| Name | Tiziana |
| Surname | Squillaro |
| Institution | Università degli Studi della Campania Luigi Vanvitelli |
| tiziana.squillaro@unicampania.it | |
| Address | Department of Medical, Surgical, Neurological, Metabolic Sciences, and Aging, 2nd Division of Neurology, Center for Rare Diseases, University of Campania "Luigi Vanvitelli", Napoli, Italy |
We focused our attention on brahma-related gene 1 (BRG1), the ATPase subunit of the SWItch/Sucrose NonFermentable (SWI/SNF) chromatin remodeling complex, and analyzed its role in mesenchymal stem cell (MSC) biology. We hypothesized that deviation from the correct concentration of these proteins, which act at the highest level of gene regulation, may be deleterious for cells. We wanted to know what would happen if a cell had to cope with altered regulation of gene expression, either by upregulation or downregulation of BRG1. We assumed that cells would try to restore homeostasis or, alternatively, that the event could trigger senescence/apoptosis phenomena. To this end, in MSCs, we silenced BRG1gene. Knockdown of BRG1 expression induced a significant increase in senescent cells and decrease in apoptotic cells. It is interesting that BRG1 downregulation also induced an increase in heterochromatin. At the molecular level, these phenomena were associated with activation of retinoblastoma-like protein 2 (RB2)/P130- and P53-related pathways. Senescence was accompanied by reduced expression of some stemness-related genes. This is consistent with our previous research, which showed that BRG1 upregulation by ectopic expression also induced senescence processes. Together, these data suggest that BRG1 belongs to a class of genes whose expression is tightly regulated; hence, subtle alterations in BRG1 activity seem to negatively affect mechanisms regulating chromatin status and, in turn, impair cellular physiology.
DNA methylation is an epigenetic modification that occurs almost exclusively on CpG dinucleotides. MECP2 is a member of a family of proteins that preferentially bind to methylated CpGs. We analyzed the contribution of MECP2 to the physiology of mesenchymal stem cells (MSCs). Partial silencing of MECP2 in human MSCs induced a significant reduction of S-phase cells, along with an increase in G(1) cells. These changes were accompanied by a reduction of apoptosis, the triggering of senescence, a decrease in telomerase activity, and the down-regulation of genes involved in maintaining stem cell properties. Senescence appeared to rely on impairment of DNA damage repair and seemed to occur through RB- and P53-related pathways. The effects of MECP2 silencing could be related to the modification of the DNA methylation status. Our results indicate that the silencing of MECP2 induces an increase in methylated cytosines in the genome. Nevertheless, MECP2 partial silencing did not change the methylation of promoters, whose expression is affected by MECP2 down-regulation.
Hematopoietic stem cells derive regulatory information also from parathyroid hormone (PTH). To explore the possibility that PTH may have a role in regulation of other stem cells residing in bone marrow, such as mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) we assessed the effect of this hormone on the in vitro behavior of MSCs and EPCs. We evidenced that MSCs were much more responsive to PTH than EPCs. PTH increased the proliferation rate of MSCs with a diminution of senescence and apoptosis. Taken together, our results may suggest a protective effect of PTH on MSCs that reduces stress phenomena and preserve genome integrity. At the opposite, PTH did not modify the fate of EPCs in culture.
Mesenchymal stem cells (MSCs) are of particular interest because they are being tested using cell and gene therapies for a number of human diseases. MSCs represent a rare population in tissues. Therefore, it is essential to grow MSCs in vitro before putting them into therapeutic use. This is compromised by senescence, limiting the proliferative capacity of MSCs. We analyzed the in vitro senescence of rat MSCs, because this animal is a widespread model for preclinical cell therapy studies. After initial expansion, MSCs showed an increased growth doubling time, lost telomerase activity, and expressed senescence-associated beta-galactosidase. Senescence was accompanied by downregulation of several genes involved in stem cell self-renewal. Of interest, several genes involved in DNA repair also showed a significant downregulation. Entry into senescence occurred with characteristic changes in Retinoblastoma (RB) expression patterns. Rb1 and p107 genes expression decreased during in vitro cultivation. In contrast, pRb2/p130 became the prominent RB protein. This suggests that RB2/P130 could be a marker of senescence or that it even plays a role in triggering the process in MSCs.
Cancer stem cells have been isolated from many tumors. Several evidences prove that neuroblastoma contains its own stem cell-like cancer cells. We chose to analyze 20 neuroblastoma tumor samples in the expression of 13 genes involved in the regulation of stem cell properties to evaluate if their misregulation could have a clinical relevance. In several specimens we detected the expression of genes belonging to the OCT3/SOX2/NANOG/KLF4 core circuitry that acts at the highest level in regulating stem cell biology. This result is in agreement with studies showing the existence of malignant stem cells in neuroblastoma. We also observed differences in the expression of some stemness-related genes that may be useful for developing new prognostic analyses. In fact, preliminary data suggests that the presence/absence of UTF1 along with differences in BMI1 mRNA levels could distinguish low grade neuroblastomas from IV stage tumors.
Histone deacetylase inhibitors (HDACi) have received a great amount of attention for their antitumoral properties. Suberoyl anilide hydroxamic acid (SAHA) and MS-275 are among the more promising HDACi for cancer treatments. Although these HDACi compounds exert low toxicity on normal cells, the therapies based on these molecules can cause side effects that can greatly impair the functions of the bone marrow microenvironment. This is a complex system that contains several types of stem cells, such as mesenchymal stem cells (MSCs). We conducted comparative studies on the effects of SAHA and MS-275 on human MSCs in order to ascertain if these compounds can impair the physiology of MSCs. Both SAHA and MS-275 induced an arrest in the cell cycle along with the induction of apoptotic pathways as evidenced by flow cytometry, annexin assay, detection of activated caspase 9, and molecular analysis of Bax/Bcl-2 expression. The MS-275 treatment induced an increase of senescent cells, whereas in cells treated with SAHA, we detected a reduction of senescent cells compared to the control. We hypothesize that SAHA preferentially transactivates apoptotic genes, thereby inducing a great majority of the damaged cells to die by programmed cell death rather than senescence. Following the HDACi treatment, we observed a decrease in the expression of some genes that are involved in the regulation of stem cell properties. This suggests that SAHA and MS-275 could also be involved in the impairment of the stemness characteristics of MSCs. The phenomena that were induced by HDACi treatment were associated with an upregulation of several cyclin kinase inhibitors. By contrast, the p53-p21 pathway is apparently not involved in these processes.
Rett syndrome (RTT) is one of the most common genetic diseases responsible for a progressive disabling neurodevelopmental disorder. Mutations in the MeCP2 gene were identified in the great majority of RTT patients. MeCP2 protein binds to methylated DNA and produces changes in chromatin structure. This is a key event in regulation of gene expression. It has been suggested that MeCP2 might be important for neuronal development. Moreover, the frequent occurrence of osteoporosis and scoliosis in RTT patients suggests impaired bone formation and/or remodeling. Mesenchymal stem cells (MSCs) can differentiate as mesodermal cells such as bone, cartilage cells, and adipocytes. MSCs have been shown to possess great somatic plasticity; in fact, they can differentiate as neurons and astrocytes. We studied RTT patients' MSCs because they are progenitors of osteocytes, and it has been suggested that RTT patients' osteogenesis could be impaired. Moreover, MSCs might represent a useful model for the study of neurogenesis. MSCs from RTT patient showed precocious signs of senescence in a comparison with the MSCs of healthy-patient control groups. This was in agreement with the reduced gene-expression in the control of stem cell self-renewal and upregulation of lineage specific genes, such as those involved in osteogenesis and neural development. Control groups enabled us to observe a lower degree of apoptosis in RTT patient cells. This means that aberrant stem/progenitor cells, instead of being eliminated, can survive and become senescent. Our research provides a new insight into RTT syndrome. Senescence phenomena could be involved in triggering RTT syndrome-associated diseases.
Mutations in the gene Leucine-Rich Repeat Kinase 2 (LRRK2) have been identified in both dominant and sporadic cases affected by Parkinson's disease (PD). The LRRK2 G2019S mutation (c.6055G>A) is the most frequent substitution in Caucasians, accounting for approximately 5-6% of familial and 0.5-2.0% of apparently sporadic PD cases. We investigated the frequency of the LRRK2 G2019S mutation in 98 unrelated Italian PD patients, including 12 probands belonging to families compatible with autosomal dominant inheritance (12%) and 86 sporadic cases (88%). We detected the G2019S mutation in one sporadic female patient (1.2%). These results confirm that the G2019S mutation is a relevant cause of sporadic PD cases in the Italian population and stress the importance of performing this genetic test, which has important implications for genetic counselling.