 healthy controls, 48 subjects with moderate AD (GDS4), and 47 with severe AD (GDS6). We analyzed postmortem brain samples from the hippocampus of 11 healthy controls and 11 severe AD patients. Fatty acid (FA) was determined in serum and brain by gas chromatography. Blood and brain MFSD2a protein expression was analyzed by Western blotting. We found a significant and progressive decline of MFSD2a levels in blood of AD patients (Control 0.83 ± 0.13, GDS4 0.72 ± 0.09, GDS6 0.48 ± 0.05*, p ˂ 0.01). We also corroborated a significant reduction of DHA and other n-3 long-chain polyunsaturated FA in serum of AD. No differences were found in MFSD2a expression or FA levels in brain of controls and AD subjects. MFSD2A carrier was analyzed in AD patients for the first time and the level of MFSD2a in the whole blood could be a potential biomarker of this disease.

Genome-wide significant risk factors on chromosome 19 and the APOE locus.

The apolipoprotein E (APOE) gene on chromosome 19q13.32, was the first, and remains the strongest, genetic risk factor for Alzheimer's disease (AD). Additional signals associated with AD have been located in chromosome 19, including ABCA7 (19p13.3) and CD33 (19q13.41). The ABCA7 gene has been replicated in most populations. However, the contribution to AD of other signals close to APOE gene remains controversial. Possible explanations for inconsistency between reports include long range linkage disequilibrium (LRLD). We analysed the contribution of ABCA7 and CD33 loci to AD risk and explore LRLD patterns across APOE region. To evaluate AD risk conferred by ABCA7 rs4147929:G>A and CD33 rs3865444:C>A, we used a large Spanish population (1796 AD cases, 2642 controls). The ABCA7 rs4147929:G>A SNP effect was nominally replicated in the Spanish cohort and reached genome-wide significance after meta-analysis (odds ratio (OR)=1.15, 95% confidence interval (95% CI)=1.12-1.19; P = 1.60 x 10-19). CD33 rs3865444:C>A was not associated with AD in the dataset. The meta-analysis was also negative (OR=0.98, 95% CI=0.93-1.04; P=0.48). After exploring LRLD patterns between APOE and CD33 in several datasets, we found significant LD (D' >0.20; P <0.030) between APOE-Ɛ2 and CD33 rs3865444C>A in two of five datasets, suggesting the presence of a non-universal long range interaction between these loci affecting to some populations. In conclusion, we provide here evidence of genetic association of the ABCA7 locus in the Spanish population and also propose a plausible explanation for the controversy on the contribution of CD33 to AD susceptibility.

One-Year Reference Norms of Cognitive Change in Spanish Old Adults: Data from the NEURONORMA Sample.

OBJECTIVE: Serial cognitive assessments are useful for many purposes, such as monitoring cognitive decline or evaluating the result of an intervention. In order to determine if an observed change is reliable and meaningful, longitudinal reference data from non-clinical samples are needed. Since neuropsychological outcomes are affected by language and cultural background, cognitive tests should be adapted, and country-based norms collected. The lack of cross-sectional normative data for Spanish population has been partially remediated, but there is still a need of reliable change norms. This paper aims to give an initial response to this need by providing several reliable change indices (RCI) for 1-year follow-up in a Spanish sample. METHOD: A longitudinal observational study was designed. A total of 122 healthy subjects over age 50 were evaluated twice (M = 369.5, SD= 10.7 days) with the NEURONORMA battery. Scores changes were analyzed, and simple discrepancy scores, standard deviation indices, RCI, and standardized regression-based scores were calculated. RESULTS: Significant improvements were observed in variables related to memory, both verbal and visual, visuospatial function, and the completion time of complex problems. Reference tables for several RCI are provided for their use in clinical settings. CONCLUSIONS: Our results confirm the existence of heterogeneous practice effects after 1 year, and support the recommendation of using reliable change norms to avoid misdiagnosis in repeated assessments. This study provides with initial, preliminary norms of cognitive change for its use in Spanish elders. Further studies on larger samples and different inter-visit intervals are still needed.

Amyloid precursor protein metabolism and inflammation markers in preclinical Alzheimer disease.

OBJECTIVE: To investigate CSF markers involved in amyloid precursor protein processing, neuronal damage, and neuroinflammation in the preclinical stages of Alzheimer disease (AD) and participants with suspected non-Alzheimer pathology (SNAP). METHODS: We collected CSF from 266 cognitively normal volunteers participating in a cross-sectional multicenter study (the SIGNAL study) to investigate markers involved in amyloid precursor protein processing (Aß42, sAPPß, ß-secretase activity), neuronal damage (total-tau [t-tau], phospho-tau [p-tau]), and neuroinflammation (YKL-40). We analyzed the relationship among biomarkers, clinical variables, and the APOE genotype, and compared biomarker levels across the preclinical stages of the National Institute on Aging-Alzheimer's Association classification: stage 0, 1, 2, 3, and SNAP. RESULTS: The median age in the whole cohort was 58.8 years (range 39.8-81.6). Participants in stages 2-3 and SNAP had higher levels of YKL-40 than those in stages 0 and 1. Participants with SNAP had higher levels of sAPPß than participants in stage 0 and 1. No differences were found between stages 0, 1, and 2-3 in sAPPß and ß-secretase activity in CSF. Age correlated with t-tau, p-tau, and YKL-40. It also correlated with Aß42, but only in APOE ε4 carriers. Aß42 correlated positively with t-tau, sAPPß, and YKL-40 in participants with normal Aß42. CONCLUSIONS: Our findings suggest that inflammation in the CNS increases in normal aging and is intimately related to markers of neurodegeneration in the preclinical stages of AD and SNAP. sAPPß and ß-secretase activity are not useful diagnostic or staging markers in preclinical AD.

A blood-based, 7-metabolite signature for the early diagnosis of Alzheimer's disease.

Accurate blood-based biomarkers of Alzheimer's disease (AD) could constitute simple, inexpensive, and non-invasive tools for the early diagnosis and treatment of this devastating neurodegenerative disease. We sought to develop a robust AD biomarker panel by identifying alterations in plasma metabolites that persist throughout the continuum of AD pathophysiology. Using a multicenter, cross-sectional study design, we based our analysis on metabolites whose levels were altered both in AD patients and in patients with amnestic mild cognitive impairment (aMCI), the earliest identifiable stage of AD. UPLC coupled to mass spectrometry was used to independently compare the levels of 495 plasma metabolites in aMCI (n = 58) and AD (n = 100) patients with those of normal cognition controls (NC, n = 93). Metabolite alterations common to both aMCI and AD patients were used to generate a logistic regression model that accurately distinguished AD from NC patients. The final panel consisted of seven metabolites: three amino acids (glutamic acid, alanine, and aspartic acid), one non-esterified fatty acid (22:6n-3, DHA), one bile acid (deoxycholic acid), one phosphatidylethanolamine [PE(36:4)], and one sphingomyelin [SM(39:1)]. Detailed analysis ruled out the influence of potential confounding variables, including comorbidities and treatments, on each of the seven biomarkers. The final model accurately distinguished AD from NC patients (AUC, 0.918). Importantly, the model also distinguished aMCI from NC patients (AUC, 0.826), indicating its potential diagnostic utility in early disease stages. These findings describe a sensitive biomarker panel that may facilitate the specific detection of early-stage AD through the analysis of plasma samples.

Evaluation of Candidate Genes Related to Neuronal Apoptosis in Late-Onset Alzheimer's Disease.

The objective of this study was to identify genetic variation in genes encoding death receptors and signals that modulate their activity. After conducting a meta-analysis with five previous genome-wide association studies and aggregated data, the most significant signals, (TNF locus: rs2395488, rs2534672, and rs9267445; and FASLG locus: rs730278), were replicated in 1,046 cases and 372 controls. The rs2395488 and rs2534672 markers showed a modest protective effect (OR = 0.849, p = 0.49780;OR= 0.687, p = 0.11335), in contrast to rs730278 marker (OR = 1.146, p = 0.17212), which did not follow the previous effect direction; in any case it reached the significance level. Final meta-analysis, adding the replication sample, confirmed these observations. We concluded that FASLG marker is not etiologically linked to Alzheimer's disease. However, single nucleotide polymorphisms around TNF locus require further analyses in order to explain the association between Alzheimer's disease and human leukocyte antigen.

Cognitive and neuroimaging profiles in mild cognitive impairment and Alzheimer's disease: data from the Spanish Multicenter Normative Studies (NEURONORMA Project).

The aim of this study was to characterize the neuropsychological and neuroimaging profiles of mild cognitive impairment (MCI) and Alzheimer's disease (AD) patients, and to study the magnitude of the differences by comparing both outcomes with healthy subjects in a cross-sectional manner. Five hundred and thirty-five subjects (356 cognitively normal adults (CONT), 79 MCI, and 100 AD) were assessed with the NEURONORMA neuropsychological battery. Thirty CONT, 23 MCI, and 23 AD subjects from this sample were included in the neuroimaging substudy. Patients' raw cognitive scores were converted to age and education-adjusted scaled ones (range 2-18) using co-normed reference values. Medians were plotted to examine the cognitive profile. MRIs were processed by means of FreeSurfer. Effect size indices (Cohen's d) were calculated in order to compare the standardized differences between patients and healthy subjects. Graphically, the observed cognitive profiles for MCI and AD groups produced near to parallel lines. Verbal and visual memories were the most impaired domains in both groups, followed by executive functions and linguistic/semantic ones. The largest effect size between AD and cognitively normal subjects was found for the FCSRT (d = 4.05, AD versus CONT), which doubled the value obtained by the best MRI measure, the right hippocampus (d = 1.65, AD versus CONT). Our results support the notion of a continuum in cognitive profile between MCI and AD. Neuropsychological outcomes, in particular the FCSRT, are better than neuroimaging ones at detecting differences among subjects.

Spanish multicenter normative studies (NEURONORMA project): normative data and equivalence of four BNT short-form versions.

The application of the Boston Naming Test (BNT) is time-consuming and shortened versions need to be developed for screening purposes. The aims of this study were to develop four equivalent 15-item forms of a Spanish adaptation of the BNT, to test the equivalence of the new versions in a clinical sample, and to provide normative data. The normative sample consisted of 340 subjects. The clinical sample included 172 patients (76 Mild Cognitive Impairment and 96 Alzheimer's disease). An empirical procedure was used to develop the shortened versions. All new versions demonstrated satisfactory internal consistency. Pearson's coefficient analysis showed strong relationships among the four short-form versions as well as between each of them and the 60-item test. The inferential confidence interval method demonstrated the equivalence between the four shortened versions. Age and education affected the score of all short-form versions, but sex was found to be unrelated to the performance. Normative data were calculated for midpoint age groups. This paper proposes four 15-item equivalent versions that could be useful and time-saving tools for screening purposes.

Phasic and tonic alerting in mild cognitive impairment: A preliminary study.

In this preliminary study we assessed the functioning of the different attentional networks in mild cognitive impairment (MCI) patients, taking as theoretical framework the Posner's cognitive neuroscience approach. Two groups of participants were tested in a single short experiment: 20 MCI patients (6 amnestic, 6 non-amnestic and 8 multiple-domain) and 18 healthy matched controls (HC). For attentional assessment we used a version of the Attention Network Test (the ANTI-V) that provided not only a score of the orienting, the executive, and the alerting networks and their interactions, but also an independent measure of vigilance (tonic alerting). The results showed that all subtypes of MCI patients exhibited a selective impairment in the tonic component of alerting, as indexed by a decrease in the d' sensitivity index, and their performance in executive network increased up to the HC group level when phasic alerting was provided by a warning tone. Our findings suggest that a core attentional deficit, especially the endogenous component of alerting, may significantly contribute to the behavioral and cognitive deficits associated with MCI.

Genetic study of neurexin and neuroligin genes in Alzheimer's disease.

The interaction between neurexins and neuroligins promotes the formation of functional synaptic structures. Recently, it has been reported that neurexins and neuroligins are proteolytically processed by presenilins at synapses. Based on this interaction and the role of presenilins in familial Alzheimer's disease (AD), we hypothesized that dysfunction of the neuroligin-neurexin pathway might be associated with AD. To explore this hypothesis, we carried out a meta-analysis of five genome-wide association studies (GWAS) comprising 1, 256 SNPs in the NRXN1, NRXN2, NRXN3, and NLGN1 genes (3,009 cases and 3,006 control individuals). We identified a marker in the NRXN3 gene (rs17757879) that showed a consistent protective effect in all GWAS, however, the statistical significance obtained did not resist multiple testing corrections (OR = 0.851, p = 0.002). Nonetheless, gender analysis revealed that this effect was restricted to males. A combined meta-analysis of the former five GWAS together with a replication Spanish sample consisting of 1,785 cases and 1,634 controls confirmed this observation (rs17757879, OR = 0.742, 95% CI = 0.632-0.872, p = 0.00028, final meta-analysis). We conclude that NRXN3 might have a role in susceptibility to AD in males.

Improving delayed face recognition in Alzheimer's disease by differential outcomes.

OBJECTIVE: Previous studies have demonstrated the benefit of the differential outcomes procedure (DOP) in human learning. In the present study we aimed to explore whether the DOP might also help to overcome the face recognition memory deficit commonly observed in Alzheimer's disease (AD) patients. METHOD: A delayed matching-to-sample task was used. Participants were instructed to choose which of the 4 alternative faces (comparison stimuli) matched the previously seen face (sample stimulus). Either short (5 seconds) or long (25 seconds) delays were interposed between the sample and the comparison stimuli. In the differential outcomes condition each sample face was paired with its own outcome. In contrast, in the nondifferential condition, outcomes were randomly arranged. RESULTS: The differential outcomes effect (DOE) was evident in the AD patients with both accuracy and latency data. That is, they showed a significantly better and faster delayed face recognition when differential outcomes were arranged. The analyses also revealed a significant main effect of delay; participants were slower in the 25 seconds condition than in the 5 seconds condition, but the difference was higher in the patients than in the controls. CONCLUSIONS: These findings demonstrate, to our knowledge for the first time, that face recognition memory in patients with Alzheimer is improved when differential outcomes are used and draw attention to the potential of this procedure as a therapeutic technique.

Neurotrophic bone marrow cellular nests prevent spinal motoneuron degeneration in amyotrophic lateral sclerosis patients: a pilot safety study.

The objective of this article is to assess the safety of intraspinal infusion of autologous bone marrow mononuclear cells (BMNCs) and, ultimately, to look for histopathological signs of cellular neurotrophism in amyotrophic lateral sclerosis (ALS) patients. We conducted an open single arm phase I trial. After 6 months observation, autologous BMNCs were infused into the posterior spinal cord funiculus. Safety was the primary endpoint and was defined as the absence of serious transplant-related adverse events. In addition, forced vital capacity (FVC), ALS-functional rating scale (ALS-FRS), Medical Research Council scale for assessment of muscle power (MRC), and Norris scales were assessed 6 and 3 months prior to the transplant and quarterly afterward for 1 year. Pathological studies were performed in case of death. Eleven patients were included. We did not observe any severe transplant-related adverse event, but there were 43 nonsevere events. Twenty-two (51%) resolved in ≤2 weeks and only four were still present at the end of follow-up. All were common terminology criteria for adverse events grade ≤2. No acceleration in the rate of decline of FVC, ALS-FRS, Norris, or MRC scales was observed. Four patients died on days 359, 378, 808, and 1,058 post-transplant for reasons unrelated to the procedure. Spinal cord pathological analysis showed a greater number of motoneurons in the treated segments compared with the untreated segments (4.2 ± 0.8 motoneurons per section [mns per sect] and 0.9 ± 0.3 mns per sect, respectively). In the treated segments, motoneurons were surrounded by CD90+ cells and did not show degenerative ubiquitin deposits. This clinical trial confirms not only the safety of intraspinal infusion of autologous BMNC in ALS patients but also provides evidence strongly suggesting their neurotrophic activity.

Using artificial neural networks in clinical neuropsychology: high performance in mild cognitive impairment and Alzheimer's disease.

Mild cognitive impairment (MCI) is a transitional state between normal aging and Alzheimer disease (AD). Artificial neural networks (ANNs) are computational tools that can provide valuable support to clinical decision making, classification, and prediction of cognitive functioning. The aims of this study were to develop, train, and explore and develop the ability of ANNs to differentiate MCI and AD, and to study the relevant variables in MCI and AD diagnosis. The sample consisted of 346 controls and 79 MCI and 97 AD patients. A linear discriminant analysis (LDA) and ANNs with 12 input neurons (10 subtests of a neuropsychological test, the abbreviated Barcelona Test; age; and education), 4 hidden neurons, and output neuron (diagnosis) were used to classify the patients. The ANNs were superior to LDA in its ability to classify correctly patients (100-98.33% vs. 96.4-80%, respectively) and showed better predictive performance. Semantic fluency, working and episodic memory and education showed up as the most significant and sensitive variables for classification. Our results indicate that ANNs have an excellent capacity to discriminate MCI and AD patients from healthy controls. These findings provide evidence that ANNs can be a useful tool for the analysis of neuropsychological profiles related to clinical syndromes.

Estrogen receptor alpha gene variants are associated with Alzheimer's disease.

The present research is aimed at assessing the role of 3 estrogen receptor alpha (ESR1) gene variants in late onset Alzheimer's disease (AD) susceptibility. One thousand one hundred thirteen unrelated late onset sporadic AD patients, 1109 healthy controls and 121 neurologically healthy elderly controls were used to carry out case-control genetic association studies with ESR1 rs3844508, rs2234693, and ESR1 noncoding deletion 1 (ESR1-NCD1) polymorphisms. Thirty-five healthy male samples were used for molecular analyses. The rs2234693 polymorphism is associated with AD in our population (odds ratio [OR], 1.29; p = 0.008). The rs3844508 marker confers protection against AD in males (OR, 0.57; p = 0.001) and the deletion ESR1-NCD1 is a risk factor for AD in women (OR, 1.67; p < 0.001). Molecular analyses on ESR1-NCD1 indicate that this deletion confers a higher response to estradiol activity on ESR1 receptor and it is also associated with differential expression of ESR1 isoforms. Our results support the involvement of ESR1 gene in AD and point to the existence of sexual dimorphism for ESR1 markers. In addition, carriers of ESR1-NCD1 deletion could overrespond to estradiol action.

Activation of PKR causes amyloid ß-peptide accumulation via de-repression of BACE1 expression.

BACE1 is a key enzyme involved in the production of amyloid ß-peptide (Aß) in Alzheimer's disease (AD) brains. Normally, its expression is constitutively inhibited due to the presence of the 5'untranslated region (5'UTR) in the BACE1 promoter. BACE1 expression is activated by phosphorylation of the eukaryotic initiation factor (eIF)2-alpha, which reverses the inhibitory effect exerted by BACE1 5'UTR. There are four kinases associated with different types of stress that could phosphorylate eIF2-alpha. Here we focus on the double-stranded (ds) RNA-activated protein kinase (PKR). PKR is activated during viral infection, including that of herpes simplex virus type 1 (HSV1), a virus suggested to be implicated in the development of AD, acting when present in brains of carriers of the type 4 allele of the apolipoprotein E gene. HSV1 is a dsDNA virus but it has genes on both strands of the genome, and from these genes complementary RNA molecules are transcribed. These could activate BACE1 expression by the PKR pathway. Here we demonstrate in HSV1-infected neuroblastoma cells, and in peripheral nervous tissue from HSV1-infected mice, that HSV1 activates PKR. Cloning BACE1 5'UTR upstream of a luciferase (luc) gene confirmed its inhibitory effect, which can be prevented by salubrinal, an inhibitor of the eIF2-alpha phosphatase PP1c. Treatment with the dsRNA analog poly (I∶C) mimicked the stimulatory effect exerted by salubrinal over BACE1 translation in the 5'UTR-luc construct and increased Aß production in HEK-APPsw cells. Summarizing, our data suggest that PKR activated in brain by HSV1 could play an important role in the development of AD.

Genetic association of complement receptor 1 polymorphism rs3818361 in Alzheimer's disease.

Complement receptor 1 gene polymorphism rs3818361 was recently shown to increase the risk of Alzheimer's disease (AD). We performed an independent replication study of this genetic variant in 2,470 individuals from Spain. By applying an allelic model, we observed a trend toward an association between this marker and late-onset AD susceptibility in our case-control study (odds ratio = 1.114, 95% confidence interval: 0.958-1.296, P = .16). Meta-analysis of available studies (n = 31,771 individuals), including previous studies and public genome-wide association study resources (Alzheimer's Disease Neuroimaging Initiative, Translational Genomics Research Institute, and Multi-site Collaborative Study for Genotype-Phenotype Associations in Alzheimer's Disease), strongly supports the effect of rs3818361 (odds ratio = 1.180, 95% confidence interval: 1.113-1.252, P < 2.99E-8) and suggests the existence of between-study heterogeneity (P < .05). We concluded that the complement receptor 1 gene may contribute to AD risk, although its effect size could be smaller than previously estimated.