Evaluation of the transcriptomic response of an Alzheimer's disease murine model induced at different ages: searching for predictors

Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disease, characterized by the impairment of mnesic and cognitive functions, that represents the most frequent type of dementia in older people worldwide. Aging is the most important risk factor for the sporadic form of the pathology and it is associated to the progressive impairment of the proteostasis network. The endoplasmic reticulum (ER), the main cellular actor involved in proteostasis, appears significantly compromised in AD due to the accumulation of β-amyloid (Aβ) protein and phosphorylated-tau protein. Increasing proteins misfolding activates a specific cellular response known as Unfolded Protein response (UPR) which orchestrates the recovery of ER function. The aim of the present study was to investigate the role of UPR and aging process in a murine model of AD induced by intracerebroventricular (i.c.v.) injection of Aβ1-42 oligomers at 3 or 18 months. The oligomers injection in aged animals caused the increased of memory impairment, oxidative stress, and the depletion of glutathione reserve. Furthermore, the RNA-sequencing analysis was performed and the bioinformatic analysis showed the enrichment of several pathways involved in neurodegeneration and protein regulations. The following analysis highlighted the significant dysregulation of the three branches of the UPR, the protein kinase RNA-like ER kinase (PERK), inositol-requiring protein 1α (IRE1α) and activating transcription factor 6 (ATF-6). In turn, ER stress affected the PI3K/Akt/Gsk3β and MAPK/ERK pathways, highlighting Mapkapk5 as a potential marker of the neurodegenerative process, which regulation could lead to the definition of new pharmacological and neuroprotective strategies to counteract AD.

Design and synthesis of novel non peptidomimtic beta-secretase inhibitors in the treatment of Alzheimer's disease

The aspartic protease BACE1 (β-amyloid precursor protein cleaving enzyme, β-secretase) is recognized as one of the most promising targets in the treatment of Alzheimer's disease (AD). The accumulation of β-amyloid peptide (Aβ) in the brain is a major factor in the pathogenesis of AD. Aβ is formed by initial cleavage of β-amyloid precursor protein (APP) by β-secretase, therefore BACE1 inhibition represents one of the therapeutic approaches to control progression of AD, by preventing the abnormal generation of Aβ. For this reason, in the last decade, many research efforts have focused at the identification of new BACE1 inhibitors as drug candidates. Generally, BACE1 inhibitors are grouped into two families: substrate-based inhibitors, designed as peptidomimetic inhibitors, and non-peptidomimetic ones. The research on non-peptidomimetic small molecules BACE1 inhibitors remains the most interesting approach, since these compounds hold an improved bioavailability after systemic administration, due to a good blood-brain barrier permeability in comparison to peptidomimetic inhibitors. Very recently, our research group discovered a new promising lead compound for the treatment of AD, named lipocrine, a hybrid derivative between lipoic acid and the AChE inhibitor (AChEI) tacrine, characterized by a tetrahydroacridinic moiety. Lipocrine is one of the first compounds able to inhibit the catalytic activity of AChE and AChE-induced amyloid-β aggregation and to protect against reactive oxygen species. Due to this interesting profile, lipocrine was also evaluated for BACE1 inhibitory activity, resulting in a potent lead compound for BACE1 inhibition. Starting from this interesting profile, a series of tetrahydroacridine analogues were synthesised varying the chain length between the two fragments. Moreover, following the approach of combining in a single molecule two different pharmacophores, we designed and synthesised different compounds bearing the moieties of known AChEIs (rivastigmine and caproctamine) coupled with lipoic acid, since it was shown that dithiolane group is an important structural feature of lipocrine for the optimal inhibition of BACE1. All the tetrahydroacridines, rivastigmine and caproctamine-based compounds, were evaluated for BACE1 inhibitory activity in a FRET (fluorescence resonance energy transfer) enzymatic assay (test A). With the aim to enhancing the biological activity of the lead compound, we applied the molecular simplification approach to design and synthesize novel heterocyclic compounds related to lipocrine, in which the tetrahydroacridine moiety was replaced by 4-amino-quinoline or 4-amino-quinazoline rings. All the synthesized compounds were also evaluated in a modified FRET enzymatic assay (test B), changing the fluorescent substrate for enzymatic BACE1 cleavage. This test method guided deep structure-activity relationships for BACE1 inhibition on the most promising quinazoline-based derivatives. By varying the substituent on the 2-position of the quinazoline ring and by replacing the lipoic acid residue in lateral chain with different moieties (i.e. trans-ferulic acid, a known antioxidant molecule), a series of quinazoline derivatives were obtained. In order to confirm inhibitory activity of the most active compounds, they were evaluated with a third FRET assay (test C) which, surprisingly, did not confirm the previous good activity profiles. An evaluation study of kinetic parameters of the three assays revealed that method C is endowed with the best specificity and enzymatic efficiency. Biological evaluation of the modified 2,4-diamino-quinazoline derivatives measured through the method C, allow to obtain a new lead compound bearing the trans-ferulic acid residue coupled to 2,4-diamino-quinazoline core endowed with a good BACE1 inhibitory activity (IC50 = 0.8 mM). We reported on the variability of the results in the three different FRET assays that are known to have some disadvantages in term of interference rates that are strongly dependent on compound properties. The observed results variability could be also ascribed to different enzyme origin, varied substrate and different fluorescent groups. The inhibitors should be tested on a parallel screening in order to have a more reliable data prior to be tested into cellular assay. With this aim, preliminary cellular BACE1 inhibition assay carried out on lipocrine confirmed a good cellular activity profile (EC50 = 3.7 mM) strengthening the idea to find a small molecule non-peptidomimetic compound as BACE1 inhibitor. In conclusion, the present study allowed to identify a new lead compound endowed with BACE1 inhibitory activity in submicromolar range. Further lead optimization to the obtained derivative is needed in order to obtain a more potent and a selective BACE1 inhibitor based on 2,4-diamino-quinazoline scaffold. A side project related to the synthesis of novel enzymatic inhibitors of BACE1 in order to explore the pseudopeptidic transition-state isosteres chemistry was carried out during research stage at Università de Montrèal (Canada) in Hanessian's group. The aim of this work has been the synthesis of the δ-aminocyclohexane carboxylic acid motif with stereochemically defined substitution to incorporating such a constrained core in potential BACE1 inhibitors. This fragment, endowed with reduced peptidic character, is not known in the context of peptidomimetic design. In particular, we envisioned an alternative route based on an organocatalytic asymmetric conjugate addition of nitroalkanes to cyclohexenone in presence of D-proline and trans-2,5-dimethylpiperazine. The enantioenriched obtained 3-(α-nitroalkyl)-cyclohexanones were further functionalized to give the corresponding δ-nitroalkyl cyclohexane carboxylic acids. These intermediates were elaborated to the target structures 3-(α-aminoalkyl)-1-cyclohexane carboxylic acids in a new readily accessible way.

New Synthetic Polyamines as Multi-Target-Directed Ligands for the Treatment of Alzheimer's Disease and Cancer: Design, Synthesis and Biological Evaluation

Alzheimer's disease (AD) and cancer represent two of the main causes of death worldwide. They are complex multifactorial diseases and several biochemical targets have been recognized to play a fundamental role in their development. Basing on their complex nature, a promising therapeutical approach could be represented by the so-called "Multi-Target-Directed Ligand" approach. This new strategy is based on the assumption that a single molecule could hit several targets responsible for the onset and/or progression of the pathology. In particular in AD, most currently prescribed drugs aim to increase the level of acetylcholine in the brain by inhibiting the enzyme acetylcholinesterase (AChE). However, clinical experience shows that AChE inhibition is a palliative treatment, and the simple modulation of a single target does not address AD aetiology. Research into newer and more potent anti-AD agents is thus focused on compounds whose properties go beyond AChE inhibition (such as inhibition of the enzyme β-secretase and inhibition of the aggregation of beta-amyloid). Therefore, the MTDL strategy seems a more appropriate approach for addressing the complexity of AD and may provide new drugs for tackling its multifactorial nature. In this thesis, it is described the design of new MTDLs able to tackle the multifactorial nature of AD. Such new MTDLs designed are less flexible analogues of Caproctamine, one of the first MTDL owing biological properties useful for the AD treatment. These new compounds are able to inhibit the enzymes AChE, beta-secretase and to inhibit both AChE-induced and self-induced beta-amyloid aggregation. In particular, the most potent compound of the series is able to inhibit AChE in subnanomolar range, to inhibit β-secretase in micromolar concentration and to inhibit both AChE-induced and self-induced beta-amyloid aggregation in micromolar concentration. Cancer, as AD, is a very complex pathology and many different therapeutical approaches are currently use for the treatment of such pathology. However, due to its multifactorial nature the MTDL approach could be, in principle, apply also to this pathology. Aim of this thesis has been the development of new molecules owing different structural motifs able to simultaneously interact with some of the multitude of targets responsible for the pathology. The designed compounds displayed cytotoxic activity in different cancer cell lines. In particular, the most potent compounds of the series have been further evaluated and they were able to bind DNA resulting 100-fold more potent than the reference compound Mitonafide. Furthermore, these compounds were able to trigger apoptosis through caspases activation and to inhibit PIN1 (preliminary result). This last protein is a very promising target because it is overexpressed in many human cancers, it functions as critical catalyst for multiple oncogenic pathways and in several cancer cell lines depletion of PIN1 determines arrest of mitosis followed by apoptosis induction. In conclusion, this study may represent a promising starting pint for the development of new MTDLs hopefully useful for cancer and AD treatment.

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

Cross-Talk tra Bifidobacterium e intestino umano: impatto sulle attività health promoting

Bifidobacteria constitute up to 3% of the total microbiota and represent one of the most important healthpromoting bacterial groups of the human intestinal microflora. The presence of Bifidobacterium in the human gastrointestinal tract has been directly related to several health-promoting activities; however, to date, no information about the specific mechanisms of interaction with the host is available. The first health-promoting activities studied in these job was the oxalate-degrading activity. Oxalic acid occurs extensively in nature and plays diverse roles, especially in pathological processes. Due to its highly oxidizing effects, hyper absorption or abnormal synthesis of oxalate can cause serious acute disorders in mammals and be lethal in extreme cases. Intestinal oxalate-degrading bacteria could therefore be pivotal in maintaining oxalate homeostasis, reducing the risk of kidney stone development. In this study, the oxalate-degrading activity of 14 bifidobacterial strains was measured by a capillary electrophoresis technique. The oxc gene, encoding oxalyl-CoA decarboxylase, a key enzyme in oxalate catabolism, was isolated by probing a genomic library of B. animalis subsp. lactis BI07, which was one of the most active strains in the preliminary screening. The genetic and transcriptional organization of oxc flanking regions was determined, unravelling the presence of other two independently transcribed open reading frames, potentially responsible for B. animalis subsp. lactis ability to degrade oxalate. Transcriptional analysis, using real-time quantitative reverse transcription PCR, revealed that these genes were highly induced in cells first adapted to subinhibitory concentrations of oxalate and then exposed to pH 4.5. Acidic conditions were also a prerequisite for a significant oxalate degradation rate, which dramatically increased in oxalate pre-adapted cells, as demonstrated in fermentation experiments with different pH-controlled batch cultures. These findings provide new insights in the characterization of oxalate-degrading probiotic bacteria and may support the use of B. animalis subsp. lactis as a promising adjunct for the prophylaxis and management of oxalate-related kidney disease. In order to provide some insight into the molecular mechanisms involved in the interaction with the host, in the second part of the job, we investigated whether Bifidobacterium was able to capture human plasminogen on the cell surface. The binding of human plasminogen to Bifidobacterium was dependent on lysine residues of surface protein receptors. By using a proteomic approach, we identified six putative plasminogen-binding proteins in the cell wall fraction of three strain of Bifidobacterium. The data suggest that plasminogen binding to Bifidobactrium is due to the concerted action of a number of proteins located on the bacterial cell surface, some of which are highly conserved cytoplasmic proteins which have other essential cellular functions. Our findings represent a step forward in understanding the mechanisms involved in the Bifidobacterium-host interaction. In these job w studied a new approach based on to MALDI-TOF MS to measure the interaction between entire bacterial cells and host molecular target. MALDI-TOF (Matrix Assisted Laser Desorption Ionization-Time of Flight)—mass spectrometry has been applied, for the first time, in the investigation of whole Bifidobacterium cells-host target proteins interaction. In particular, by means of this technique, a dose dependent human plasminogen-binding activity has been shown for Bifidobacterium. The involvement of lysine binding sites on the bacterial cell surface has been proved. The obtained result was found to be consistent with that from well-established standard methodologies, thus the proposed MALDI-TOF approach has the potential to enter as a fast alternative method in the field of biorecognition studies involving in bacterial cells and proteins of human origin.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Molecular approach to Neurodegenerative Disorders: Role of Nociceptin/Orphanin FQ - NOP System in Parkinson and Epigenetic Mechanism in Alzheimer's Disease

With life expectancies increasing around the world, populations are getting age and neurodegenerative diseases have become a global issue. For this reason we have focused our attention on the two most important neurodegenerative diseases: Parkinson’s and Alzheimer’s. Parkinson’s disease is a chronic progressive neurodegenerative movement disorder of multi-factorial origin. Environmental toxins as well as agricultural chemicals have been associated with PD. Has been observed that N/OFQ contributes to both neurotoxicity and symptoms associated with PD and that pronociceptin gene expression is up-regulated in rat SN of 6-OHDA and MPP induced experimental parkinsonism. First, we investigated the role of N/OFQ-NOP system in the pathogenesis of PD in an animal model developed using PQ and/or MB. Then we studied Alzheimer's disease. This disorder is defined as a progressive neurologic disease of the brain leading to the irreversible loss of neurons and the loss of intellectual abilities, including memory and reasoning, which become severe enough to impede social or occupational functioning. Effective biomarker tests could prevent such devastating damage occurring. We utilized the peripheral blood cells of AD discordant monozygotic twin in the search of peripheral markers which could reflect the pathology within the brain, and also support the hypothesis that PBMC might be a useful model of epigenetic gene regulation in the brain. We investigated the mRNA levels in several genes involve in AD pathogenesis, as well DNA methylation by MSP Real-Time PCR. Finally by Western Blotting we assess the immunoreactivity levels for histone modifications. Our results support the idea that epigenetic changes assessed in PBMCs can also be useful in neurodegenerative disorders, like AD and PD, enabling identification of new biomarkers in order to develop early diagnostic programs.

Genetic analysis of tumour initiation and progression in a Drosophila model of epithelial cancer

Neoplastic overgrowth depends on the cooperation of several mutations ultimately leading to major rearrangements in cellular behaviour. The molecular crosstalk occurring between precancerous and normal cells strongly influences the early steps of the tumourigenic process as well as later stages of the disease. Precancerous cells are often removed by cell death from normal tissues but the mechanisms responsible for such fundamental safeguard processes remain in part elusive. To gain insight into these phenomena I took advantage of the clonal analysis methods available in Drosophila for studying the phenotypes due to loss of function of the neoplastic tumour suppressor lethal giant larvae (lgl). I found that lgl mutant cells growing in wild-type imaginal wing discs are subject to the phenomenon of cell competition and are eliminated by JNK-dependent cell death because they express very low levels of dMyc oncoprotein compared to those in the surrounding tissue. Indeed, in non-competitive backgrounds lgl mutant clones are able to overgrow and upregulate dMyc, overwhelming the neighbouring tissue and forming tumourous masses that display several cancer hallmarks. These phenotypes are completely abolished by reducing dMyc abundance within mutant cells while increasing it in lgl clones growing in a competitive context re-establishes their tumourigenic potential. Similarly, the neoplastic growth observed upon the oncogenic cooperation between lgl mutation and activated Ras/Raf/MAPK signalling was found to be characterised by and dependent on the ability of cancerous cells to upregulate dMyc with respect to the adjacent normal tissue, through both transcriptional and post-transcriptional mechanisms, thereby confirming its key role in lgl-induced tumourigenesis. These results provide first evidence that the dMyc oncoprotein is required in lgl mutant tissue to promote invasive overgrowth in developing and adult epithelial tissues and that dMyc abundance inside versus outside lgl mutant clones plays a key role in driving neoplastic overgrowth.

Design and synthesis of multipotent drugs: application to Alzheimer's disease and benign prostatic hyperplasia

The aim of this thesis was to synthesize multipotent drugs for the treatment of Alzheimer’s disease (AD) and for benign prostatic hyperplasia (BPH), two diseases that affect the elderly. AD is a neurodegenerative disorder that is characterized, among other factors, by loss of cholinergic neurons. Selective activation of M1 receptors through an allosteric site could restore the cholinergic hypofunction, improving the cognition in AD patients. We describe here the discovery and SAR of a novel series of quinone derivatives. Among them, 1 was the most interesting, being a high M1 selective positive allosteric modulator. At 100 nM, 1 triplicated the production of cAMP induced by oxotremorine. Moreover, it inhibited AChE and it displayed antioxidant properties. Site-directed mutagenesis experiments indicated that 1 acts at an allosteric site involving residue F77. Thus, 1 is a promising drug because the M1 activation may offer disease-modifying properties that could address and reduce most of AD hallmarks. BPH is an enlargement of the prostate caused by increased cellular growth. Blockade of α1-ARs is the predominant form of medical therapy for the treatment of the symptoms associated with BPH. α1-ARs are classified into three subtypes. The α1A- and α1D-AR subtypes are predominant in the prostate, while α1B-ARs regulate the blood pressure. Herein, we report the synthesis of quinazoline-derivatives obtained replacing the piperazine ring of doxazosin and prazosin with (S)- or (R)-3-aminopiperidine. The presence of a chiral center in the 3-C position of the piperidine ring allowed us to exploit the importance of stereochemistry in the binding at α1-ARs. It turned out that the S configuration at the 3-C position of the piperidine increases the affinity of the compounds at all three α1-AR subtypes, whereas the configuration at the benzodioxole ring of doxazosin derivatives is not critical for the interaction with α1-ARs.

Regulation of wake-sleep states and state-dependent cardiovascular function in diet-induced obesity rats

Obesity often predisposes to coronary heart disease, heart failure, and sudden death. Also, several studies suggest a reciprocal enhancing interaction between obesity and sleep curtailment. Aim of the present study was to go deeper in the understanding of sleep and cardiovascular regulation in an animal model of diet-induced obesity (DIO). According to this, Wake-Sleep (W-S) regulation, and W-S dependent regulation of cardiovascular and metabolic/thermoregulatory function was studied in DIO rats, under normal laboratory conditions and during sleep deprivation and the following recovery period, enhancing either wake or sleep, respectively. After 8 weeks of the delivery of a hypercaloric (HC) diet, treated animals were heavier than those fed a normocaloric (NC) diet (NC: 441 ±17g; HC: 557±17g). HC rats slept more than NC ones during the activity period (Dark) of the normal 12h:12h light-dark (LD) cycle (Wake: 67.3±1.2% and 57.2 ±1.6%; NREM sleep (NREMS): 26.8±1.0% and 34.0±1.4%; REM sleep (REMS): 5.7±0. 6% and 8.6±0.7%; for NC and HC, respectively; p<0.05 for all). HC rats were hypertensive throughout the W-S states, as shown by the mean arterial blood pressure values across the 24-h period (Wake: 90.0±5.3 and 97.3±1.3; NREMS: 85.1±5.5 and 92.2±1.2; REMS: 87.2±4.5 and 96.5±1.1, mmHg for NC and HC, respectively; p<0.05 for all). Also, HC rats appeared to be slightly bradycardic compared to NC ones (Wake: 359.8±9.3 and 352.4±7.7; NREMS: 332.5±10.1 and 328.9±5.4; REMS: 338.5±9.3 and 334.4±5.8; bpm for NC and HC, respectively; p<0.05 for Wake). In HC animals, sleep regulation was not apparently altered during the sleep rebound observed in the recovery period following sleep deprivation, although REMS rebound appeared to be quicker in NC animals. In conclusion, these results indicate that in the rat obesity interfere with W-S and cardiovascular regulation and that DIO rats are suitable for further studies aimed at a better understanding of obesity comorbidities.

Molecular basis of Osteoarthritis and aspects of cellular senescence in disease

The aim of this study is to investigate on some molecular mechanisms contributing to the pathogenesis of osteoarthritis (OA) and in particular to the senescence of articular chondrocytes. It is focused on understanding molecular events downstream GSK3β inactivation or dependent on the activity of IKKα, a kinase that does not belong to the phenotype of healthy articular chondrocytes. Moreover, the potential of some nutraceuticals on scavenging ROS thus reducing oxidative stress, DNA damage, and chondrocyte senescence has been evaluated in vitro. The in vitro LiCl-mediated GSK3β inactivation resulted in increased mitochondrial ROS production, that impacted on cellular proliferation, with S-phase transient arrest, increased SA-β gal and PAS staining, cell size and granularity. ROS are also responsible for the of increased expression of two major oxidative lesions, i.e. 1) double strand breaks, tagged by γH2AX, that associates with activation of GADD45β and p21, and 2) 8-oxo-dG adducts, that associate with increased IKKα and MMP-10 expression. The pattern observed in vitro was confirmed on cartilage from OA patients. IKKa dramatically affects the intensity of the DNA damage response induced by oxidative stress (H2O2 exposure) in chondrocytes, as evidenced by silencing strategies. At early time point an higher percentage of γH2AX positive cells and more foci in IKKa-KD cells are observed, but IKKa KD cells proved to almost completely recover after 24 hours respect to their controls. Telomere attrition is also reduced in IKKaKD. Finally MSH6 and MLH1 genes are up-regulated in IKKαKD cells but not in control cells. Hydroxytyrosol and Spermidine have a great ROS scavenging capacity in vitro. Both treatments revert the H2O2 dependent increase of cell death and γH2AX-foci formation and senescence, suggesting the ability of increasing cell homeostasis. These data indicate that nutraceuticals represent a great challenge in OA management, for both therapeutical and preventive purposes.

Multimodal (EEG-fMRI) functional connectivity study of levodopa effect in Parkinson’s disease

Aim: To assess if the intake of levodopa in patients with Parkinson’s Disease (PD) changes cerebral connectivity, as revealed by simultaneous recording of hemodynamic (functional MRI, or fMRI) and electric (electroencephalogram, EEG) signals. Particularly, we hypothesize that the strongest changes in FC will involve the motor network, which is the most impaired in PD. Methods: Eight patients with diagnosis of PD “probable”, therapy with levodopa exclusively, normal cognitive and affective status, were included. Exclusion criteria were: moderate-severe rest tremor, levodopa induced dyskinesia, evidence of gray or white matter abnormalities on structural MRI. Scalp EEG (64 channels) were acquired inside the scanner (1.5 Tesla) before and after the intake of levodopa. fMRI functional connectivity was computed from four regions of interest: right and left supplementary motor area (SMA) and right and left precentral gyrus (primary motor cortex). Weighted partial directed coherence (w-PDC) was computed in the inverse space after the removal of EEG gradient and cardioballistic artifacts. Results and discussion: fMRI group analysis shows that the intake of levodopa increases hemodynamic functional connectivity among the SMAs / primary motor cortex and: sensory-motor network itself, attention network and default mode network. w-PDC analysis shows that EEG connectivity among regions of the motor network has the tendency to decrease after the intake the levodopa; furthermore, regions belonging to the DMN have the tendency to increase their outflow toward the rest of the brain. These findings, even if in a small sample of patients, suggest that other resting state physiological functional networks, beyond the motor one, are affected in patients with PD. The behavioral and cognitive tasks corresponding to the affected networks could benefit from the intake of levodopa.