SACM - United States of America
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Item Restricted Modulation of Autistic-Related Factors in Hippocampal Neurons: Role of Oxytocin(2023-04-24) Alfaifi, Hassan; Castejon, AnaBackground: Autism spectrum disorder (ASD) is a neurodevelopmental disorder associated with dysregulation of several cellular processes. Accumulating evidence links ASD to the abnormality of cellular growth and programmed cell death (apoptosis). According to many postmortem and animal studies, abnormalities of several apoptotic signaling pathways have been linked to the induction of ASD, such as the ERK and p53 signaling pathways. Besides, the participation of neuroinflammation and oxidative stress in ASD induction and perpetuation has been identified. It has been reported that the levels of ROS and interleukin-1β are abnormally increased in neuronal brain cells in individuals with ASD. Therefore, agents that can improve cellular growth, regulate apoptosis, and reduce oxidative stress and neuroinflammation, like the neuropeptide oxytocin (OXT), may be effective in managing ASD. Objective: Our main goal was to investigate the effects of OXT on autistic-related factors, including cellular growth, oxidative stress, and neuroinflammation, as well as the intracellular signaling pathways involved in these effects. Methodology: We evaluated the effect of OXT on cell growth and death by performing cell counting (hemocytometer), MTT assay, and Bresto blue assay in hippocampal neurons (mHippoE-2). The proliferative effect mechanisms were evaluated using western blotting and MTT assay. In the survival experiments, viability was assessed by MTT assay in cells incubated in the presence or absence of OXT 1000 nM and/or 1000nM OXTA with oxidative stress inducers (H2O2, DMNQ, and CPT) and neuroinflammatory inducer (LPS). The mechanisms of the protective effect were evaluated using western blotting, ELISA. Also, we used the DCFDA kit to evaluate the antioxidant effect of OXT. Moreover, we employed the immunocytochemistry technique to assess the effect of 1000 nM OXT and/or 1000 nM OXTA against the induced morphological alterations. v Results: This study revealed that OXT significantly induced cell growth in hippocampal neurons (mHippoE-2). The OXTA (L-371,257) significantly reduced cell growth. The proliferative effect of OXT is mediated through MEK/ERK signaling pathway. In addition, treatment with 1000 nM OXT significantly reduced the reduction in cell viability induced by oxidative stress inducers (H2O2, CPT, and DMNQ) but not inflammatory inducer (LPS). In addition, OXT significantly reduced ROS generation when the cells were exposed to H2O2 and DMNQ but not CPT. The western blotting technique demonstrated that OXT significantly reduced the protein levels of p53-caspase 3 and increased the levels of Mdm2 induced by H2O2 and DMNQ, but not CPT. Our morphological studies showed that OXT countered the reduction in cellular projection length induced by H2O2, CPT, and DMNQ. Furthermore, OXT significantly reduced the protein levels of PI3K and p-AKT but not the NLRP3-caspase 1 signaling pathway. Conclusion: Our results indicate that OXT has a proliferative effect by activating the ERK signaling pathway. Furthermore, we revealed that the protective effect of OXT was mediated through the modulation of oxidative stress and mitochondrial apoptosis pathway. Moreover, OXT decreased the levels of some inflammatory-mediated proteins. On the other hand, these effects were lacking in the presence of OXTA. These results will contribute to a better understanding of OXT’s potential role in autistic-related factors associated with cell loss, oxidative stress, and neuroinflammation.5 0Item Restricted TARGETING SPERMINE OXIDASE TO REDUCE NEURODEGENERATION AND VISION LOSS IN DIABETIC RETINOPATHY(2023) Alfarhan, Moaddey; Narayanan, S. PriyaDiabetic retinopathy, a major cause of vision loss, is characterized by neurodegenerative and vascular changes in the retina. Current treatments target the late phase of the disease and have undesirable side effects. Previous studies from our laboratory have shown that spermine oxidase (SMOX) is critically involved in retinal neurovascular changes. However, the molecular mechanisms of SMOX-induced neuronal damage and the impact of the long-term effects of SMOX inhibition on the diabetic retina have not been investigated yet. This present study investigated the molecular mechanisms regulated by SMOX in causing retinal neuronal damage and determined the impact of long-term inhibition of SMOX on neuro-glial injury and vision loss. Utilizing experimental models of retinal excitotoxicity, our results demonstrated upregulation in the number of microglia/macrophages in excitotoxic retinas, while MDL 72527 (a pharmacological inhibitor of SMOX) treatment reduced these changes. When retinal excitotoxicity upregulated several pro-inflammatory genes, MDL72527 treatment reduced many of them and increased anti-inflammatory genes. Furthermore, SMOX inhibition upregulated antioxidant signaling and reduced the level of conjugated acrolein in excitotoxic retinas. In vitro studies using a microglia cell line showed activated morphology and increased reactive oxygen species formation in response to the treatment with conjugated acrolein (a product of SMOX function). Streptozotocin-induced mouse model of diabetes was used to assess the effectiveness of long-term SMOX inhibition in visual acuity (VA), contrast sensitivity (CS), retinal function, and neuro-glial injury. Significant reductions in measures of VA and CS were observed in diabetic mice following 8, 16, and 24 weeks of diabetes. However, treatment with MDL 72527 improved both VA and CS at all the time points studied, with a significant increase at 24 weeks post-diabetes. Diabetic mice indicated marked reductions in the scotopic electroretinography (ERG) amplitudes, while MDL 72527 treatment significantly improved these reductions. Further, using neuronal and glial markers, MDL 72527 treatment reduced neuronal and glial injury in the diabetic retina, indicated by changes in neuronal and glial markers, respectively. Overall, our findings suggest that SMOX plays a critical role in retinal neurodegeneration and targeting SMOX signaling using MDL 72527 may provide a new strategy for reducing vision loss in diabetes.10 0