Melatonin Rescues the Dendrite Collapse Induced by the Pro-Oxidant Toxin Okadaic Acid in Organotypic Cultures of Rat Hilar Hippocampus.

The pro-oxidant compound okadaic acid (OKA) mimics alterations found in Alzheimer's disease (AD) as oxidative stress and tau hyperphosphorylation, leading to neurodegeneration and cognitive decline. Although loss of dendrite complexity occurs in AD, the study of this post-synaptic domain in chemical-induced models remains unexplored. Moreover, there is a growing expectation for therapeutic adjuvants to counteract these brain dysfunctions. Melatonin, a free-radical scavenger, inhibits tau hyperphosphorylation, modulates phosphatases, and strengthens dendritic arbors. Thus, we determined if OKA alters the dendritic arbors of hilar hippocampal neurons and whether melatonin prevents, counteracts, or reverses these damages. Rat organotypic cultures were incubated with vehicle, OKA, melatonin, and combined treatments with melatonin either before, simultaneously, or after OKA. DNA breaks were assessed by TUNEL assay and nuclei were counterstained with DAPI. Additionally, MAP2 was immunostained to assess the dendritic arbor properties by the Sholl method. In hippocampal hilus, OKA increased DNA fragmentation and reduced the number of MAP2(+) cells, whereas melatonin protected against oxidation and apoptosis. Additionally, OKA decreased the dendritic arbor complexity and melatonin not only counteracted, but also prevented and reversed the dendritic arbor retraction, highlighting its role in post-synaptic domain integrity preservation against neurodegenerative events in hippocampal neurons.

Tridimensional Retinoblastoma Cultures as Vitreous Seeds Models for Live-Cell Imaging of Chemotherapy Penetration.

A preclinical model could aid in understanding retinoblastoma vitreous seeds behavior, drug penetration, and response to chemotherapy to optimize patient treatment. Our aim was to develop a tridimensional in vitro model of retinoblastoma vitreous seeds to assess chemotherapy penetration by means of live-cell imaging. Cell cultures from patients with retinoblastoma who underwent upfront enucleation were established and thoroughly characterized for authentication of human tumor origin. The correlation of the in vitro tridimensional structures resembling human spheres and dusts vitreous seeds was established. Confocal microscopy was used to quantify real-time fluorescence of topotecan as a measure of its penetration into different sizes of spheres. Cell viability was determined after chemotherapy penetration. The in vitro spheres and dusts models were able to recapitulate the morphology, phenotype, and genotype of patient vitreous seeds. The larger the size of the spheres, the longer the time required for the drug to fully penetrate into the core (p < 0.05). Importantly, topotecan penetration correlated with its cytotoxic activity. Therefore, the studied tridimensional cell model recapitulated several characteristics of vitreous seeds observed in patients with retinoblastoma and were successfully used to assess live-cell imaging of chemotherapy penetration for drug distribution studies.

Short term changes in the proteome of human cerebral organoids induced by 5-MeO-DMT.

Dimethyltryptamines are entheogenic serotonin-like molecules present in traditional Amerindian medicine recently associated with cognitive gains, antidepressant effects, and changes in brain areas related to attention. Legal restrictions and the lack of adequate experimental models have limited the understanding of how such substances impact human brain metabolism. Here we used shotgun mass spectrometry to explore proteomic differences induced by 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT) on human cerebral organoids. Out of the 6,728 identified proteins, 934 were found differentially expressed in 5-MeO-DMT-treated cerebral organoids. In silico analysis reinforced previously reported anti-inflammatory actions of 5-MeO-DMT and revealed modulatory effects on proteins associated with long-term potentiation, the formation of dendritic spines, including those involved in cellular protrusion formation, microtubule dynamics, and cytoskeletal reorganization. Our data offer the first insight about molecular alterations caused by 5-MeO-DMT in human cerebral organoids.

The effect of laminin and its peptide SIKVAV on a human salivary gland adenoid cystic carcinoma cell line.

We have previously demonstrated that laminin modulates the expression of adhesion molecules in an adenoid cystic carcinoma cell line (CAC2 cells). We are currently studying whether laminin can induce modifications in the overall morphology of CAC2 cells. These cells were grown in a three-dimensional preparation of laminin-1. Phenotype differences were assessed by light and transmission electron microscopy. CAC2 cells grown inside laminin-1 formed ductlike and pseudocystic structures. Based on our findings we suggest that laminin is a key regulator of tubular and pseudocystic patterns of adenoid cystic carcinoma. We also analyzed the effect of a molecular domain of laminin-1, the peptide SIKVAV (Ser-Ile-Lys-Val-Ala-Val) on CAC2 cells. This peptide was chosen because it is effective in cell proliferation and differentiation, and because it has never been tested before in salivary gland neoplasms. When CAC2 cells were grown inside SIKVAV-enriched laminin-1, only pseudocystic structures were observed. Since no ductlike structures were observed in samples treated with SIKVAV, we may assume that this peptide is at least one of the molecular domains of laminin responsible for the pseudocystic pattern observed in adenoid cystic carcinoma. Function disturbing experiments strongly suggested that the integrin alpha3beta1 play a role in the effect of laminin on CAC2 cells.