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SARA D'ALESSANDRO

Dottorando
Dipartimento di Scienze Biomediche, Metaboliche e Neuroscienze

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Pubblicazioni

2024 - Molecular mechanisms underlying inherited photoreceptor degeneration as targets for therapeutic intervention [Articolo su rivista]
Bighinati, A.; Adani, E.; Stanzani, A.; D'Alessandro, S.; Marigo, V.
abstract

Retinitis pigmentosa (RP) is a form of retinal degeneration characterized by primary degeneration of rod photoreceptors followed by a secondary cone loss that leads to vision impairment and finally blindness. This is a rare disease with mutations in several genes and high genetic heterogeneity. A challenging effort has been the characterization of the molecular mechanisms underlying photoreceptor cell death during the progression of the disease. Some of the cell death pathways have been identified and comprise stress events found in several neurodegenerative diseases such as oxidative stress, inflammation, calcium imbalance and endoplasmic reticulum stress. Other cell death mechanisms appear more relevant to photoreceptor cells, such as high levels of cGMP and metabolic changes. Here we review some of the cell death pathways characterized in the RP mutant retina and discuss preclinical studies of therapeutic approaches targeting the molecular outcomes that lead to photoreceptor cell demise.

2023 - Intracellular cGMP increase is not involved in thyroid cancer cell death [Articolo su rivista]
Alessandro, Sara D'; Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Perri, Carmela; Antoniani, Francesco; Righi, Sara; Simoni, Manuela; Brigante, Giulia; Casarini, Livio
abstract

Introduction: Type 5 phosphodiesterase (PDE5) inhibitors (PDE5i) lead to intracellular cyclic-guanosine monophosphate (cGMP) increase and are used for clinical treatment of erectile dysfunction. Studies found that cGMP may up/downregulate the growth of certain endocrine tumor cells, suggesting that PDE5i could impact cancer risk. Aim: We evaluated if PDE5i may modulate thyroid cancer cell growth in vitro. Materials and methods: We used malignant (K1) and benign (Nthy-ori 3-1) thyroid cell lines, as well as the COS7 cells as a reference model. Cells were treated 0-24 h with the PDE5i vardenafil or the cGMP analog 8-br-cGMP (nM-μM range). cGMP levels and caspase 3 cleavage were evaluated by BRET, in cGMP or caspase 3 biosensor-expressing cells. Phosphorylation of the proliferation-associated extracellularly-regulated kinases 1 and 2 (ERK1/2) was evaluated by Western blotting, while nuclear fragmentation by DAPI staining. Cell viability was investigated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results: Both vardenafil and 8-br-cGMP effectively induced dose-dependent cGMP BRET signals (p≤0.05) in all the cell lines. However, no differences in caspase 3 activation occurred comparing PDE5i-treated vs untreated cells, at all concentrations and time-points tested (p>0.05). These results match those obtained upon cell treatment with 8-br-cGMP, which failed in inducing caspase 3 cleavage in all the cell lines (p>0.05). Moreover, they reflect the lack of nuclear fragmentation. Interestingly, the modulation of intracellular cGMP levels with vardenafil or the analog did not impact cell viability of both malignant and benign thyroid tumor cell lines, nor the phosphorylation of ERK1/2 (p>0.05). Conclusions: This study demonstrates that increased cGMP levels are not linked to cell viability or death in K1 and Nthy-ori 3-1 cell lines, suggesting that PDE5i do not impact the growth of thyroid cancer cells. Since different results were previously published, further investigations are recommended to clarify the impact of PDE5i on thyroid cancer cells.

2023 - Lack of GPER-TSHR heteromers is a hallmark of thyroid cancer [Abstract in Rivista]
Perri, Carmela; D'Alessandro, Sara; Paradiso, Elia; Lazzaretti, Clara; Mascolo, Elisa; Baschieri, Lara; Roy, Neena; Sperduti, Samantha; Simoni, Manuela; Brigante, Giulia; Casarini, Livio
abstract

2023 - LH increases the response to FSH in granulosa-lutein cells from sub/poor-responder patients in vitro [Articolo su rivista]
Sperduti, Samantha; Paradiso, Elia; Anzivino, Claudia; Lazzaretti, Clara; Limoncella, Silvia; D'Alessandro, Sara; Roy, Neena; Reggianini, Francesca; Ferrari, Tommaso; Melli, Beatrice; La Sala, Giovanni Battista; Nicoli, Alessia; Daolio, Jessica; Villani, Maria Teresa; Tagliavini, Simonetta; Trenti, Tommaso; Potì, Francesco; Sandhowe, Reinhild; Centonze, Chiara; Lispi, Monica; Simoni, Manuela; Casarini, Livio
abstract

STUDY QUESTION Does LH addition to FSH in vitro recover the human primary granulosa lutein cell (hGLC) sub/poor-response? SUMMARY ANSWER A picomolar concentration of LH may recover the FSH-induced cAMP and progesterone production of hGLC from sub/poor-responder women. WHAT is KNOWN ALREADY Clinical studies suggested that FSH and LH co-treatment may be beneficial for the ovarian response of sub/poor-responders undergoing ovarian stimulation during ART. STUDY DESIGN, SIZE, DURATION hGLC samples from 286 anonymous women undergoing oocyte retrieval for ART were collected from October 2017 to February 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS hGLCs from women undergoing ovarian stimulation during ART were blindly purified, cultured, genotyped and treated in vitro by increasing concentrations of FSH (nM) +/- 0.5 nM LH. cAMP and progesterone levels produced after 3 and 24 h, respectively, were measured. In vitro data were stratified a posteriori, according to the donors' ovarian response, into normo-, sub- and poor-responder groups and statistically compared. The effects of LH addition to FSH were compared with those obtained by FSH alone in all the groups as well. MAIN RESULTS AND THE ROLE of CHANCE hGLCs from normo-responders were shown to have higher sensitivity to FSH treatment than sub-/poor-responders in vitro. Equimolar FSH concentrations induced higher cAMP (about 2.5- to 4.2-fold), and progesterone plateau levels (1.2- to 2.1-fold), in cells from normo-responder women than those from sub-/poor-responders (ANOVA; P < 0.05). The addition of LH to the cell treatment significantly increased overall FSH efficacy, indicated by cAMP and progesterone levels, within all groups (P > 0.05). Interestingly, these in vitro endpoints, collected from the normo-responder group treated with FSH alone, were similar to those obtained in the sub-/poor-responder group under FSH + LH treatment. No different allele frequencies and FSH receptor (FSHR) gene expression levels between groups were found, excluding genetics of gonadotropin and their receptors as a factor linked to the normo-, sub- and poor-response. In conclusion, FSH elicits phenotype-specific ovarian lutein cell response. Most importantly, LH addition may fill the gap between cAMP and steroid production patterns between normo- and sub/poor-responders. LIMITATIONS, REASONS FOR CAUTION Although the number of experimental replicates is overall high for an in vitro study, clinical trials are required to demonstrate if the endpoints evaluated herein reflect parameters of successful ART. hGLC retrieved after ovarian stimulation may not fully reproduce the response to hormones of granulosa cells from the antral follicular stage. WIDER IMPLICATIONS of THE FINDINGS This in vitro assay may describe the individual response to personalize ART stimulation protocol, according to the normo-, sub- and poor-responder status. Moreover, this in vitro study supports the need to conduct optimally designed, randomized clinical trials exploring the personalized use of LH in assisted reproduction. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by Merck KGaA. M.L. and C.C. are employees of Merck KGaA or of the affiliate Merck Serono SpA. Other authors have no competing interests to declare.

2023 - Protein kinase B (Akt) blockade inhibits LH/hCG-mediated 17,20-lyase, but not 17[alpha]-hydroxylase activity of CYP17a1 in mouse Leydig cell steroidogenesis [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Melli, Beatrice; Perri, Carmela; D'Alessandro, Sara; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Casarini, Livio
abstract

2023 - Reprogramming of reproductive signals via human luteinizing hormone/choriogonadotropin receptor (LHCGR)/G protein-coupled estrogen receptor (GPER) heteromers [Abstract in Rivista]
Lazzaretti, Clara; Paradiso, Elia; Sperduti, Samantha; Sayers, Niamh; Pelagatti, Ginevra; D'Alessandro, Sara; Perri, Carmela; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Simoni, Manuela; Hanyaloglu, Aylin; Casarini, Livio
abstract

2022 - Analysis of follicle-stimulating hormone receptor (FSHR)/g-protein coupled estrogen receptor (GPER) complex internalization through early and late endosomes [Abstract in Atti di Convegno]
Lazzaretti, Clara; Casadei Garofani, Beatrice; Paradiso, Elia; D'Alessandro, Sara; Sperduti, Samantha; Roy, Neena; Mascolo, Elisa; Baschieri, Lara; Anzivino, Claudia; Simoni, Manuela; Casarini, Livio
abstract

2022 - cGMP is not involved in thyroid cancer cell death [Abstract in Atti di Convegno]
D'Alessandro, Sara; Paradiso, Elia; Lazzaretti, Clara; Sperduti, Samantha; Baschieri, Lara; Mascolo, Elisa; Roy, Neena; Anzivino, Claudia; Righi, Sara; Santi, Daniele; Brigante, Giulia; Simoni, Manuela; Casarini, Livio
abstract

2022 - Luteinizing hormone (LH)- and choriogonadotropin (hCG)-induced internalization of the receptor (LHCGR) is responsible for hormone-specific signaling [Abstract in Rivista]
Paradiso, Elia; Lazzaretti, Clara; D'Alessandro, Sara; Sperduti, Samantha; Roy, Neena; Mascolo, Elisa; Baschieri, Lara; Anzivino, Claudia; Simoni, Manuela; Casarini, Livio
abstract

2022 - Phosphodiesterase (PDE) 5 inhibitors sildenafil, tadalafil and vardenafil impact cAMP-specific PDE8 isoforms-linked second messengers and steroid production in a mouse Leydig tumor cell line [Articolo su rivista]
Limoncella, S.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Barbagallo, F.; Pacifico, S.; Guerrini, R.; Tagliavini, S.; Trenti, T.; Santi, D.; Simoni, M.; Sola, M.; Di Rocco, G.; Casarini, L.
abstract

Type 5 phosphodiesterase (PDE5) blockade by inhibitors (PDE5i) results in intracellular cyclic guanosine monophosphate (cGMP) increase and smooth muscle relaxation and are used for the treatment of men erectile dysfunction. Although they have high specificity for PDE5, these inhibitors are suspected to cross-interact also with cyclic adenosine monophosphate (cAMP)-specific PDEs, inducing the intracellular accumulation of this cyclic nucleotide and related testosterone increase, positively impacting male reproductive parameters. However, the link between the use of PDE5i and the activation of cAMP-mediated steroidogenesis is still unclear. We have investigated whether three PDE5i, sildenafil, tadalafil and vardenafil, cross-interacts with the high affinity cAMP-specific enzymes type 8A and 8B PDEs (PDE8A and PDE8B), in live, transfected mouse Leydig tumor (mLTC1) and human embryonic kidney (HEK293) cell lines in vitro. The PDE5i-induced production of cAMP-dependent testosterone and its precursor progesterone was evaluated as well. We have developed PDE8A/B biosensors and modified cyclic nucleotides confirming enzyme binding to cAMP, but not to cGMP, in our cell models. cAMP binding to PDE8A/B was displaced upon cell treatment with PDE5i, revealing that sildenafil, tadalafil and vardenafil have similar effectiveness in live cells, in vitro. The cross-interaction between PDE5i and PDE8A/B supports the gonadotropin-enhanced intracellular cAMP increase, occurring together with cGMP increase, as well as steroid synthesis. Indeed, we found that Leydig cell treatment by PDE5i increases progesterone and testosterone production triggered by gonadotropins. We demonstrated that PDE5i may interact with the cAMP-specific PDE8A and PDE8B, possibly inducing intracellular cAMP and sex steroid hormone increase. These findings support clinical data suggesting that PDE5i might increase testosterone levels in men.

2022 - Regulation of antral follicular growth by an interplay between gonadotropins and their receptors [Articolo su rivista]
Casarini, L.; Paradiso, E.; Lazzaretti, C.; D'Alessandro, S.; Roy, N.; Mascolo, E.; Zareba, K.; Garcia-Gasca, A.; Simoni, M.
abstract

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors’ functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The “heteromer hypothesis” explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle.

2022 - The endocrine disruptor Benzo[a]Pyrene inhibits gonadotropin-mediated steroidogenesis in a mouse Leydig tumor cell line [Abstract in Atti di Convegno]
Sperduti, Samantha; Roy, Neena; Lazzaretti, Clara; Paradiso, Elia; D'Alessandro, Sara; Mascolo, Elisa; Baschieri, Lara; Santi, Daniele; Simoni, Manuela; Casarini, Livio
abstract

2021 - Endocrine Disruption of the Follicle-Stimulating Hormone Receptor Signaling During the Human Antral Follicle Growth [Articolo su rivista]
Roy, N.; Mascolo, E.; Lazzaretti, C.; Paradiso, E.; D'Alessandro, S.; Zareba, K.; Simoni, M.; Casarini, L.
abstract

An increasing number of pollutants with endocrine disrupting potential are accumulating in the environment, increasing the exposure risk for humans. Several of them are known or suspected to interfere with endocrine signals, impairing reproductive functions. Follicle-stimulating hormone (FSH) is a glycoprotein playing an essential role in supporting antral follicle maturation and may be a target of disrupting chemicals (EDs) likely impacting female fertility. EDs may interfere with FSH-mediated signals at different levels, since they may modulate the mRNA or protein levels of both the hormone and its receptor (FSHR), perturb the functioning of partner membrane molecules, modify intracellular signal transduction pathways and gene expression. In vitro studies and animal models provided results helpful to understand ED modes of action and suggest that they could effectively play a role as molecules interfering with the female reproductive system. However, most of these data are potentially subjected to experimental limitations and need to be confirmed by long-term observations in human.