Astrocytes specifically synthesize and release endozepines, a family of regulatory peptides including octadecaneuropeptide (ODN). We have previously reported that ODN rescues neurons and astrocytes from 6-OHDA-induced oxidative stress and cell death. The purpose of this study was to examine the potential implication of miR-34b, miR-29a, and miR-21 in the protective activity of ODN on 6-OHDA-induced oxidative stress and cell death in cultured rat astrocytes. Flow cytometry analysis showed that 6-OHDA increased the number of early apoptotic and apoptotic dead cells while treatment with the subnanomolar dose of ODN significantly reduced the number of apoptotic cells induced by 6-OHDA. 6-OHDA-treated astrocytes exhibited the over-expression of miR-21 (+118%) associated with a knockdown of miR-34b (−61%) and miR-29a (−49%). Co-treatment of astrocytes with ODN blocked the 6-OHDA-stimulated production of ROS and NO and stimulation of Bax and caspase-3 gene transcription. Concomitantly, ODN down-regulated the expression of miR-34b and miR-29a and rescued the 6-OHDA-associated reduced expression of miR21, indicating that ODN regulates their expression during cell death. Transfection with miR-21-3p inhibitor prevented the effect of 6-OHDA against cell death. In conclusion, our study indicated that (i) the expression of miRNAs miR-34b, miR-29a, and miR-21 is modified in astrocytes under 6-OHDA injury and (ii) that ODN prevents this deregulation to induce its neuroprotective action. The present study identified miR-21 as an emerging candidate and as a promising pharmacological target that opens new neuroprotective therapeutic strategies in neurodegenerative diseases, especially in Parkinson’s disease.

High concentrations of pituitary adenylate cyclase-activating polypeptide (PACAP) and a high density of PACAP binding sites have been detected in the developing rat cerebellum. In particular, PACAP receptors are actively expressed in immature granule cells, where they activate both adenylyl cyclase and phospholipase C. The aim of the present study was to investigate the ability of PACAP to induce calcium mobilization in cerebellar granule neurons. Administration of PACAP-induced a transient, rapid, and monophasic rise of the cytosolic calcium concentration ([Ca²⁺]_i), while vasoactive intestinal peptide was devoid of effect, indicating the involvement of the PAC1 receptor in the Ca²⁺ response. Preincubation of granule cells with the Ca²⁺ ATPase inhibitor, thapsigargin, or the D-myo-inositol 1,4,5-trisphosphate (IP₃) receptor antagonist, 2-aminoethoxydiphenyl borate, markedly reduced the stimulatory effect of PACAP on [Ca²⁺]_i. Furthermore, addition of the calcium chelator, EGTA, or exposure of cells to the non-selective Ca²⁺ channel blocker, NiCl₂, significantly attenuated the PACAP-evoked [Ca²⁺]_i increase. Preincubation of granule neurons with the N-type Ca²⁺ channel blocker, ω-conotoxin GVIA, decreased the PACAP-induced [Ca²⁺]_i response, whereas the L-type Ca²⁺ channel blocker, nifedipine, and the P- and Q-type Ca²⁺ channel blocker, ω-conotoxin MVIIC, had no effect. Altogether, these findings indicate that PACAP, acting through PAC1 receptors, provokes an increase in [Ca²⁺]_i in granule neurons, which is mediated by both mobilization of calcium from IP₃-sensitive intracellular stores and activation of N-type Ca²⁺ channel. Some of the activities of PACAP on proliferation, survival, migration, and differentiation of cerebellar granule cells could thus be mediated, at least in part, through these intracellular and/or extracellular calcium fluxes.

STUDY QUESTION: Does exposure to first-line chemotherapies affect the density and maturity status of testicular somatic cells from prepubertal to postpubertal males at the time of cryopreservation? SUMMARY ANSWER: Chemotherapy exposures, particularly high doses of alkylating agents, are associated with alterations in immunohistochemical markers of testicular somatic cell density and maturation, including SOX9 for Sertoli cell density, COUP-TFII for peritubular myoid cell maturation, and CYP11A1 and INSL3 for Leydig cell maturation, with the related effects becoming more nuanced when age and underlying pathology are considered. WHAT IS KNOWN ALREADY: The increased survival rates following cancer treatment highlight the importance of integrating fertility preservation, whether through sperm cryopreservation in pubertal and postpubertal males or testicular tissue freezing (TTF), which is now established for prepubertal and peripubertal boys. Although induction chemotherapy is known to impair germ cells, its effects on somatic cells (Sertoli, peritubular myoid, and Leydig cells) within testicular tissue collected after exposure to chemotherapeutic drugs remain largely unexplored. STUDY DESIGN, SIZE, DURATION: This retrospective study included testicular tissue samples from 51 patients from prepubertal to postpubertal males diagnosed with cancer, aged between 6 months and 33 years. Their medical diagnoses and previous chemotherapy exposures were recorded. We examined histological sections of testicular tissue to assess the impact of chemotherapy on somatic cell density and maturity status. PARTICIPANTS/MATERIALS, SETTING, METHODS: The density and maturity status of testicular somatic cells were assessed by immunofluorescent staining. The analyses were performed on fresh testicular tissue fragments from prepubertal and postpubertal males with cancer who had undergone TTF prior to conditioning for hematopoietic stem cell transplantation. All had received first-line chemotherapies. In addition, the functionality of somatic cells was assessed using hormone serum levels that reflect their endocrine activity, to provide a better understanding of the functional integrity of the testicular environment. MAIN RESULTS AND THE ROLE OF CHANCE: This study investigated the impact of chemotherapy drug exposure and cancer type on testicular somatic cells in boys. Patients were categorized based on their exposure to specific chemotherapeutic drugs, including carboplatin, other alkylating agents, anthracyclines, vincristine, and cytarabine. High cumulative exposure to alkylating agents, defined as a cyclophosphamide equivalent dose (CED) ≥ 8000 mg/m², led to a decrease in COUP-TFII expression in peritubular myoid cells and CYP11A1 in Leydig cells (P = 0.038 and P = 0.013, respectively). Additionally, INSL3 expression in Leydig cells declined with increasing CED (r = −0.33, P = 0.019), independently of the patients’ age. Exposure to anthracyclines was also associated with reduced INSL3 (P = 0.027), although this association did not persist after CED adjustment. Combined exposure to alkylating agents and anthracyclines further decreased SOX9+ Sertoli cells (P = 0.021), whereas vincristine showed no additive deleterious effect. An alteration in Leydig cell maturation was observed in patients treated for neuroblastoma, highlighting the impact of the disease itself on testicular tissue. Hormonal analyses indicated no significant impact of induction chemotherapy on somatic cell functionality. LIMITATIONS, REASONS FOR CAUTION: Histological data were obtained from testicular tissue of patients exposed to chemotherapy treatments, mostly prepubertal fragments, with no healthy controls being available. Analyses included histology and hormonal profiling; molecular studies were not performed due to limited tissue cryopreserved for future fertility restoration. WIDER IMPLICATIONS OF THE FINDINGS: This study provides valuable information on the impact of chemotherapy on testicular somatic cells in patients. Because somatic cells are essential for supporting spermatogenesis, the preservation of the testicular microenvironment after chemotherapy is a reassuring prospect for patients considering future fertility restoration approaches. FUNDING: This work was funded by a PhD grant by La Ligue Nationale contre le Cancer [to V.R.] as well as financial contributions from University of Rouen Normandy, Rouen University Hospital, and the Institute for Research and Innovation in Biomedicine (IRIB). Additional fundings were provided by French National Research Agency (ANR-21-CE14-0068) [to N.R.], French Biomedicine Agency [to A.F.], GIRCI NO [to A.F.], Laurette Fugain’s Association [to L.D.], Rouen Normandy Metropolis [to L.D.], and Priority Research Programs and Equipment (PEPR): Women’s Health, Couples’ Health (SAFE). The funding sources had no role in the design of the study, collection, analysis and interpretation of data, and writing of the manuscript. DISCLOSURES: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Background/Objectives: Endozepines known as the endogenous ligands of benzodiazepine-binding sites, include the diazepam binding inhibitor (DBI) and its processing products, the triakontatetraneuropeptide (TTN) and the octadecaneuropeptide (ODN). Despite indisputable evidence of the binding of ODN on GABA_AR-BZ-binding sites, their action on this receptor lacks compelling electrophysiological observations, with some studies reporting that ODN acts as a negative allosteric modulator (NAM) of GABA_AR while others suggest the opposite (positive allosteric modulation, PAM effect). All these studies were carried out in vitro with various neuronal cell types. To further elucidate the role of ODN in neuronal excitability, we tested its effect in vivo in the cerebral cortex of the anesthetized mouse. Methods: Spontaneous neuronal spikes were recorded by means of an extracellular pipette, in the vicinity of which ODN was micro-infused, either at a high dose (10⁻⁵ M) or low dose (10⁻¹¹ M). Results: ODN at a high dose induced a significant increase in neuronal spiking. This effect could be antagonized by the GABA_AR-BZ-binding site blocker flumazenil. In sharp contrast, at low concentrations, ODN reduced neuronal spiking with a magnitude similar to GABA itself. Interestingly, this decrease in neuronal activity by low dose of ODN was not flumazenil-dependent, suggesting that this effect is mediated by another receptor. Finally, we show that astrocytes in culture, known to be stimulated by picomolar doses of ODN via a GPCR, increased their export of GABA when stimulated by low dose of ODN. Conclusion: Our results confirm the versatility of ODN in the control of GABA transmission, but suggest that its PAM-like effect is, at least in part, mediated via an astrocytic non-GABA_AR ODN receptor release of GABA.

Apnea of prematurity (AOP) affects 50% of preterm infants causing intermittent hypoxia (IH), which can lead to long-term neurodevelopmental deficits. Cerebellar abnormalities have been observed in AOP but the relationship between vascular alterations and neural development remains unclear. This study investigates how IH affects cerebellar angiogenesis using a murine model of AOP. We developed an innovative 3D imaging workflow combining IMARIS and VesselVio software to quantitatively analyze cerebellar vascularization at different postnatal stages (P4, P8, P12, P21, and P70). We correlate these results with a transcriptomic analysis of 23 angiogenesis-related genes in the same stages to uncover the associated molecular pathways. We found that IH induced significant vascular changes, particularly at P4, with a global increase in vascular-network dimensions. By P8, the vascular network normalized, but genes were downregulated in all pathways studied. After P12, at the end of the IH protocol, transcriptional regulations vary but persist long-term. Moreover, differential analysis showed distinct effects on superficial versus deep vascular networks, allowing for a more precise understanding of remodeling patterns throughout development. Overall, transcriptomic changes were associated with morphological alterations in a time-dependent manner, suggesting a multiphasic IH response through development with lasting effects. Key regulations included VEGF, angiopoietin, and matrix metalloprotease signaling. These findings demonstrate that IH disrupts cerebellar angiogenesis in parallel with neurogenesis, potentially contributing to the neurodevelopmental deficits observed in AOP. Thus, the interconnected nature of angio- and neurogenesis during cerebellar development makes it crucial to take vascular aspects into account in therapeutic approaches to neurodevelopmental disorders. Highlights: Novel 3D imaging workflow reveals cerebellar vascular changes in apnea of prematurity mouse model Intermittent hypoxia induces early hypervascularization at P4 followed by later adaptation Superficial and deep vascular networks show distinct responses to intermittent hypoxia Vascularization-related genes are affected during cerebellar development Alterations in angiogenesis go together with neurodevelopmental defects