Investigation of Sirtuins as Therapeutic Targets in Neurodegenerative Disorders: Studies on Mechanisms and Possible Combined Therapy

Up to now, current therapies for Alzheimer’s disease (AD) can treat the symptoms with modest effect and have little impact on the overall progression. AD is a complex, multifactorial disorder, featured by aggregation of toxic proteins, inflammation, oxidative stress, synaptic deficits, and cognitive decline. Thus, the multi-target therapeutic strategy is of particular interest as it uses a combination of drugs to affect different molecular targets and converge on neuroprotection or even disease modification. In drug discovery for neurodegenerative processes, an interesting role emerged for the histone-deacetylase enzymes Sirtuins (SIRTs), and SIRT1 and SIRT2 have been associated with neuroprotection and neurodegeneration, respectively. The availability of SIRTs small molecule modulators allowed the achievement of good results of SIRT1 activation or SIRT2 inhibition in models of neurodegeneration and AD, as both SIRTs are actively involved, to different extent, in the regulation of amyloidogenic processing, inflammatory and oxidative cascades. A direct intervention on such molecular players could be beneficial in obtaining cognitive or pathologic phenotype improvement deriving from multiple pathways targeting. In this work, we aimed at combining SIRT1 activation and SIRT2 inhibition to study a possible multi-target approach on AD models, both in vitro and in vivo. An initial drug dose-response and biochemical assessment was performed on the AD in vitro model H4-sw, focusing on the increase of the neuroprotective sAPPα fragment. Next, 3xTg-AD mice received the single or the combined treatment with SIRT1 activator SRT2104 and SIRT2 inhibitor AK7 for two weeks, and the effects on cognitive performance and key biochemical parameters were assessed. Results showed that both SIRT1 and SIRT2 single modulation independently improved cognitive performance in treated mice. Combined treatment showed complete memory recovery too. Some differences between single and double treatment emerged in biochemical assessments. Double-treated mice had increased NRF2, sAPPα and reduced Aβ oligomers in hippocampus, already seen upon SIRT2 inhibition, suggesting that cognitive improvement given by SIRT2 modulation could be attributed to those underlying effects. Together with memory recovery, SIRT1 activator treated mice showed decreased hippocampal CD11b and GFAP, increased synaptophysin, SOD1, sAPPα and NRF2, all seen upon double treatment too. Thus, SIRT1 and SIRT2 independent modulations could improve memory deficit acting both on peculiar or common protein targets. In conclusion, the combined treatment resumed all the behavioural and biochemical effects of single modulations. Our data support the concept that, with a multi-target strategy, it could be possible to take full advantage of the complementarity of SIRT1/SIRT2 treatments and obtain cognitive improvement based on changes on several underlying biochemical mechanisms that with single modulation could only be partially achieved. All these observations could open up new insights for research on SIRTs involvement in AD and neurodegeneration

The star formation history in the local group

In this thesis, I focused on the study of the dwarf galaxies in the Local Group (LG). Since they can be resolved into stars, this group of galaxies offers a perfect laboratory in addressing the problems still open in galaxy formation and evolution. In addition, the LG is composed of a unique mixture of all the morphological types of dwarfs, namely dwarf irregulars (dIrr), spheroidals (dSph), and the recently discovered ultra faints (UFD). The star formation histories (SFH) of these systems are fundamental to obtain deeper insights into their stellar populations, and especially on the presence of radial gradients. The sample of galaxies analysed in the present study is composed of a dIrr, two dSphs, and a UFD. These are NGC 6822, Draco, Ursa Minor (UMi), and Hercules, respectively. In all the cases the photometry was obtained using DAOPHOT/ ALLSTAR/ ALLFRAME (Stetson 1987, 1994), which resulted in the derivation of very accurate colour-magnitude diagrams (CMD). The study of NGC 6822 is based on HST data, whereas in all the other cases Subaru data have been used. In addition, in the case of NGC 6822 and Draco the accurate star formation history was derived using the IAC method composed of IACstar/ IAC-pop/ MinnIAC (Aparicio & Gallart 2004; Aparicio & Hidalgo 2009; Hidalgo et al. 2011). The data sample of NGC 6822 is divided into six fields, which cover the whole bar of this dwarf galaxy. The SFH solutions we derived show an enhanced star formation rate (SFR) in Fields 1 and 3 during the past 500 Myr, whereas the SFRs in the other fields are almost extinguished at very recent epochs. In addition, I studied the radial gradients of the populations by considering the total mass converted into stars in two time intervals (between 0 and 0.5 Gyr ago and between 0.5 and 13.5 Gyr ago). The scale lengths of the young and intermediate-to-old populations result perfectly compatible, with the exception of the young populations in Fields 1 and 3. The recent SFR in these two fields is greater than in the other ones. This might be an indication that in these two fields we are sampling incipient spiral arms. In addition, the age-metallicity relations were derived. As expected, the metallicity increases with time for all of the fields. No radial gradient in the metallicity is observed. The data available for the dSph Draco are composed of five fields, which cover as far out as 3 core radii. The SFH of Draco is characterized by a unique burst which took place ∼ 12.9 Gyr ago, with a width of ∼ 0.5 Gyr. This is compatible with a quenching of the SF activity due to the effect of reionization, even though a signature in the spectroscopic measurement of the metallicity rises doubts about this interpretation. In addition, I studied the presence of radial gradients in Draco. I calculated the scale lengths of different evolutionary features of this galaxy. All the scale lengths are compatible, hence there is evidence that Draco is composed of a unique stellar population. For UMi the SFH was not obtained, because of a significant difference between the predictions of the evolutionary models and the observed CMD. A real peculiarity in this galaxy is excluded if archival HST photometry is considered, since it agrees with the models. Hence I requested two additional sets of data, obtained with the IAC80 and NOT telescopes. The first dataset was used in order to verify the calibration equations obtained. The second one was used to investigate if a non linearity between the NOT and Subaru photometries was present. None of the two data sets were useful in solving the discrepancy, hence I performed a study of the populations in this galaxy based on star counts. In particular, I compared the width of the observed sub-giant branch with that predicted by stellar evolutionary models. This comparison sheds light on the duration of the SF burst in UMi, which results to be between 0.15 and 2 Gyr. Moreover, I investigated the radial gradients in this dSph. As in the previous cases I derived the scale lengths for different evolutionary features in this galaxy, which result compatible. Hence, UMi appears to be dominated by only one stellar population. The photometry of Hercules resulted to be too shallow to derive the accurate SFH, therefore, I compared it to a set of GGC and isochrones belonging to the BaSTI library (Pietrinferni et al. 2004). This comparison discloses that this galaxy hosts a population of age ≥ 12 Gyr and of very low metal content.Mi tesis est´a focalizada en el estudio de las galaxias enanas del Grupo Local (GL). Ya que las galaxias del GL se pueden resolver en estrellas, este grupo ofrece un laboratorio perfecto para estudiar la formaci´on y la evoluci´on estelar y gal´actica. Las galaxias enanas del GL son principalmente de tres tipos morfol´ogicos, irregulares, esferoidales, y ultra debiles (UFD). Adem´as, las historias de formaci´on estelar (HFE) son fundamentales para obtener informaci´on sobre las poblaciones estelares de que est´an compuestas. Las galaxias que he estudiado en mi tesis son una irregular, NGC 6822, dos esferoidales, Draco y Ursa Minor (UMi), y una ultra d´ebil, Hercules. Para todas las galaxias hice la fotometr´ıa aplicando el paquete DAOPHOT/ ALLSTAR/ ALLFRAME (Stetson 1987, 1994). De esta manera obtuve el diagramas color-magnitud (DCM) de cada galaxia. Los datos de NGC 6822 han sido obtenidos con el HST; los dem´as, con el telescopio Subaru. Para NGC 6822 y Draco deriv´e las HFEs usando el paquete desarrollado en el IAC espec´ıficamente para estudiar las poblaciones resueltas de las galaxias cercanas. Este paquete incluye IAC-star/ IAC-pop/ MinnIAC (Aparicio & Gallart 2004; Aparicio & Hidalgo 2009; Hidalgo et al. 2011). Los datos de NGC 6822 est´an divididos en seis campos, que cubren todo el cuerpo principal de esta galaxia. La HFE de NGC 6822 est´a caracterizada por una subida de la tasa de formaci´on estelar en los ultimos 500 Ma˜nos en los campos 1 y 3, mientras los dem´as campos tienen un tasa aproximadamente cero en ´epocas m´as recientes. Para NGC 6822 he estudiado tambi´en los gradientes de poblaciones estelares en dos intervalos temporales, uno joven entre 0 y 0.5 Ga˜nos y otro entre 0.5 y 13.5 Ga˜nos. Las escalas de longitud en los dos intervalos temporales resultan perfectamente compatibles, con la excepci´on de las poblaciones j´ovenes en los campos 1 y 3. Este resultado podr´ıa indicar que en estos dos campos hay brazos espirales en formaci´on. Las relaciones edad-metalicidad muestran que la metalicidad crece con el tiempo y que no hay dependencia con la distancia desde el centro de la galaxia. Draco ha sido observada en cinco campos, que cubren hasta tres veces su radio del n´ucleo. La HFE est´a caracterizada por un episodio de formaci´on estelar, con edad de ∼ 12.9 Gyr y anchura de 0.5 Gyr. Este resultado es compatible con la extinci´on de la tasa de formaci´o estelar por la reionizaci´on cosmica. No obstante, los estudios espectrosc´opicos arrojan dudas sobre esta interpretaci´on. Para concluir el estudio de Draco me focalic´e en los gradientes radiales de las poblaciones en esta galaxia. Calcul´e las escalas de longitud caracter´ısticas de distintas fases evolutivas y los valores obtenidos son todos compatibles, dentro de los errores. Esto es una evidencia de que no hay gradientes radiales en las poblaciones de esta galaxia y por lo tanto se puede considerar compuesta de una sola poblaci´on estelar muy antigua. En el caso de UMi no he derivado la HFE, porque se ha encontrado una fuerte discrepancia entre los datos observados y los modelos de evoluci´on estelar. Se excluye una peculiaridad real en esta galaxia, ya que los datos del HST son compatibles con los modelos evolutivos. Por esta raz´on he pedido dos observaciones adicionales, que se han realizado una con el IAC80 y otra con el NOT. Los datos del IAC80 han sido usados para comprobar los coeficientes derivados en la calibraci´on fotom´etrica. En cambio, los datos del NOT, que llegan a magnitudes m´as profundas, fueron usados para excluir la posibilidad de que los datos del Subaru no sean lineales a magnitudes d´ebiles. Ninguno de los dos grupos de datos ha ayudado a solucionar la discrepancia encontrada. Por esta raz´on compar´e la anchura de la rama de las sub-gigantes observada con la de la rama de las sub-gigantes de los modelos, considerando distintos intervalos de muestreo de edad. Usando esta comparaci´on se obtiene que el brote de formaci´on estelar en UMi ha durado entre 0.15 y 2 Ga˜nos. He seleccionado distintas fases evolutivas y, para estas he calculado las escalas de longitud. Tambi´en en este caso todos resultan compatibles dentro de los errores. Lo mismo que Draco, UMi posee una poblaci´on estelar ´unica. Para Hercules no deriv´e la HFE porque la fotometr´ıa no es bastante profunda. De todas formas, para obtener informaciones sobre las poblaciones estelares en esta galaxia, hice una comparaci´on con c´umulos globulares e is´ocronas de la libreria BaSTI (Pietrinferni et al. 2004). Hercules est´a dominada por una poblaci´on estelar muy antigua y de baja metalicidad

Abnormal hippocampal melatoninergic system: a potential link between absence epilepsy and depression-like behavior in WAG/Rij rats?

Absence epilepsy and depression are comorbid disorders, but the molecular link between the two disorders is unknown. Here, we examined the role of the melatoninergic system in the pathophysiology of spike and wave discharges (SWDs) and depression-like behaviour in the Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat model of absence epilepsy. In WAG/Rij rats, SWD incidence was higher during the dark period of the light-dark cycle, in agreement with previous findings. However, neither pinealectomy nor melatonin administration had any effect on SWD incidence, suggesting that the melatoninergic system was not involved in the pathophysiology of absence-like seizures. Endogenous melatonin levels were lower in the hippocampus of WAG/Rij rats as compared to non-epileptic control rats, and this was associated with higher levels of melatonin receptors in the hippocampus, but not in the thalamus. In line with the reduced melatonin levels, cell density was lower in the hippocampus ofWAG/Rij rats and was further reduced by pinealectomy. As expected, WAG/Rij rats showed an increased depression-like behaviour in the sucrose preference and forced swim tests, as compared to non-epileptic controls. Pinealectomy abolished the difference between the two strains of rats by enhancing depression-like behaviour in non-epileptic controls. Melatonin replacement displayed a significant antidepressant-like effect in bothWAG/Rij and control rats. These findings suggest that a defect of hippocampal melatoninergic system may be one of the mechanisms underlying the depression-like phenotype inWAG/Rij rats and that activation of melatonin receptors might represent a valuable strategy in the treatment of depression associated with absence epilepsy

Assessing agricultural eco-efficiency in Italian Regions

none5Agriculture plays a key role in providing a wide range of ecosystem services, such as food, feed, fiber and biofuel, thus taking part in the economic development of countries. On the other hand, this sector also gives rise to negative externalities. The eco-efficiency has been considered as a meaningful index for assessing how efficient economic activities are in terms of resource-use and environmental pressures: measuring eco-efficiency provides policy makers with important information for developing policies focused on sustainable management and ef- ficient use of natural resources in the agricultural sector. In this context, sustainable development is now one of the most important objectives of the European Union Common Agricultural Policy (CAP) that has a key role in facing the challenges of the new paradigm of sustainability of agriculture. In this direction, the aim of this paper is to evaluate the eco-efficiency of the Italian agricultural sector, as an index useful for emphasizing the dif- ferences among some national geographical areas. This paper tries to fill the lack of scientific studies on agri- cultural eco-efficiency in Italy, despite the strategic role played by Italy in Europe. For this purpose, the Data Envelopment Analysis (DEA) methodology was used, focusing on the integration between agricultural pro- ductivity and resource conservation, in order to develop a support tool for policy makers and managers. The analysis had shown a better orientation in saving resources for the Southern Regions and a greater orientation in productivity for the Northern Regions. Overall, Italy seems to have a good capacity for sustainable management of agricultural resources although there is still space for improvement. In this regard, the measurement of eco- efficiency provides a useful index for policy makers to achieve better performances in terms of agricultural sustainability. This means that CAP subsidies should be granted in exchange for specific environmental ex- ternalities provided by farmers as a result of more ecologically friendly management with a land use planning avoiding the depleting of Ecosystem Services rich areas, allowing for the achievement of a balance between economic growth and ecosystem protection. Although the paper has expanded the literature on agricultural eco- efficiency, this work has some limitations that could serve as a reference for future studies that can include other ecological variables such as the provision of some ecosystem services that can be enhanced or impacted by agricultural development. Finally, the challenge to realize sustainable agriculture can represent a long-term guarantee of food security as well as societal well-being.openColuccia, Benedetta; Valente, Donatella; Fusco, Giulio; De Leo, Federica; Porrini, DonatellaColuccia, Benedetta; Valente, Donatella; Fusco, Giulio; De Leo, Federica; Porrini, Donatell

Albendazole negatively regulates keratinocyte proliferation

Abstract Background: Increased keratinocyte proliferation occurs in the skin of psoriatic patients and is supposed to play a role in the pathogenesis of this disorder. Compounds interfering with keratinocyte proliferation could be useful in the management of psoriatic patients. Aim: To investigate whether albendazole, an anti-helmintic drug that regulates epithelial cell function in various systems, inhibits keratinocyte proliferation in models of psoriasis. Methods: Aldara-treated mice received daily topical application of albendazole. Keratinocyte proliferation and keratin (K) 6 and K16 expression were evaluated by immunohistochemistry and Western blotting and inflammatory cells/mediators were analysed by immunohistochemistry and real-time PCR. In human keratinocytes (HEKa and HaCaT) treated with albendazole, cell cycle and proliferation, keratins and cell cycle-associated factors were evaluated by flow cytometry, colorimetric assay and Western blotting respectively. Results: Aldara-treated mice given albendazole exhibited reduced epidermal thickness, decreased number of proliferating keratinocytes and K6/K16 expression. Reduction of CD3- and Ly6G-positive cells in the skin of albendazole-treated mice associated with inhibition of IL-6, TNF-α, IL-1β, IL-17A, IL-36, CCL17, CXCL1, CXCL2 and CXCL5 expression. Treatment of keratinocytes with albendazole reduced K6/K16 expression and reversibly inhibited cell growth by promoting accumulation of cells in S-phase. This phenomenon was accompanied by down-regulation of CDC25A, a phosphatase regulating progression of cell cycle through S-phase, and PKR-dependent hyper-phosphorylation of eIF2α, an inhibitor of CDC25 translation. In Aldara-treated mice, albendazole activated PKR, enhanced eIF2α phosphorylation and reduced CDC25A expression. Conclusions: Data show that albendazole inhibits keratinocyte proliferation and exerts therapeutic effect in a murine model of psoriasis

West Nile Virus lineage 1 in Italy: newly introduced or a re-occurrence of a previously circulating strain?

In Italy, West Nile virus (WNV) appeared for the first time in the Tuscany region in 1998. After 10 years of absence, it re-appeared in the areas surrounding the Po River delta, affecting eight provinces in three regions. Thereafter, WNV epidemics caused by genetically divergent isolates have been documented every year in the country. Since 2018, only WNV Lineage 2 has been reported in the Italian territory. In October 2020, WNV Lineage 1 (WNV-L1) re-emerged in Italy, in the Campania region. This is the first occurrence of WNV-L1 detection in the Italian territory since 2017. WNV was detected in the internal organs of a goshawk (Accipiter gentilis) and a kestrel (Falco tinnunculus). The RNA extracted in the goshawk tissue samples was sequenced, and a Bayesian phylogenetic analysis was performed by a maximum-likelihood tree. Genome analysis, conducted on the goshawk WNV complete genome sequence, indicates that the strain belongs to the WNV-L1 Western-Mediterranean (WMed) cluster. Moreover, a close phylogenetic similarity is observed between the goshawk strain, the 2008–2011 group of Italian sequences, and European strains belonging to the Wmed cluster. Our results evidence the possibility of both a new re-introduction or unnoticed silent circulation in Italy, and the strong importance of keeping the WNV surveillance system in the Italian territory activ

Metabolomics Fingerprint Induced by the Intranigral Inoculation of Exogenous Human Alpha-Synuclein Oligomers in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is considered a synucleinopathy because of the intraneuronal accumulation of aggregated α-synuclein (αSyn). Recent evidence points to soluble αSyn-oligomers (αSynO) as the main cytotoxic species responsible for cell death. Given the pivotal role of αSyn in PD, αSyn-based models are crucial for the investigation of toxic mechanisms and the identification of new therapeutic targets in PD. By using a metabolomics approach, we evaluated the metabolic profile of brain and serum samples of rats infused unilaterally with preformed human αSynOs (HαSynOs), or vehicle, into the substantia nigra pars compacta (SNpc). Three months postinfusion, the striatum was dissected for striatal dopamine (DA) measurements via High Pressure Liquid Chromatography (HPLC) analysis and mesencephalon and serum samples were collected for the evaluation of metabolite content via gas chromatography mass spectrometry analysis. Multivariate, univariate and correlation statistics were applied. A 40% decrease of DA content was measured in the HαSynO-infused striatum as compared to the contralateral and the vehicle-infused striata. Decreased levels of dehydroascorbic acid, myo-inositol, and glycine, and increased levels of threonine, were found in the mesencephalon, while increased contents of fructose and mannose, and a decrease in glycine and urea, were found in the serum of HαSynO-infused rats. The significant correlation between DA and metabolite content indicated that metabolic variations reflected the nigrostriatal degeneration. Collectively, the metabolomic fingerprint of HαSynO-infused rats points to an increase of oxidative stress markers, in line with PD neuropathology, and provides hints for potential biomarkers of PD

Increased transcription of transglutaminase 1 mediates neuronal death in in vitro models of neuronal stress and Aβ1–42-mediated toxicity

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