Clinical and biomarker profiling of prodromal Alzheimer's disease in workpackage 5 of the Innovative Medicines Initiative PharmaCog project: a 'European ADNI study'.

BACKGROUND: In the field of Alzheimer's disease (AD), the validation of biomarkers for early AD diagnosis and for use as a surrogate outcome in AD clinical trials is of considerable research interest. OBJECTIVE: To characterize the clinical profile and genetic, neuroimaging and neurophysiological biomarkers of prodromal AD in amnestic mild cognitive impairment (aMCI) patients enrolled in the IMI WP5 PharmaCog (also referred to as the European ADNI study). METHODS: A total of 147 aMCI patients were enrolled in 13 European memory clinics. Patients underwent clinical and neuropsychological evaluation, magnetic resonance imaging (MRI), electroencephalography (EEG) and lumbar puncture to assess the levels of amyloid ß peptide 1-42 (Aß42), tau and p-tau, and blood samples were collected. Genetic (APOE), neuroimaging (3T morphometry and diffusion MRI) and EEG (with resting-state and auditory oddball event-related potential (AO-ERP) paradigm) biomarkers were evaluated. RESULTS: Prodromal AD was found in 55 aMCI patients defined by low Aß42 in the cerebrospinal fluid (Aß positive). Compared to the aMCI group with high Aß42 levels (Aß negative), Aß positive patients showed poorer visual (P = 0.001), spatial recognition (P < 0.0005) and working (P = 0.024) memory, as well as a higher frequency of APOE4 (P < 0.0005), lower hippocampal volume (P = 0.04), reduced thickness of the parietal cortex (P < 0.009) and structural connectivity of the corpus callosum (P < 0.05), higher amplitude of delta rhythms at rest (P = 0.03) and lower amplitude of posterior cingulate sources of AO-ERP (P = 0.03). CONCLUSION: These results suggest that, in aMCI patients, prodromal AD is characterized by a distinctive cognitive profile and genetic, neuroimaging and neurophysiological biomarkers. Longitudinal assessment will help to identify the role of these biomarkers in AD progression.

Neuronal cell adhesion genes and antidepressant response in three independent samples.

Drug-effect phenotypes in human lymphoblastoid cell lines recently allowed to identify CHL1 (cell adhesion molecule with homology to L1CAM), GAP43 (growth-associated protein 43) and ITGB3 (integrin beta 3) as new candidates for involvement in the antidepressant effect. CHL1 and ITGB3 code for adhesion molecules, while GAP43 codes for a neuron-specific cytosolic protein expressed in neuronal growth cones; all the three gene products are involved in synaptic plasticity. Sixteen polymorphisms in these genes were genotyped in two samples (n=369 and 90) with diagnosis of major depressive episode who were treated with antidepressants in a naturalistic setting. Phenotypes were response, remission and treatment-resistant depression. Logistic regression including appropriate covariates was performed. Genes associated with outcomes were investigated in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) genome-wide study (n=1861) as both individual genes and through a pathway analysis (Reactome and String databases). Gene-based analysis suggested CHL1 rs4003413, GAP43 rs283393 and rs9860828, ITGB3 rs3809865 as the top candidates due to their replication across the largest original sample and the STAR*D cohort. GAP43 molecular pathway was associated with both response and remission in the STAR*D, with ELAVL4 representing the gene with the highest percentage of single nucleotide polymorphisms (SNPs) associated with outcomes. Other promising genes emerging from the pathway analysis were ITGB1 and NRP1. The present study was the first to analyze cell adhesion genes and their molecular pathways in antidepressant response. Genes and biomarkers involved in neuronal adhesion should be considered by further studies aimed to identify predictors of antidepressant response.

PPP3CC gene: a putative modulator of antidepressant response through the B-cell receptor signaling pathway.

Antidepressant pharmacogenetics represents a stimulating, but often discouraging field. The present study proposes a combination of several methodologies across three independent samples. Genes belonging to monoamine, neuroplasticity, circadian rhythm and transcription factor pathways were investigated in two samples (n=369 and 88) with diagnosis of major depression who were treated with antidepressants. Phenotypes were response, remission and treatment-resistant depression. Logistic regression including appropriate covariates was performed. Genes associated with outcomes were investigated in the STAR*D (Sequenced Treatment Alternatives to Relieve Depression) genome-wide study (n=1861). Top genes were further studied through a pathway analysis. In both original samples, markers associated with outcomes were concentrated in the PPP3CC gene. Other interesting findings were particularly in the HTR2A gene in one original sample and the STAR*D. The B-cell receptor signaling pathway proved to be the putative mediator of PPP3CC's effect on antidepressant response (P=0.03). Among innovative candidates, PPP3CC, involved in the regulation of immune system and synaptic plasticity, seems promising for further investigation.

A novel on-a-chip system with a 3D-bioinspired gut mucus suitable to investigate bacterial endotoxins dynamics.

The possible pathogenic impact of pro-inflammatory molecules produced by the gut microbiota is one of the hypotheses considered at the basis of the biomolecular dialogue governing the microbiota-gut-brain axis. Among these molecules, lipopolysaccharides (LPS) produced by Gram-negative gut microbiota strains may have a potential key role due to their toxic effects in both the gut and the brain. In this work, we engineered a new dynamic fluidic system, the MINERVA device (MI-device), with the potential to advance the current knowledge of the biological mechanisms regulating the microbiota-gut molecular crosstalk. The MI-device supported the growth of bacteria that are part of the intestinal microbiota under dynamic conditions within a 3D moving mucus model, with features comparable to the physiological conditions (storage modulus of 80 ± 19 Pa, network mesh size of 41 ± 3 nm), without affecting their viability (∼ 109 bacteria/mL). The integration of a fluidically optimized and user-friendly design with a bioinspired microenvironment enabled the sterile extraction and quantification of the LPS produced within the mucus by bacteria (from 423 ± 34 ng/mL to 1785 ± 91 ng/mL). Compatibility with commercially available Transwell-like inserts allows the user to precisely control the transport phenomena that occur between the two chambers by selecting the pore density of the insert membrane without changing the design of the system. The MI-device is able to provide the flow of sterile medium enriched with LPS directly produced by bacteria, opening up the possibility of studying the effects of bacteria-derived molecules on cells in depth, as well as the assessment and characterization of their effects in a physiological or pathological scenario.

Associations of the plasma interleukin 6 (IL-6) levels with disability and mortality in the elderly in the Treviso Longeva (Trelong) study.

IL-6 expression is regulated by the interplay of several transcriptional and hormonal factors, including sex steroids and glucocorticoids. In late life IL-6 expression increases as a result from loss of the normally inhibiting sex steroids. IL-6 is one of several proinflammatory cytokines. It has been proposed that many chronic inflammatory diseases are the result of a dysregulation of IL-6 expression. In this work we demonstrate that increased IL-6 levels in elderly are associated with higher disability and mortality, also independently of age and comorbidity.

The Treviso Longeva (Trelong) study: a biomedical, demographic, economic and social investigation on people 70 years and over in a typical town of North-East of Italy.

Longevity is a complex process resulting from genetic and environmental factors, as well as their interaction. These factors are poorly understood, and the comparison among health status, socio-economics, demographic and other characteristics of the elderly people can help in understanding these complex interactions. Such an interdisciplinary approach is necessary to allow an appropriate evaluation of longevity. Here we report the methodology and the first results of a representative study performed in 2003-2004 on people of 70 years and over, living in a typical town of North-East of Italy. In the research we collected biomedical, demographic, socio-economic and quality of life (QoL) data.

PEN-2 gene mutation in a familial Alzheimer's disease case.

Genetic evidence indicates a central role of cerebral accumulation of beta-amyloid (Abeta) in the pathogenesis of Alzheimer's disease (AD). Beside presenilin 1 and 2, three other recently discovered proteins (Aph 1, PEN 2 and nicastrin) are associated with gamma-secretase activity, the enzymatic complex generating Abeta. Alterations in genes encoding these proteins were candidates for a role in AD. The PEN 2 gene was examined for unknown mutations and polymorphisms in sporadic and familial Alzheimer patients. Samples from age-matched controls (n=253), sporadic AD (SAD, n=256) and familial AD (FAD, n=140) were screened with DHPLC methodology followed by sequencing. Scanning the gene identified for the first time a missense mutation (D90N) in a patient with FAD. Three intronic polymorphisms were also identified, one of which had a higher presence of the mutated allele in AD subjects carrying the allele epsilon4 of apolipoprotein E than controls. The pathogenic role of the PEN-2 D90N mutation in AD is not clear, but the findings might lead to new studies on its functional and genetic role.

In vivo anti-inflammatory effect of statins is mediated by nonsterol mevalonate products.

This study set out to clarify whether the inhibition of sterol or nonsterol derivatives arising from mevalonate biotransformation plays a major role in the in vivo anti-inflammatory action of statins. Hepatic synthesis of all these derivatives was inhibited in mice by administered statins, whereas squalestatin inhibited only sterol derivatives. Using a short-term treatment schedule, we found that statins reduced the hepatic activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase without affecting blood cholesterol. This treatment inhibited lipopolysaccharide- and carrageenan-induced pouch leukocyte recruitment and the exudate production of interleukin-6, monocyte chemotactic protein-1, and RANTES. Coadministration of mevalonate reversed the effect of statin on leukocyte recruitment. The inhibition of sterol synthesis by squalestatin did not have any anti-inflammatory effect, indicating that the biosynthesis of nonsterol compounds arising from mevalonate is crucial for the in vivo regulation of cytokine and chemokine production by statins. Their inhibition by statins may account for the reported anti-inflammatory effects of these drugs and may provide a biochemical basis for the recently reported effects of statins in the prevention of cardiovascular disease and mortality.

Proliferation-dependent pattern of expression of a dihydrofolate reductase-thymidylate synthase gene from Daucus carota.

The pattern of expression of a carrot dhfr-ts gene was evaluated in different plant organs, in somatic embryos, and in hypocotyl explants induced to dedifferentiate in vitro by the addition of the synthetic auxin 2,4 dichorophenoxyacetic acid. The promoter of this gene was also placed upstream of a uidA (GUS) reporter gene and, using biolistic and protoplasts transient expression assays, was shown to drive a particularly high level of expression in actively growing suspension cells. The results from these expression analyses combined with the presence of putative cell cycle-related cis-acting elements in the dhfr-ts promoter, strongly point to a cell division-dependent expression of this gene.

Cloning and characterization of a Brassica napus gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding factor.

The CCAAT motif present in the promoter of several genes is recognized in yeast and animals by a highly specific heteromeric factor (variously called HAP, CBF, CP1 or NF-Y) which is composed of a minimum of three subunits. A plant homologue of the CBF-B/HAP2 subunit is described for the first time in this report. Sequence comparison of the Brassica napus (Bn) CCAAT-binding factor (CBF) B subunit with the homologous yeast and animal proteins revealed that the critical amino-acid domains involved in DNA binding and subunit assembly are also conserved in plants. Interestingly, the Gln-rich regions found in the animal and yeast proteins, which may be involved in transcriptional activation, are absent in the Bn CBF-B subunit. The analysis of various cDNAs and of a genomic clone revealed the presence of alternatively spliced transcripts which could originate from different promoters.

Development of an enzyme-linked immunosorbent assay for human plasma inter-alpha-trypsin inhibitor (ITI) using specific antibodies against each of the H1 and H2 heavy chains.

Inter-alpha-trypsin inhibitor (ITI) is a serine-proteinase inhibitor of human plasma enzymes. ITI is composed of three polypeptide chains covalently linked: bikunin, responsible for the antiprotease activity and two heavy chains H1 and H2. Human plasma also contains other components immunologically related to ITI such as pre-alpha-trypsin inhibitor (paI), inter-alpha-like inhibitor (IalphaLI) and free bikunin. The ELISA procedure we propose exclusively measures native ITI within the range 12.5-200 microgram/l. The intra- and interassay coefficients of variation were less than 5.6% and 8.7%, respectively. When ITI was added to plasma samples, full recovery was obtained. EDTA-plasma from 30 healthy individuals revealed a mean level of 241.5 mg/l (range 145.5-506). The high specificity, sensitivity, reproducibility and accuracy of the present assay should facilitate the specific measurement of native ITI in blood and thus might represent a useful tool for further physiopathological studies.

Endotoxin regulates the maturation of sterol regulatory element binding protein-1 through the induction of cytokines.

Endotoxin (LPS), by raising the levels of cytokines, markedly influences lipid metabolism. To clarify the molecular mechanism of this effect, we examined the action of endotoxin in vitro and in vivo on the regulation of sterol regulatory element binding protein-1 (SREBP-1). In HepG2 cells stimulated with LPS, a dose-dependent increase in the level of the mature form of SREBP-1 was observed. For in vivo studies, endotoxin was administered intraperitoneally to CD1 mice fed with a standard or a cholesterol-enriched diet to increase the basal levels of circulating and liver cholesterol. Endotoxin raised cholesterol levels and stimulated the maturation of hepatic SREBP-1 in both normal and cholesterol-fed mice, indicating that the lipogenic effect of LPS was independent of endogenous sterol levels. To assess whether the lipogenic effect of endotoxin was linked to cytokine production, we administered LPS to C57Bl/6J endotoxin-sensitive and to C3H/HeJ endotoxin-resistant mice, which do not produce tumor necrosis factor in response to LPS. Significant induction of cholesterol levels and SREBP-1 activation was observed only in C57Bl/6J mice, indicating that cytokine production is crucial for the regulation of SREBP-1, and that the transcriptional activation of cholesterol biosynthesis may be part of the acute-phase response.

The promoter of aBrassica napus polygalacturonase gene directs pollen expression ofß-glucuronidase in transgenicBrassica plants.

A 647-bp 5'-flanking fragment obtained from genomic clone Sta 44G(2) belonging to a family of polygalacturonase genes expressed inBrassica napus pollen was fused to theß-glucuronidase (GUS) marker gene. This fusion construct was introduced intoB. napus plants viaAgrobacterium tumefaciens transformation. Analysis of the transgenicB. napus plants revealed that this promoter fragment is sufficient to direct GUS expression specifically in the anther and that GUS activity increases in pollen during maturation.

Body mass index, lifestyles, physical performance and cognitive decline: the "Treviso Longeva (TRELONG)" study.

OBJECTIVES: The relative contributions of risk factors, as body mass index (BMI), depression, chronic diseases, smoking, and lifestyles (as physical and performance activity, social contacts and reading habit) to cognitive decline in the elderly are unclear. We explored these variables in relation to 7-year cognitive decline in long-lived Italian elderly. DESIGN: Secondary data analysis of a longitudinal study of a representative, age-stratified, population sample. SETTING: The TREVISO LONGEVA (TRELONG) Study, in Treviso, Italy. PARTICIPANTS: 120 men and 189 women, age 77 years and older (mean age 80.2 ± 6.9 years) survivors after seven years of follow up. MEASUREMENTS: Cognitive decline measured as difference between Mini-Mental State Examination (MMSE) score in 2003 and in 2010; Body mass index (BMI), handgrip, Short Physical Performance Battery (SPPB) score, social contacts, reading habit, sight, hearing, schooling, mediterranean diet and multiple clinical and survey data recorded at baseline in 2003. RESULTS: In separate univariate analyses, age, SPPB score < 5, depressive symptoms (GDS) and more comorbidities (CCI) were associated with greater cognitive decline. Otherwise higher BMI, higher handgrip, reading habit, non-deteriorated sight and hearing, and schooling were protective. In a final multivariate model, age and higher BMI were associated with greater cognitive decline while reading habits was protective. SPPB score < 5 tends, though weakly, to be associated with greater cognitive decline. These associations remained with multivariate adjustment for gender, schooling, Charlson co-morbidity index (CCI) and baseline MMSE. CONCLUSION: Age and higher baseline BMI, independent of gender, and other confounding factors, are risk factors for cognitive decline. Reading habit plays a protective role seven years later among northern Italian adults aged 70 years or older. Low physical performance tends, though weakly, to be associated with greater cognitive decline.

The Treviso Dementia (TREDEM) Study: A Biomedical, Neuroradiological, Neuropsychological and Social Investigation of Dementia in North-Eastern Italy.

BACKGROUND: The incidence of dementia increases exponentially with age but knowledge of real disease-modifying interventions is still limited. OBJECTIVES: To describe the study design and methods of a large prospective cohort study aimed at exploring the complex underlying relationships existing among cognition, frailty, and health-related events in older persons with cognitive impairment. DESIGN: Prospective cohort study of a representative population of outpatients attending the Treviso Cognitive Impairment Center between 2000 and 2010. SETTING: The TREVISO DEMENTIA (TREDEM) Study conducted in Treviso, Italy. PARTICIPANTS: 490 men and 874 women, mean age 79.1 ± 7.8 years (range 40.2-100 years). MEASUREMENTS: Physiological data, biochemical parameters, clinical conditions, neuroradiological parameters (e.g., brain atrophy and cerebral vascular lesions identified by computerized tomography scans), neuropsychological assessment, and physical function markers were measured at baseline. Patients were followed-up to 10 years. RESULTS: The final sample included in the study was predominantly composed of women and characterized by an initial physical function impairment and increased vascular risk profile. Cognitive function of the sample population showed moderate cognitive impairment (Mini Mental State Examination 20.2 ± 6.3; Clinical Dementia Rating 1.2 ± 0.7), and a prevalence of vascular dementia of 26.9%. Cortical, subcortical and hippocampus atrophy were all significantly correlated with age and cognitive function. CONCLUSION: Results obtained from the preliminary analyses conducted in the TREDEM study suggest that the database will support the accomplishment of important goals in understanding the nature of cognitive frailty and neurodegenerative diseases.

Macroautophagy and the proteasome are differently involved in the degradation of alpha-synuclein wild type and mutated A30P in an in vitro inducible model (PC12/TetOn).

Many data suggest that alpha synuclein (α-syn) aggregation is involved in Parkinson's disease (PD) neurotoxicity and is accelerated by the pathogenetic point mutation A30P. The triplication of α-syn gene has been linked to early-onset familial PD, suggesting that the cellular dosage of α-syn is an important modulator of its toxicity. To verify this point, we developed an inducible model of α-syn expression (both wild type [WT] and mutated A30P) in rat PC12/TetOn cells. At low expression level, both α-syn(WT) and (A30P) did not aggregate, were not toxic, and displayed a protective action against oxidative stress triggered by hydrogen peroxide (H(2)O(2)). By increasing α-syn expression, its antioxidant function was no longer detectable as for the A30P form, but again no aggregation and cell death were present both for the WT and the mutated protein. To clarify why α-syn did not accumulate at high expression level, we inhibited macroautophagy by 3-methyladenine (3-MA) and the proteasome by MG132. In presence of 3-MA, α-syn(WT) accumulated, A11 anti-oligomer antibody-positive aggregates were detectable, and cell toxicity was evident, while proteasome inhibition did not increase α-syn(WT) accumulation. Macroautophagy or proteasome inhibition slightly increased α-syn(A30P) toxicity, with no detectable aggregation. This model can provide useful details about α-syn function, aggregation, and degradation pathways.

Factors related to disability: evidence from the "Treviso Longeva (TRELONG) study".

Prolongation of life is an important public health goal as long as there is an emphasis on the quality of life (QoL) and independent living. Diminishing abilities to ambulate and participate in activities of daily living point to a serious decline in functional health, increasing the risk of institutionalization and death. In our work we found a pattern of factors associated with disability, especially cognitive impairment, as well as stroke, physical activity and performance, reading, and the nutritional biomarkers, blood albumin and high-density lipoprotein cholesterol (HDL-C). The attention to this cluster of markers, suggesting multidimensional prevention, may have unexpected good effects against disability.

Physical activity, socialization and reading in the elderly over the age of seventy: what is the relation with cognitive decline? Evidence from "The Treviso Longeva (TRELONG) study".

Evidence in the literature suggests that physical activity, social contacts and cognitively stimulating activity, such as reading, often considered individually, may improve cognitive performance. Our work examines their interactions and confirms their positive effects on cognitive functions. The correlations between physical activity, socialization, reading and improved cognitive performance remained significant after adjusting for confounding factors, such as comorbidity and hearing function. Our work suggests that these factors are important for the prevention of cognitive decline in the elderly.

Dihydrofolate reductase from Daucus carota cell suspension cultures: purification, molecular and kinetic characterization.

The purification of dihydrofolate reductase (5, 6, 7, 8 tetrahydrofolate: NADP(+) oxidoreductase, E.C.: 1.5.1.3) from Daucus carota to apparent homogeneity, is described. The enzyme is a soluble protein with a molecular weight of 183 000±2 500, composed of identical subunits of 58 400±1 000. The enzyme is only weakly recognized by antibodies against human DHFR. The carrot DHFR is characterized by a pH optimum of 5.9, Km values for dihydrofolate and NADPH of 3.7 µM and 2.2 µM, respectively and a turnover number of 4 750 or 1 500 when referring to the 183 K form or the 58 K monomer, respectively. Molecular and kinetic properties are remarkably different from those reported for the soybean enzyme. Sensitivity to methotrexate is similar to that of bacterial and mammalian enzymes while sensitivity to trimethoprim and dihydrotriazine is intermediate between the two groups of organisms.

Inter-alpha-inhibitor as marker for neutrophil proteinase activity: an in vitro investigation.

Human neutrophil proteinases have been implicated in the pathogenesis of a wide variety of inflammatory diseases. The degradation of plasma proteins such as coagulation and fibrinolysis factors has been attributed to the excessive release of elastase in septicemia and in other conditions in which heightened proteolysis occurs. Inter-alpha-inhibitor (IalphaI) is particularly sensitive to cleavage by leukocyte proteinases. For this reason, the determination of IalphaI has been proposed as a method for evaluating plasma protein proteolysis by neutrophil enzymes. In this article we provide evidence that intact residual IalphaI can be accurately quantified by enzyme-linked immunosorbent assay (ELISA) determination without interference from fragments released from IalphaI by incubation with triggered neutrophils. We demonstrate that under these conditions IalphaI was quickly and steadily proteolyzed in a cell dose-dependent manner. Alpha-1 proteinase inhibitor (alpha1PI) partially protected IalphaI; however, the proteolysis persisted when IalphaI was incubated with stimulated neutrophils in the presence of a large relative excess of alpha1PI over the amount of elastase theoretically present in cells. For the same amount of alpha1PI, serum provided a better protection than alpha1PI alone but did not completely inhibit the IalphaI degradation. Therefore, ELISA determination of IalphaI might be useful for monitoring the in vivo activity of neutrophil proteinases in systemic proteolytic states.