Rare A360T Mutation Alters GSK3ß(Ser9) Binding in the Cytosolic Loop of Presenilin 1, Influencing ß-Catenin Nuclear Localization and Pro-Death Gene Expression in Alzheimer's Disease Case.

Presenilin 1 (PS1) forms, via its large cytosolic loop, a trimeric complex with N-cadherin and ß-catenin, which is a key component of Wnt signaling. PS1 undergoes phosphorylation at 353 and 357 serines upon enhanced activity and elevated levels of the GSK3ß isoform. PS1 mutations surrounding these serines may alter the stability of the ß-catenin complex. Such mutations are found in some cases of familial early-onset Alzheimer's disease (fEOAD), but their functional impact remains obscure. One of such variants of PS1, the A360T substitution, is located close to GSK3ß-targeted serine residues. This variant was recently demonstrated in the French population, but more detail is needed to understand its biological effects. To assess the significance of this variant, we employed functional studies using a fibroblast cell line from an Alzheimer's disease case (a female proband) carrying the A360T mutation. Based on functional transcriptomic, cellular, and biochemical assays, we demonstrated atypically impaired ß-catenin/GSK3ß signaling in the A360T patient's fibroblasts. In detail, this was characterized by a decreased level of active cytosolic ß-catenin and bound by PS1, an increased level of nuclear ß-catenin, an increased level of inhibited GSK3ß phosphorylated on Ser9, and enhanced interaction of GSK3ß(Ser9) with PS1. Based on the transcriptomic profile of the A360T fibroblasts, we proposed a dysregulated transcriptional activity of ß-catenin, exemplified by increased expression of various cyclin-dependent kinases and cyclins, such as cyclin D1, potentially inducing neurons' cell cycle re-entry followed by apoptosis. The A360T cells did not exhibit significant amyloid pathology. Therefore, cell death in this PS1 cytosolic loop mutation may be attributed to impaired ß-catenin/GSK3ß signaling rather than amyloid deposition per se. We further estimated the biological and clinical relevance of the A360T variant by whole exome sequencing (WES). WES was performed on DNA from the blood of an A360T female proband, as well as an unrelated male patient carrying the A360T mutation and his mutation-free daughter (both unavailable for the derivation of the fibroblast cell lines). WES confirmed the highest-priority AD causality of the A360T variant in PS1 and also profiled the pathways and processes involved in the A360T case, highlighting the greatest importance of altered Wnt signaling.

Paraoxonase 1 decline and lipid peroxidation rise reflect a degree of brain atrophy and vascular impairment in dementia.

BACKGROUND: Paraoxonase 1 (PON1) is an enzyme with the capability to protect against lipid oxidation and atherosclerotic lesions formation. Impaired antioxidative capacity and enhanced lipid peroxidation (reflected by malondialdehyde rise) accompany dementias. OBJECTIVES: The aim of this study was to discern the possible differences in the activity and phenotype distribution of PON1, and lipid peroxidation level in dementias of neurodegenerative and vascular pathology, to assess whether they reflect structural changes in the brain, and to evaluate their potential as dementia markers. MATERIAL AND METHODS: Paraoxonase 1 arylesterase activity and polymorphisms (dual-substrate method), and malondialdehyde/thiobarbituric acid reactive substances (MDA/TBARS) levels were determined spectrophotometrically in 257 serum samples derived from 136 dementive patients (with Alzheimer's disease (AD; n = 63), vascular dementia (VaD; n = 40) and mixed-type dementia (MD; n = 33), as well as from 121 non-dementive individuals. The results were analyzed with reference to dementia type and severity (assessed with Mini Mental State Examination (MMSE) and Clinical Dementia Rating (CDR) scales), structural brain changes (estimated with magnetic resonance imaging (MRI) - Global Cortical Atrophy (GCA), Medial Temporal lobe Atrophy (MTA) and Fazekas scales)) and brain ischemia (Hachinski Ischemic Scale (HIS) index), and evaluated using receiver operating characteristic (ROC) analysis. RESULTS: Malondialdehyde/thiobarbituric acid reactive substances were increased in dementia (more in VaD than AD). In patients with vascular involvement, MDA/TBARS elevation reflected a degree of global cortical atrophy. Paraoxonase 1 activity was decreased in patients with dementia, especially in patients with severe cognitive deficits. In VaD, a drop in PON1 reflected a degree of MTA and brain ischemia. MDA/TBARS displayed 75% accuracy as a general dementia marker, but, similarly to PON1, were a poor differential marker. CONCLUSIONS: Both indices were more associated with vascular involvement and the severity of brain atrophy or ischemia rather than with degree of cognitive decline.

Targeted metabolomic analysis of nitric oxide/L-arginine pathway metabolites in dementia: association with pathology, severity, and structural brain changes.

L-Arginine/NO pathway is altered in Alzheimer disease (AD). Its clinical relevance and pathway status in vascular dementia (VaD) are unknown. Using targeted metabolomics (a liquid chromatography-mass spectrometry) we assessed L-arginine, L-citrulline, dimethylamine (DMA), asymmetric dimethyl arginine (ADMA) and symmetric dimethylarginine (SDMA) in AD (n = 48), mixed-type dementia (MD; n = 34), VaD (n = 40) and non-demented individuals (n = 140) and determined their clinical relevance (the association with dementia pathology, cognitive impairment, and structural brain damage). L-Arginine, ADMA, L-arginine/ADMA, and L-citrulline levels were decreased in dementia and L-arginine, L-citrulline, age and sex were its independent predictors correctly classifying 91% of cases. L-Arginine and L-arginine/ADMA were differentiating between VaD and AD with moderate accuracy. L-Arginine, L-arginine/ADMA, SDMA, and DMA reflected structural brain changes. DMA and L-citrulline were elevated in patients with strategic infarcts and SDMA, L-arginine/ADMA, and DMA were independent predictors of Hachinski ischemic score. ADMA and SDMA accumulation reflected severity of cognitive impairment. In summary, L-Arginine/NO pathway is altered in neurodegenerative and vascular dementia in association with neurodegenerative and vascular markers of brain damage and severity of cognitive impairment.

Systemic hepcidin quantified with LC-MS/MS in dementia in association with disease pathology and severity and with structural changes in the brain.

Hepcidin is a peptide hormone regulating iron metabolism, the dyshomeostasis of which has been implicated in dementia. Yet, data on hepcidin status in dementia are scanty, limited to Alzheimer's disease (AD) and inconsistent due to methodological problems with its determination using immunoassays and/or lack of homogeneity of evaluated groups. Hepcidin association with vascular dementia (VaD) remains unknown. We proposed a mass spectrometry method of hepcidin quantification in sera and aimed at determining hepcidin systemic status in patients with dementia of AD, VaD, or mixed (MD) pathology, with reference to the degree of cognitive loss and structural changes in the brain as well as at evaluating the diagnostic potential of hepcidin as a biomarker. We found that hepcidin concentrations were significantly elevated in VaD and insignificantly so in AD or MD and that they positively correlated with the Clinical Dementia Rating and inversely with the Mini Mental State Examination. Hepcidin tended to be more pronouncedly elevated in patients with advanced cortical atrophy and white matter lesions. It displayed a biphasic relationship with the Hachinski Ischemic Scale and a good accuracy as dementia but not differential marker. Taken together, our results demonstrated that dementia of vascular and not neurodegenerative pathology is associated with significant elevation of systemic hepcidin. Hepcidin elevation reflects the degree of cognitive loss as well as the severity of structural changes in the brain. If confirmed in a prospective study, hepcidin quantification may hold promise as a diagnostic marker; its accuracy as a differential marker of VaD is insufficient.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease.

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

Association of Cerebral Amyloid-ß Aggregation With Cognitive Functioning in Persons Without Dementia.

Importance: Cerebral amyloid-ß aggregation is an early event in Alzheimer disease (AD). Understanding the association between amyloid aggregation and cognitive manifestation in persons without dementia is important for a better understanding of the course of AD and for the design of prevention trials. Objective: To investigate whether amyloid-ß aggregation is associated with cognitive functioning in persons without dementia. Design, Setting, and Participants: This cross-sectional study included 2908 participants with normal cognition and 4133 with mild cognitive impairment (MCI) from 53 studies in the multicenter Amyloid Biomarker Study. Normal cognition was defined as having no cognitive concerns for which medical help was sought and scores within the normal range on cognitive tests. Mild cognitive impairment was diagnosed according to published criteria. Study inclusion began in 2013 and is ongoing. Data analysis was performed in January 2017. Main Outcomes and Measures: Global cognitive performance as assessed by the Mini-Mental State Examination (MMSE) and episodic memory performance as assessed by a verbal word learning test. Amyloid aggregation was measured with positron emission tomography or cerebrospinal fluid biomarkers and dichotomized as negative (normal) or positive (abnormal) according to study-specific cutoffs. Generalized estimating equations were used to examine the association between amyloid aggregation and low cognitive scores (MMSE score ≤27 or memory z score≤-1.28) and to assess whether this association was moderated by age, sex, educational level, or apolipoprotein E genotype. Results: Among 2908 persons with normal cognition (mean [SD] age, 67.4 [12.8] years), amyloid positivity was associated with low memory scores after age 70 years (mean difference in amyloid positive vs negative, 4% [95% CI, 0%-7%] at 72 years and 21% [95% CI, 10%-33%] at 90 years) but was not associated with low MMSE scores (mean difference, 3% [95% CI, -1% to 6%], P = .16). Among 4133 patients with MCI (mean [SD] age, 70.2 [8.5] years), amyloid positivity was associated with low memory (mean difference, 16% [95% CI, 12%-20%], P < .001) and low MMSE (mean difference, 14% [95% CI, 12%-17%], P < .001) scores, and this association decreased with age. Low cognitive scores had limited utility for screening of amyloid positivity in persons with normal cognition and those with MCI. In persons with normal cognition, the age-related increase in low memory score paralleled the age-related increase in amyloid positivity with an intervening period of 10 to 15 years. Conclusions and Relevance: Although low memory scores are an early marker of amyloid positivity, their value as a screening measure for early AD among persons without dementia is limited.

The Central Biobank and Virtual Biobank of BIOMARKAPD: A Resource for Studies on Neurodegenerative Diseases.

Biobanks are important resources for biomarker discovery and assay development. Biomarkers for Alzheimer's and Parkinson's disease (BIOMARKAPD) is a European multicenter study, funded by the EU Joint Programme-Neurodegenerative Disease Research, which aims to improve the clinical use of body fluid markers for the diagnosis and prognosis of Alzheimer's disease (AD) and Parkinson's disease (PD). The objective was to standardize the assessment of existing assays and to validate novel fluid biomarkers for AD and PD. To support the validation of novel biomarkers and assays, a central and a virtual biobank for body fluids and associated data from subjects with neurodegenerative diseases have been established. In the central biobank, cerebrospinal fluid (CSF) and blood samples were collected according to the BIOMARKAPD standardized pre-analytical procedures and stored at Integrated BioBank of Luxembourg. The virtual biobank provides an overview of available CSF, plasma, serum, and DNA samples at each site. Currently, at the central biobank of BIOMARKAPD samples are available from over 400 subjects with normal cognition, mild cognitive impairment (MCI), AD, frontotemporal dementia (FTD), vascular dementia, multiple system atrophy, progressive supranuclear palsy, PD, PD with dementia, and dementia with Lewy bodies. The virtual biobank contains information on over 8,600 subjects with varying diagnoses from 21 local biobanks. A website has been launched to enable sample requests from the central biobank and virtual biobank.

Evaluation of visinin-like protein 1 concentrations in the cerebrospinal fluid of patients with mild cognitive impairment as a dynamic biomarker of Alzheimer's disease.

BACKGROUND: The correlations between pathology of neurodegenerative diseases, especially Alzheimer's disease (AD), and concentrations of neuronal calcium sensor proteins, such as visinin-like protein 1 (VILIP-1), in cerebrospinal fluid (CSF) have been discussed in the literature but its utility as biomarker of AD in comparison with mild cognitive impairment (MCI) has not been studied yet. OBJECTIVE: Therefore, the aim of our study was to assess the clinical utility of the measurement of CSF concentrations of VILIP-1 in patients with AD, MCI subjects, and non-demented controls. The clinical and neuropsychological diagnoses were supported by CSF biomarkers of neurochemical dementia diagnostics: decreased concentrations of Aß1-42 and/or Aß42/40 ratio and increased concentrations of Tau and pTau181 proteins. METHODS: The study included 33 AD patients, 15 subjects with MCI, and 18 elderly individuals without cognitive deficits. The CSF concentrations of biomarkers tested were determined by using the ELISA method. RESULTS: Concentrations of VILIP-1 in CSF were significantly higher in AD patients compared to the MCI subjects and elderly individuals without cognitive impairment. Increased concentrations of VILIP-1 correlated significantly with reduced Aß42/40 ratio and higher pTau181 in AD group. CONCLUSION: Our findings suggest that VILIP-1 may play a role in the AD pathophysiology and is a good candidate for dynamic biomarker of AD, although this issue requires further investigation.

Concentrations of matrix metalloproteinases and their tissue inhibitors in the cerebrospinal fluid of patients with Alzheimer's disease.

BACKGROUND: A growing body of evidence shows the involvement of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in neurodegeneration processes, but reports of their concentrations in the cerebrospinal fluid (CSF) are inconsistent. OBJECTIVE: Therefore, the aim of our study was to evaluate the CSF concentrations of MMP-2, MMP-3, MMP-9, and their inhibitors (TIMP-1 and TIMP-2) in carefully selected groups of patients with Alzheimer's disease (AD), mild cognitive impairment (MCI), and non-demented controls, whose clinical and neuropsychological diagnoses were confirmed by the corresponding CSF biomarkers of neurochemical dementia diagnostics: decreased concentrations of Aß1-42 and/or Aß42/40 ratio, and increased concentrations of Tau and pTau181 proteins. METHODS: The study included 33 AD patients, 15 subjects with MCI, and 18 elderly individuals without cognitive deficits. The CSF concentrations of MMPs and TIMPs were determined with ELISAs. RESULTS: CSF concentrations of MMP-9 were significantly lower, and the concentrations of MMP-3 significantly higher in AD patients compared to the controls. Neither MMP-2 nor TIMPs showed significant changes among the groups investigated. CONCLUSION: Altered concentrations of two out of three MMPs investigated in this study suggest that this family of biomolecules may play a role in the AD pathophysiology. Further studies are needed to establish their potential diagnostic utility.