Interaction between APOE4 genotype and environmental risk factors in Alzheimer's disease

Alzheimer's disease (AD) is probably caused by both genetic and environmental risk factors. The major genetic risk factor is the E4 variant of apolipoprotein E gene called apoE4. Several risk factors for developing AD have been identified including lifestyle, such as dietary habits. The mechanisms behind the AD pathogenesis and the onset of cognitive decline in the AD brain are presently unknown. In this study we wanted to characterize the effects of the interaction between environmental risk factors and apoE genotype on neurodegeneration processes, with particular focus on behavioural studies and neurodegenerative processes at molecular level. Towards this aim, we used 6 months-old apoE4 and apoE3 Target Replacement (TR) mice fed on different diets (high intake of cholesterol and high intake of carbohydrates). These mice were evaluated for learning and memory deficits in spatial reference (Morris Water Maze (MWM)) and contextual learning (Passive Avoidance) tasks, which involve the hippocampus and the amygdala, respectively. From these behavioural studies we found that the initial cognitive impairments manifested as a retention deficit in apoE4 mice fed on high carbohydrate diet. Thus, the genetic risk factor apoE4 genotype associated with a high carbohydrate diet seems to affect cognitive functions in young mice, corroborating the theory that the combination of genetic and environmental risk factors greatly increases the risk of developing AD and leads to an earlier onset of cognitive deficits. The cellular and molecular bases of the cognitive decline in AD are largely unknown. In order to determine the molecular changes for the onset of the early cognitive impairment observed in the behavioural studies, we performed molecular studies, with particular focus on synaptic integrity and Tau phosphorylation. The most relevant finding of our molecular studies showed a significant decrease of Brain-derived Neurotrophic Factor (BDNF) in apoE4 mice fed on high carbohydrate diet. Our results may suggest that BDNF decrease found in apoE4 HS mice could be involved in the earliest impairment in long-term reference memory observed in behavioural studies. The second aim of this thesis was to study possible involvement of leptin in AD. There is growing evidence that leptin has neuroprotective properties in the Central Nervous System (CNS). Recent evidence has shown that leptin and its receptors are widespread in the CNS and may provide neuronal survival signals. However, there are still numerous questions, regarding the molecular mechanism by which leptin acts, that remain unanswered. Thus, given to the importance of the involvement of leptin in AD, we wanted to clarify the function of leptin in the pathogenesis of AD and to investigate if apoE genotype affect leptin levels through studies in vitro, in mice and in human. Our findings suggest that apoE4 TR mice showed an increase of leptin in the brain. Leptin levels are also increased in the cerebral spinal fluid of AD patients and apoE4 carriers with AD have higher levels of leptin than apoE3 carriers. Moreover, leptin seems to be expressed by reactive glial cells in AD brains. In vitro, ApoE4 together with Amyloid beta increases leptin production by microglia and astrocytes. Taken together, all these findings suggest that leptin replacement might not be a good strategy for AD therapy. Our results show that high leptin levels were found in AD brains. These findings suggest that, as high leptin levels do not promote satiety in obese individuals, it might be possible that they do not promote neuroprotection in AD patients. Therefore, we hypothesized that AD brain could suffer from leptin resistance. Further studies will be critical to determine whether or not the central leptin resistance in SNC could affect its potential neuroprotective effects.

Risk factors for postoperative delirium in the elderly

Background: Delirium is defined as an acute disorder of attention and cognition. Delirium is common in hospitalized elderly patient and is associated with increased morbidity, length of stay and patient care costs. Although Delirium can develop at any time during hospitalization, it typically presents early in the post-operative period (Post-Operative Delirium, POD) in the surgery context. The molecular mechanism and possible genetics basis of POD onset are not known, as well as all the risk factors are not completely defined. Our hypothesis is that genetic risk factor involving the inflammatory response could have possible effects on the immunoneuroendocrine system. Moreover, our previous data (inflamm-aging) suggest that aging is associated with an increase of inflammatory status, favouring age-related diseases such as neurodegenerative diseases, frailty, depression among other. Some pro-inflammatory or anti-inflammatory cytokines, seem to play a crucial role in increasing the inflammatory status and in the communication and regulation of immunoneuroendocrine system. Objective: this study evaluated the incidence of POD in elderly patients undergoing general surgery, clinical/physical and psychological risk factors of POD insurgency and investigated inflammatory and genetic risk factors. Moreover, this study evaluated the consequence of POD in terms of institutionalization, development of permanent cognitive dysfunction or dementia and mortality Methods: patients aged over 65 admitted for surgery at the Urgency Unit of S.Orsola-Malpighi Hospital were eligible for this case–control study. Risk factors significantly associated with POD in univariate analysis were entered into multivariate analysis to establish those independently associated with POD. Preoperative plasma level of 9 inflammatory markers were measured in 42 control subjects and 43 subjects who developed POD. Functional polymorphisms of IL-1 α , IL-2, IL-6, IL-8, IL-10 and TNF-alpha cytokine genes were determined in 176 control subjects and 27 POD subjects. Results: A total of 351 patients were enrolled in the study. The incidence of POD was 13•2 %. Independent variables associated with POD were: age, co-morbidity, preoperative cognitive impairment, glucose abnormalities. Median length of hospital stay was 21 days for patients with POD versus 8 days for control patients (P < 0•001). The hospital mortality rate was 19 and 8•4 % respectively (P = 0•021) and mortality rate after 1 year was also higher in POD (P= 0.0001). The baseline of IL-6 concentration was higher in POD patients than patients without POD, whereas IL-2 was lower in POD patients compared to patients without POD. In a multivariate analysis only IL-6 remained associated with POD. Moreover IL-6, IL-8 and IL-2 are associated with co-morbidity, intra-hospital mortality, compromised functional status and emergency admission. No significant differences in genotype distribution were found between POD subjects and controls for any SNP analyzed in this study. Conclusion: In this study we found older age, comorbidity, cognitive impairment, glucose abnormalities and baseline of IL-6 as independent risk factors for the development of POD. IL-6 could be proposed as marker of a trait that is associated with an increased risk of delirium; i.e. raised premorbid IL-6 level predict for the development of delirium.

Biomolecular studies in Alzheimer’s Disease models: investigations in vitro and in vivo

The Alzheimer’s disease (AD), the most prevalent form of age-related dementia, is a multifactorial and heterogeneous neurodegenerative disease. The molecular mechanisms underlying the pathogenesis of AD are yet largely unknown. However, the etiopathogenesis of AD likely resides in the interaction between genetic and environmental risk factors. Among the different factors that contribute to the pathogenesis of AD, amyloid-beta peptides and the genetic risk factor apoE4 are prominent on the basis of genetic evidence and experimental data. ApoE4 transgenic mice have deficits in spatial learning and memory associated with inflammation and brain atrophy. Evidences suggest that apoE4 is implicated in amyloid-beta accumulation, imbalance of cellular antioxidant system and in apoptotic phenomena. The mechanisms by which apoE4 interacts with other AD risk factors leading to an increased susceptibility to the dementia are still unknown. The aim of this research was to provide new insights into molecular mechanisms of AD neurodegeneration, investigating the effect of amyloid-beta peptides and apoE4 genotype on the modulation of genes and proteins differently involved in cellular processes related to aging and oxidative balance such as PIN1, SIRT1, PSEN1, BDNF, TRX1 and GRX1. In particular, we used human neuroblastoma cells exposed to amyloid-beta or apoE3 and apoE4 proteins at different time-points, and selected brain regions of human apoE3 and apoE4 targeted replacement mice, as in vitro and in vivo models, respectively. All genes and proteins studied in the present investigation are modulated by amyloid-beta and apoE4 in different ways, suggesting their involvement in the neurodegenerative mechanisms underlying the AD. Finally, these proteins might represent novel potential diagnostic and therapeutic targets in AD.

Studio del possibile ruolo del complesso polycomb nel determinare la schisi del labbro e del palato

La labioschisi con o senza palatoschisi non-sindromica (NSCL/P) è tra le più frequenti alterazioni dello sviluppo embrionale, causata dall’interazione di fattori genetici e ambientali, moti dei quali ancora ignoti. L'obiettivo del mio progetto di Dottorato consiste nell’identificazione di fattori di rischio genetico in un processo a due stadi che prevede la selezione di geni candidati e la verifica del loro coinvolgimento nella determinazione della malformazione mediante studi di associazione. Ho analizzato alcuni polimorfismi a singolo nucleotide (SNPs) dei geni RFC1 e DHFR, appartenenti alla via metabolica dell’acido folico, evidenziando una debole associazione tra alcuni degli SNPs indagati e la NSCL/P nella popolazione italiana. Presso il laboratorio della Dott.ssa Mangold dell’Università di Bonn, ho valutato il ruolo di 15 diverse regioni cromosomiche nel determinare la suscettibilità alla malattia, evidenziando una significativa associazione per i marcatori localizzati in 8q24 e 1p22. Ho quindi rivolto la mia attenzione al ruolo del complesso Polycomb nell’insorgenza della schisi. Nell’uomo i due complessi Polycomb, PRC1 e PRC2, rimodellano la cromatina agendo da regolatori dei meccanismi trascrizionali alla base della differenziazione cellulare e dello sviluppo embrionale. Ho ipotizzato che mutazioni a carico di geni appartenenti a PRC2 possano essere considerati potenziali fattori di rischio genetico nel determinare la NSCL/P. Il razionale consiste nel fatto che JARID2, una proteina che interagisce con PRC2, è associata all’insorgenza della NSCL/P ed espressa a livello delle cellule epiteliali delle lamine palatine che si approssimano alla fusione. L’indagine condotta analizzando i geni di elementi o partner dei due complessi Polycomb, ha evidenziato un’associazione significativa con alcuni polimorfismi dei geni indagati, associazione ulteriormente confermata dall’analisi degli aplotipi. Le analisi condotte sui geni candidati mi hanno permesso di raccogliere dati interessanti sull’eziologia della malformazione. Studi indipendenti saranno necessari per poter validare l'associazione tra le varianti genetiche di questi geni candidati e la NSCL/P.

Genetic and environmental factors associated with the risk of cognitive decline and dementia

AD is the most common age related neurodegenerative disease in the industrialized world. Clinically AD is defined as a progressing decline of cognitive functions. Neuropathologically, AD is characterized by the aggregation of b-amyloid (Ab) peptide in the form of extracellular senile plaques, and hyperphosphorlylated tau protein in the form of intracellular neurofibrillary tangles. These neuropathological hallmarks are often accompanied by abundant microvascular damage and pronounced inflammation of the affected brain regions. In this thesis we investigated several aspects of AD focusing on the genetic aspect. We confirmed that Alpha 1 antichymotrypsin (ACT), an acute phase protein, was associated to AD subjects, being plasma levels higher in AD cases than controls. In addition, in a GWA study we demonstrated that two different gene, Clusterin and CR1 were strongly associated to AD. A single gene association not explain such a complex disease like AD. The goal should be to created a network of genetic, phenotypic and clinical data associated to AD. We used a new algorithm, the ANNs, aimed to map variables and search for connectivity among variables. We found specific variables associated to AD like cholesterol levels, the presence of variation in HMGCR enzyme and the age. Other factors such as the BMI, the amount of HDL and blood folate levels were also associated with AD. Pathogen infections, above all viral infections, have been previously associated to AD. The hypothesis suggests that virus and in particular herpes virus could enter the brain when an individual becomes older, perhaps because of a decline in the immune system. Our new hypothesis is that the presence of SNPs in our GWA gene study results in a genetic signature that might affect individual brain susceptibility to infection by herpes virus family during aging.

Exercise associated factors affecting cardiovascular health and function: from myocyte signaling to genetic variations

The exact mechanisms of the exercise induced adaptations is not lucid, but recent studies have delineated two means of signaling by which the adaptations occur (1) substrate availability signaling (metabolic stress) (2) hormone-receptor signaling. We have decided to specifically investigate two metabolic signaling enzymes [AMP-activated kinase (AMPK) and Sirtuin 1(SIRT1)] and two hormones [Adiponectin and Adrenergic stimulation].Tis based on four papers with the following conclusions: (1)Increase in SIRT1 activity and expression in H9c2 cells treated with phenylephrine is an adaptive response to the hypertrophic stress, mediated by AMPK. (2)The lack of optimal nutritional conditions (energetic substrates) due to a prolonged activation of AMPK can contrast the establishment of hypertrophy, possibly also by means of the negative modulation of ODC activity. (3) Our findings offer a possibile hypothesis as to the fact the the G allele on site 45 could lead to the increasd risk of Type II diabetes through a decrease in lean body mass. (4) Our results suggest that there is an ADIPOQ gene effect in relation to bone parameters. Statistical analysis show that the presence of the T allele in position 45 favors an increase in lumbar spine bone mineral content (BMC) when compared to subjects with a G allele substitution, which can be do the the increase in lean body mass in this genotype group.

Cellular and Molecular Biomarkers for Risk Assessment of Colorectal cancer

The detection of Colorectal Cancer (CRC), at early stages, is one of the proven strategies resulting in a higher cure rate. In recent years, several studies have appeared identifying potential cancer markers in serum, plasma and stool in an attempt to improve actual screening procedures. Thus, the aim of the study was (1) Evaluate MN frequency, (2) Evaluate plasma ultrafiltrate capacity to induce MN formation, (3) Evaluate SEPT9 and NOTCH3 promoter methylation profile in peripheral blood lymphocytes from subjects resulted positive to fecal occult blood test and examined by colonoscopy. MN frequency was significantly higher in subjects with histological diagnosis of CRC and adenoma than control (p ≤ 0.001 and p ≤ 0.01, respectively). About, CF-MN analysis, a statistically significant difference was observed between CRC and control (p ≤ 0.05). On the other hand, SEPT9 and NOTCH3 promoter methylation status was significantly lower in CRC subjects than controls; additionally, NOTCH3 promoter methylation status was significantly lower in CRC subjects than adenoma subjects (p ≤ 0.01). The results obtained allow conclude that MN frequency varies according CRC pathologic status and, together with other variables, is a valid biomarker for adenoma and CRC risk. Additionally, the plasma of patients affected with CRC not only serve as a biomarker for oxidative stress but also as biomarker of genetic damage correlated with the carcinogenic process that verifies in colon-rectum. SEPT9 and NOTCH3 promoter methylation status, at peripheral blood level, varies according hystopathological changes observed in colon-rectum, suggesting that promoter methylation profile of these genes could be a reliable biomarker for CRC risk.

Genetic and environmental factors associated with cardiovascular diseases and acute myocardial infarction

Acute myocardial infarction (AMI) is a multifactorial disease with a complex pathogenesis where lifestyle, individual genetic background and environmental risk factors are involved. Altered inflammatory responses seems to be implicated in the pathogenesis of atherosclerosis. To understand which genes may predispose to increased risk of cardiovascular disease gene polymorphism of immune regulatory genes, and clinical events from the Offs of parents with an early AMI were investigated. Genetics data from Offs were compared with those obtained from healthy subjects and an independent cohort of patients with clinical sporadic AMI. Rates of clinical events during a 24 years follow up from Offs and from an independent Italian population survey were also evaluated. This study showed that a genetic signature consisting of the concomitant presence of the CC genotype of VEGF, the A allele of IL-10 and the A allele of IFN-γ was indeed present in the Offs population. During the 24-year follow-up, Offs with a positive familiarity in spite of a relatively young age showed an increased prevalence of diabetes, ischemic heart disease and stroke. In these patients with the genetic signature the EBV and HHV-6 herpes virus were also investigated and founded. These findings reinforce the notion that subjects with a familial history of AMI are at risk of an accelerated aging of cardiovascular system resulting in cardiovascular events. These data suggest that selected genes with immune regulatory functions and envoronmental factors are part of the complex genetic background contributing to familiarity for cardiovascular diseases.N

Hypertension, hypercholesterolemia, hyperaldosteronism: a genetic perspective for personalized therapy.

Essential, primary, or idiopathic hypertension is defined as high BP in which secondary causes such as renovascular disease, renal failure, pheochromocytoma, hyperaldosteronism, or other causes of secondary hypertension are not present. Essential hypertension accounts for 80-90% of all cases of hypertension; it is a heterogeneous disorder, with different patients having different causal factors that may lead to high BP. Life-style, diet, race, physical activity, smoke, cultural level, environmental factors, age, sex and genetic characteristics play a key role in the increasing risk. Conversely to the essential hypertension, secondary hypertension is often associated with the presence of other pathological conditions such as dyslipidaemia, hypercholesterolemia, diabetes mellitus, obesity and primary aldosteronism. Amongst them, primary aldosteronism represents one of the most common cause of secondary hypertension, with a prevalence of 5-15% depending on the severity of blood pressure. Besides high blood pressure values, a principal feature of primary aldosteronism is the hypersecretion of mineralcorticoid hormone, aldosterone, in a manner that is fairly autonomous of the renin-angiotensin system. Primary aldosteronism is a heterogeneous pathology that may be divided essentially in two groups, idiopathic and familial form. Despite all this knowledge, there are so many hypertensive cases that cannot be explained. These individuals apparently seem to be healthy, but they have a great risk to develop CVD. The lack of known risk factors makes difficult their classification in a scale of risk. Over the last three decades a good help has been given by the pharmacogenetics/pharmacogenomics, a new area of the traditional pharmacology that try to explain and find correlations between genetic variation, (rare variations, SNPs, mutations), and the risk to develop a particular disease.

Identification of novel genetic alterations in pediatric cytogenetically normal acute myeloid leukemia by next-generation sequencing

Pediatric acute myeloid leukemia (AML) is a molecularly heterogeneous disease that arises from genetic alterations in pathways that regulate self-renewal and myeloid differentiation. While the majority of patients carry recurrent chromosomal translocations, almost 20% of childhood AML do not show any recognizable cytogenetic alteration and are defined as cytogenetically normal (CN)-AML. CN-AML patients have always showed a great variability in response to therapy and overall outcome, underlining the presence of unknown genetic changes, not detectable by conventional analyses, but relevant for pathogenesis, and outcome of AML. The development of novel genome-wide techniques such as next-generation sequencing, have tremendously improved our ability to interrogate the cancer genome. Based on this background, the aim of this research study was to investigate the mutational landscape of pediatric CN-AML patients negative for all the currently known somatic mutations reported in AML through whole-transcriptome sequencing (RNA-seq). RNA-seq performed on diagnostic leukemic blasts from 19 pediatric CN-AML cases revealed a considerable incidence of cryptic chromosomal rearrangements, with the identification of 21 putative fusion genes. Several of the fusion genes that were identified in this study are recurrent and might have a prognostic and/or therapeutic relevance. A paradigm of that is the CBFA2T3-GLIS2 fusion, which has been demonstrated to be a common alteration in pediatric CN-AML, predicting poor outcome. Important findings have been also obtained in the identification of novel therapeutic targets. On one side, the identification of NUP98-JARID1A fusion suggests the use of disulfiram; on the other, here we describe alteration-activating tyrosine kinases, providing functional data supporting the use of tyrosine kinase inhibitors to specifically inhibit leukemia cells. This study provides new insights in the knowledge of genetic alterations underlying pediatric AML, defines novel prognostic markers and putative therapeutic targets, and prospectively ensures a correct risk stratification and risk-adapted therapy also for the “all-neg” AML subgroup.