Altered transcriptional and epigenetic regulation affects brain cells proliferation and differentiation in the ultra-rare genetic demyelinating disease AGC1 deficiency: a study on in vitro models

AGC1 deficiency is a rare demyelinating disease caused by mutations in the SLC25A12 gene, which encodes for the mitochondrial glutamate-aspartate carrier 1 (AGC1/Alarar), highly expressed in the central nervous system. In neurons, impairment in AGC1 activity leads to reduction in N-acetyl-aspartate, the main lipid precursor for myelin synthesis (Profilo et al., 2017); in oligodendrocytes progenitors cells, AGC1 down regulation has been related to early arrest proliferation and premature differentiation (Petralla et al., 2019). Additionally, in vivo AGC1 deficiency models i.e., heterozygous mice for AGC1 knock-out and neurospheres from their subventricular zone, respectively, showed a global decrease in cells proliferation and a switch in neural stem cells (NSCs) commitment, with specific reduction in OPCs number and increase in neural and astrocytic pools (Petralla et al., 2019). Therefore, the present study aims to investigate the transcriptional and epigenetic regulation underlying the alterations observed in OPCs and NSCs biological mechanisms, in either AGC1 deficiency models of Oli-neu cells (murine immortalized oligodendrocytes precursors cells), partially silenced by a shRNA for SLC25A12 gene, and SVZ-derived neurospheres from AGC1+/- mice. Western blot and immunofluorescence analysis revealed significant variations in the expression of transcription factors involved in brain cells’ proliferation and differentiation, in association with altered histone post-translational modifications, as well as histone acetylases (HATs) and deacetylases (HDACs) activity/expression, suggesting an improper transcriptional and epigenetic regulation affecting both AGC1 deficiency in vitro models. Furthermore, given the large role of acetylation in controlling in specific time-windows OPC maturation (Hernandez and Casaccia; 2015), pharmacological HATs/HDACs inhibitions were performed, confirming the involvement of chromatin remodelling enzymes in the altered proliferation and early differentiation observed in the AGC1 deficiency models of siAGC1 Oli-neu cells and AGC1+/- mice-derived neurospheres.

Diet Effects on Activation and Maturation of Feed Control over the Gastrointestinal Defence Barrier in Piglets

Weaning is an important and complex step involving many stresses that interfere deeply with feed intake, gastro-intestinal tract (GIT) development and adaptation to the weaning diet in young pigs. The health of the pig at weaning, its nutrition in the immediate post-weaning period, and the physical, microbiological and psychological environment are all factors that interact to determine food intake and subsequent growth. GIT disorders, infections and diarrhoea increase at the time of weaning, in fact pathogens such as enterotoxigenic Escherichia coli (ETEC) are major causes of mucosal damage in post-weaning disease contributing to diarrhoea in suckling and post-weaned pigs. The European ban in 2006 put on antibiotic growth promoters (AGP) has stimulated research on the mechanisms of GIT disorders and on nutritional approaches for preventing or reducing such disturbances avoiding AGPs. Concerning these aspects here are presented five studies based on the interplay among nutrition, genomic, immunity and physiology with the aim to clarify some of these problematic issues around weaning period in piglets. The first three evaluate the effects of diets threonine or tryptophan enriched on gut defence and health as possible alternatives to AGP in the gut. The fourth is focused on the possible immunological function related with the development of the stomach. The fifth is a pilot study on the gastric sensing and orexygenic signal given by fasting or re-feeding conditions. Although some results are controversial, it appears that both tryptophan and threonine supplementation in weaning diets have a preventive role in E.coli PWD and favorable effects in the gut especially in relation to ETEC susceptible genotype. While the stomach is believed as almost aseptic organ, it shows an immune activity related with the mucosal maturation. Moreover it shows an orexygenic role of both oxyntic mucosa and pyloric mucosa, and its possible relation with nutrient sensing stimuli.

Insights in the maturation of pathogenic bacteria vaccine candidates using mass spectrometry based approaches

The study of the maturation process that occurs to a protein is of pivotal importance for the understanding of its function. This is true also in the vaccine field but in this case is also important to evaluate if inappropriate protein conformation and maturation play roles in the impairment of the functional immunogenicity of protein vaccines. Mass spectrometry (MS) is the method of choice for the study of the maturation process since each modification that occurs during the maturation will lead to a change in the mass of the entire protein. Therefore the aim of my thesis is the development of mass spectrometry-based approaches to study the maturation of proteins and the application of these methods to proteic vaccine candidates. The thesis is divided in two main parts. In the first part, I focused my attention on the study of the maturation of different vaccine candidates using native mass spectrometry. The analyses in this case have been performed using recombinant proteins produced in E. coli. In the second part I applied different MS strategies for the identification of unknown PTMs on pathogenic bacteria surface proteins since modified surface proteins are now considered for vaccine candidate selection.

Pharmacological Rescue of Dendritic Pathology in the Ts65Dn Mouse Model of Down Syndrome

Down syndrome (DS) is a genetic pathology characterized by brain hypotrophy and severe cognitive disability. Although defective neurogenesis is an important determinant of cognitive impairment, a severe dendritic pathology appears to be an equally important factor. It is well established that serotonin plays a pivotal role both on neurogenesis and dendritic maturation. Since the serotonergic system is profoundly altered in the DS brain, we wondered whether defects in the hippocampal development can be rescued by treatment with fluoxetine, a selective serotonin reuptake inhibitor and a widely used antidepressant drug. A previous study of our group showed that fluoxetine fully restores neurogenesis in the Ts65Dn mouse model of DS and that this effect is accompanied by a recovery of memory functions. The goal of the current study was to establish whether fluoxetine also restores dendritic development and maturation. In mice aged 45 days, treated with fluoxetine in the postnatal period P3-P15, we examined the dendritic arbor of newborn and mature granule cells of the dentate gyrus (DG). The granule cells of trisomic mice had a severely hypotrophic dendritic arbor, fewer spines and a reduced innervation than euploid mice. Treatment with fluoxetine fully restored all these defects. Moreover the impairment of excitatory and inhibitory inputs to CA3 pyramidal neurons was fully normalized in treated trisomic mice, indicating that fluoxetine can rescue functional connectivity between the DG and CA3. The widespread beneficial effects of fluoxetine on the hippocampal formation suggest that early treatment with fluoxetine can be a suitable therapy, possibly usable in humans, to restore the physiology of the hippocampal networks and, hence, memory functions. These findings may open the way for future clinical trials in children and adolescents with DS.

Caratterizzazione e ruolo di PKCε e PKCδ in modelli di differenziamento megacariocitario normale e patologico

La PKCε e la PKCδ, chinasi ubiquitariamente distribuite e ad azione pleiotropica, sono implicate del differenziamento, sopravvivenza e proliferazione cellulare. Esse sono coinvolte nel processo differenziativo delle cellule staminali ematopoietiche e in fenomeni patologici associati al compartimento sanguigno. In questa tesi sono presentati i risultati riguardanti lo studio in vitro del ruolo di PKCε e PKCδ nel contesto del differenziamento megacariocitario, in particolare si caratterizza l’espressione e la funzione di queste chinasi nel modello umano e nel modello murino di Megacariocitopoiesi, normale e patologica. Confrontando le cinetiche dei due modelli presi in analisi nello studio è stato possibile osservare come in entrambi PKCε e PKCδ dimostrino avere una chiara e specifica modulazione nel progredire del processo differenziativo. Questi dati, se confrontati, permettono di affermare che PKCε e PKCδ presentano un pattern di espressione opposto e, nel modello umano rispetto a quello murino, reciproco: nell’uomo i livelli di PKCε devono essere down-modulati, mentre nel topo, al contrario, i livelli della chinasi risultano up-modulati durante lo stesso processo. Analogamente, le CD34+ in differenziazione presentano una costante e maggiore espressione di PKCδ durante la maturazione MK, mentre nel modello murino tale proteina risulta down-modulata nella fase più tardiva di formazione della piastrina. Le chinasi mostrano in oltre di agire, nei due modelli, attraverso pathways distinti e cioè RhoA nel topo e Bcl-xL nell’uomo. È stato inoltre verificato che l’aberrante differenziamento MK osservato nella mielofibrosi primaria (PMF), è associato a difetti di espressione di PKCε e di Bcl-xL e che una forzata down-modulazione di PKCε porta ad un ripristino di un normale livello di espressione di Bcl-xL così come della popolazione di megacariociti formanti propiastrine. I dati ottenuti indicano quindi che PKCε e PKCδ svolgono un ruolo importante nel corretto differenziamento MK e che PKCε potrebbe essere un potenziale nuovo target terapeutico nelle PMF.

Relevance of the pig stomach for the detection of dietary factors and gut maturation and control

In this thesis two approaches were applied to achieve a double general objective. The first chapter was dedicated to the study of the distribution of the expression of genes of several bitter and fat receptor in several gastrointestinal tracts. A set of 7 genes for bitter taste and for 3 genes for fat taste was amplified with real-time PCR from mRNA extracted from 5 gastrointestinal segments of weaned pigs. The presence of gene expression for several chemosensing receptors for bitter and fat taste in different compartments of the stomach confirms that this organ should be considered a player for the early detection of bolus composition. In the second chapter we investigated in young pigs the distribution of butyrate-sensing olfactory receptor (OR51E1) receptor along the GIT, its relation with some endocrine markers, its variation with age, and after interventions affecting the gut environment and intestinal microbiota in piglets and in different tissues. Our results indicate that OR51E1 is strictly related to the normal GIT enteroendocrine activity. In the third chapter we investigated the differential gene expression between oxyntic and pyloric mucosa in seven starter pigs. The obtained data indicate that there is significant differential gene exression between oxintic of the young pig and pyloric mucosa and further functional studies are needed to confirm their physiological importance. In the last chapter, thymol, that has been proposed as an oral alternative to antibiotics in the feed of pigs and broilers, was introduced directly into the stomach of 8 weaned pigs and sampled for gastric oxyntic and pyloric mucosa. The analysis of the whole transcript expression shoes that the stimulation of gastric proliferative activity and the control of digestive activity by thymol can influence positively gastric maturation and function in the weaned pigs.