Valutazione del ruolo dell'espressione di IRS-1 nel differenziamento osteoblastico di cellule di osteosarcoma ed MSCs

L’osteosarcoma (OS) è il tumore primitivo dell’osso più comune in età pediatrica e adolescenziale. L’OS è stato recentemente riconsiderato come una patologia da de-differenziamento, legata all’interruzione del processo cui vanno incontro i precursori osteoblastici, quali le cellule staminali mesenchimali (MSCs), per trasformarsi in osteoblasti maturi. Il sistema IGF è coinvolto nella regolazione della proliferazione e del differenziamento di cellule di OS. IRS-1 è un mediatore critico di tale via di segnalazione e il suo livello di espressione modula il differenziamento di cellule ematopoietiche. Lo scopo di questa tesi è stato quello di definire il ruolo di IRS-1 nel differenziamento osteoblastico di MSCs e cellule di OS. Il potenziale differenziativo di cellule di OS umano e murino e di MSCs derivate da midollo osseo è stato valutato tramite Alizarin Red staining e Real Time-PCR. Dai dati ottenuti è emerso come i livelli di espressione di IRS-1 diminuiscano durante il differenziamento osteoblastico. Conseguentemente, i livelli di espressione di IRS-1 sono stati manipolati utilizzando shRNA per down-regolare l’espressione della proteina o un plasmide per sovra-esprimerla. Sia la down-regolazione sia la sovra-espressione di IRS-1 hanno inibito il differenziamento osteoblastico delle linee cellulari considerate. Allo scopo di valutare il contributo di IRS-1 nella via di segnalazione di IGF-1R è stato utilizzato l’inibitore di tale recettore, αIR-3. Anche in questo caso è stata osservata una riduzione della capacità differenziativa. L’inibitore del proteasoma MG-132 ha portato ad un aumento dei livelli di IRS-1, portando nuovamente all’inibizione del differenziamento osteoblastico e suggerendo che l’ubiquitinazione di questa proteina potrebbe avere un ruolo importante nel mantenimento di appropriati livelli di espressione di IRS-1. I risultati ottenuti indicano la criticità dei livelli di espressione di IRS-1 nella determinazione della capacità differenziativa sia di cellule di OS umano e murino, sia delle MSCs.

Sonic Hedgehog pathway impairment in Neural Precursor Cells of the Ts65Dn mouse, an animal model of Down syndrome

Mental retardation in Down syndrome (DS) has been imputed to the decreased brain volume, which is evident starting from the early phases of development. Recent studies in a widely used mouse model of DS, the Ts65Dn mouse, have shown that neurogenesis is severely impaired during the early phases of brain development, suggesting that this defect may be a major determinant of brain hypotrophy and mental retardation in individuals with DS. Recently, it has been found that in the cerebellum of Ts65Dn mice there is a defective responsiveness to Sonic Hedgehog (Shh), a potent mitogen that controls cell division during brain development, suggesting that failure of Shh signaling may underlie the reduced proliferation potency in DS. Based on these premises, we sought to identify the molecular mechanisms underlying derangement of the Shh pathway in neural precursor cells (NPCs) from Ts65Dn mice. We found that the expression levels of the Shh receptor Patched1 (Ptch1) were increased compared to controls both at the RNA and protein level. Partial silencing of Ptch1 expression in trisomic NPCs restored cell proliferation, indicating that proliferation impairment was due to Ptch1 overexpression. We further found that the overexpression of Ptch1 in trisomic NPCs is related to increased levels of AICD, a transcription-promoting fragment of amyloid precursor protein (APP). Increased AICD binding to the Ptch1 promoter favored its acetylated status, thus enhancing Ptch1 expression. Taken together, these data provide novel evidence that Ptch1 over expression underlies derangement of the Shh pathway in trisomic NPCs, with consequent proliferation impairment. The demonstration that Ptch1 over expression in trisomic NPCs is due to an APP fragment provides a link between this trisomic gene and the defective neuronal production that characterizes the DS brain.

Effect of loss of CDKL5 on brain development in a new Cdkl5 knockout mouse model

Rett's Syndrome (RTT) is a severe neurodevelopmental disorder, characterized by cognitive disability that appears in the first months/years of life. Recently, mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been detected in RTT patients characterized by early-onset seizures. CDKL5 is highly expressed in the brain starting from early postnatal stages to adulthood, suggesting the importance of this kinase for proper brain maturation and function. However, the role/s of CDKL5 in brain development and the molecular mechanisms whereby CDKL5 exerts its effects are still largely unknown. In order to characterize the role of CDKL5 on brain development, we created a mice carrying a targeted conditional knockout allele of Cdkl5. A first behavioral characterization shows that Cdkl5 knockout mice recapitulate several features that mimic the clinical features described in CDKL5 patients and are a useful tool to investigate phenotypic and functional aspects of Cdkl5 loss. We used the Cdkl5 knockout mouse model to dissect the role of CDKL5 on hippocampal development and to establish the mechanism/s underlying its actions. We found that Cdkl5 knockout mice showed increased precursor cell proliferation in the hippocampal dentate gyrus. Interestingly, this region was also characterized by an increased rate of apoptotic cell death that caused a reduction in the final neuron number in spite of the proliferation increase. Moreover, loss of Cdkl5 led to decreased dendritic development of new generated granule cells. Finally, we identified the Akt/GSK3-beta signaling as a target of Cdkl5 in the regulation of neuronal precursor proliferation, survival and maturation. Overall our findings highlight a critical role of CDKL5/AKT/GSK3-beta signaling in the control of neuron proliferation, survival and differentiation and suggest that CDKL5-related alterations of these processes during brain development underlie the neurological symptoms of the CDKL5 variant of RTT.

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.

Toward a 3D in vitro model based on decellularized thymus to maintain adult thymic ephitelial cells functionality

During my PhD,I have been develop an innovative technique to reproduce in vitro the 3D thymic microenvironment, to be used for growth and differentiation of thymocytes, and possible transplantation replacement in conditions of depressed thymic immune regulation. The work has been developed in the laboratory of Tissue Engineering at the University Hospital in Basel, Switzerland, under the tutorship of Prof.Ivan Martin. Since a number of studies have suggested that the 3D structure of the thymic microenvironment might play a key role in regulating the survival and functional competence of thymocytes, I’ve focused my effort on the isolation and purification of the extracellular matrix of the mouse thymus. Specifically, based on the assumption that TEC can favour the differentiation of pre-T lymphocytes, I’ve developed a specific decellularization protocol to obtain the intact, DNA-free extracellular matrix of the adult mouse thymus. Two different protocols satisfied the main characteristics of a decellularized matrix, according to qualitative and quantitative assays. In particular, the quantity of DNA was less than 10% in absolute value, no positive staining for cells was found and the 3D structure and composition of the ECM were maintained. In addition, I was able to prove that the decellularized matrixes were not cytotoxic for the cells themselves, and were able to increase expression of MHC II antigens compared to control cells grown in standard conditions. I was able to prove that TECs grow and proliferate up to ten days on top the decellularized matrix. After a complete characterization of the culture system, these innovative natural scaffolds could be used to improve the standard culture conditions of TEC, to study in vitro the action of different factors on their differentiation genes, and to test the ability of TECs to induce in vitro maturation of seeded T lymphocytes.

The constitutive activation of the DNA damage response pathway is a novel therapeutic target in aggressive B-cell lymphoma

The recent finding that MYC-driven cancers are sensitive to inhibition of the DNA damage response (DDR) pathway, prompted us to investigate the role of DDR pathway as therapeutic target in diffuse large B-cell lymphoma (DLBCL), which frequently overexpresses the MYC oncogene. In a preliminary immunohistochemical study conducted on 99 consecutive DLBCL patients, we found that about half of DLBCLs showed constitutive expression of the phosphorylated forms of checkpoint kinases (CHK) and CDC25c, markers of DDR activation, and of phosphorylated histone H2AX (γH2AX), marker of DNA damage and genomic instability. Constitutive γH2AX expression correlated with c-MYC levels and DDR activation, and defined a subset of tumors characterised by poor outcome. Next, we used the CHK inhibitor PF-0477736 as a tool to investigate whether the inhibition of the DDR pathway might represent a novel therapeutic approach in DLBCL. Submicromolar concentrations of PF-0477736 hindered proliferation in DLBCL cell lines with activated DDR pathway. These results were fully recapitulated with a different CHK inhibitor (AZD-7762). Inhibition of checkpoint kinases induced rapid DNA damage accumulation and apoptosis in DLBCL cell lines and primary cells. These data suggest that pharmacologic inhibition of DDR through targeting of CHK kinases may represent a novel therapeutic strategy in DLBCL. The second part of this work is the clinical, molecular and functional description of a paradigmatic case of primary refractory Burkitt lymphoma characterized by spatial intratumor heterogeneity for the TP53 mutational status, high expression levels of genomic instability and DDR activation markers, primary resistance to chemotherapy and exquisite sensitivity to DDR inhibitors.