RESUMEN

Estrogen plays fundamental roles in nervous system development and function. Traditional studies examining the effect of estrogen in the brain have focused on the nuclear estrogen receptors (ERs), ERα and ERß. Studies related to the extranuclear, membrane-bound G-protein-coupled ER (GPER/GPR30) have revealed a neuroprotective role for GPER in mature neurons. In this study, we investigated the differential effects of GPER activation in primary rat embryonic day 18 (E18) hippocampal and cortical neurons. Microscopy imaging, multielectrode array (MEA), and Ca2+ imaging experiments revealed that GPER activation with selective agonist, G-1, and nonselective agonist, 17ß-estradiol (E2), increased neural growth, neural firing activity, and intracellular Ca2+ more profoundly in hippocampal neurons than in cortical neurons. The GPER-mediated Ca2+ rise in hippocampal neurons involves internal Ca2+ store release via activation of phospholipase C (PLC) and extracellular entry via Ca2+ channels. Immunocytochemistry results revealed no observable difference in GPER expression/localization in neurons, yet real-time qPCR (RT-qPCR) and Western blotting showed a higher GPER expression in the cortex than hippocampus, implying that GPER expression level may not fully account for its robust physiological effects in hippocampal neurons. We used RNA sequencing data to identify distinctly enriched pathways and significantly expressed genes in response to G-1 or E2 in cultured rat E18 hippocampal and cortical neurons. In summary, the identification of differential effects of GPER activation on hippocampal and cortical neurons in the brain and the determination of key genes and molecular pathways are instrumental toward an understanding of estrogen's action in early neuronal development.

Asunto(s)

RESUMEN

Numerous studies have reported neuroprotective and procognitive effects of estrogens. The estrogen 17ß-estradiol (E2) activates both the classical nuclear estrogen receptors ERα and ERß as well as the G protein-coupled estrogen receptor (GPER). The differential effects of targeting the classical estrogen receptors over GPER are not well-understood. A limited number of selective GPER compounds have been described. In this study, 10 novel compounds were synthesized and exhibited half-maximal effective concentration values greater than the known GPER agonist G-1 in calcium mobilization assays performed in nonadherent HL-60 cells. Of these compounds, 2-cyclohexyl-4-isopropyl-N-((5-(tetrahydro-2H-pyran-2-yl)furan-2-yl)methyl)aniline, referred to as CITFA, significantly increased axonal and dendritic growth in neurons extracted from embryonic day 18 (E18) fetal rat hippocampal neurons. Confirmation of the results was performed by treating E18 hippocampal neurons with known GPER-selective antagonist G-36 and challenging with either E2, G-1, or CITFA. Results from these studies revealed an indistinguishable difference in neurite outgrowth between the treatment and control groups, exhibiting that neurite outgrowth in response to G-1 and CITFA originates from GPER activation and can be abolished with pretreatment of an antagonist. Subsequent docking studies using a homology model of GPER showed unique docking poses between G-1 and CIFTA. While docking poses differed between the ligands, CIFTA exhibited more favorable distance, bond angle, and strain for hydrogen-bonding and hydrophobic interactions.

Asunto(s)

RESUMEN

A recurrent de novo mutation in the transcriptional corepressor CTBP1 is associated with neurodevelopmental disabilities in children (Beck et al., 2016, 2019; Sommerville et al., 2017). All reported patients harbor a single recurrent de novo heterozygous missense mutation (p.R342W) within the cofactor recruitment domain of CtBP1. To investigate the transcriptional activity of the pathogenic CTBP1 mutant allele in physiologically relevant human cell models, we generated induced pluripotent stem cells (iPSC) from the dermal fibroblasts derived from patients and normal donors. The transcriptional profiles of the iPSC-derived "early" neurons were determined by RNA-sequencing. Comparison of the RNA-seq data of the neurons from patients and normal donors revealed down regulation of gene networks involved in neurodevelopment, synaptic adhesion and anti-viral (interferon) response. Consistent with the altered gene expression patterns, the patient-derived neurons exhibited morphological and electrophysiological abnormalities, and susceptibility to viral infection. Taken together, our studies using iPSC-derived neuron models provide novel insights into the pathological activities of the CTBP1 p.R342W allele.

RESUMEN

Brain functions rely critically upon the proper development of neuronal processes (axons and dendrites) and the formation of functional networks. Any genetic factors or environmental compounds that alter the morphological features of neurons may render the nervous system dysfunctional and result in neuronal disorders. In vitro cell culture is an important technique in assessing the effect of chemicals on neurite formation and growth of individual neurons in desired brain regions and has been fundamental in advancing our understanding of the nervous system development and functioning. Despite others offering excellent techniques in cell cultures (Catlin et al., 2016), there is a lack of available resources for teaching students how to analyze neurite outgrowth and run proper statistics on their data. Here, we first briefly discuss culturing cryopreserved mammalian neurons. We then give detailed options to aid upper level undergraduate neurobiology students to quantify neurite outgrowth using NeuronJ, a plugin in the free ImageJ package, Fiji, on both phase contrast and immunofluorescent images. This laboratory exercise provides students the opportunity to culture live neurons, quantify neuronal growth, experiment with the effects of common chemicals on neural development, and conduct statistical data analysis. Previous students expressed their great appreciation for the opportunity to work with live neurons and conduct data quantification and analysis like a true scientist. The ability to accurately measure and calculate the overall growth of neurons using the software ImageJ greatly enhanced students' confidence in presenting their results both in oral and written format.

RESUMEN

OBJECTIVE: The objective of this work was to test the synergistic effects of substrate stiffness, electro-conductivity, composition and electrical stimulation on the morphology, alignment and directional neurite outgrowth of neuron-like PC12 cells. The use of exogenous electrical stimulation has emerged as a promising new intervention to promote neural regeneration following injury. For critical gap size nerve injuries, a permissive biomaterial coupled to electrical stimulation may be needed to provide guidance and support for neurite outgrowth. Thus, the combinatorial effects of biomaterial composition and properties and exogenous electrical stimulation need interrogation to develop successful therapeutic interventions. Carefully designed in vitro models are ideally suited to perform such multidimensional detailed studies. APPROACH: We assembled a simple electrical stimulation device to deliver uniform electrical current with minimum voltage field variation through a hydrogel. We used polyacrylamide (PA), polyethylene glycol (PEG), and multi-walled carbon nanotubes (MWCNT)-PEG nanocomposite hydrogels of varying stiffness, resistivity and MWCNT concentration. Cells were seeded on the substrates for 24 h, stimulated for 1 h at 30 V m-1 DC, and then cultured for additional 24 h. Non-stimulated cells were used as controls. To induce neurite outgrowth, cells were primed with nerve growth factor (100 ng ml-1). MAIN RESULTS: For all substrates tested, electrical stimulation induced neurite alignment at 60-90° angle to the applied current. It also increased total neurite outgrowth by 18%-49% and mean neurite length by 20%-46% (increase dependent on the underlying substrate) compared to non-stimulated cells. The nanocomposite composed of 20% w/v PEG and 0.1% w/v MWCNTs resulted in the highest total neurite outgrowth and mean neurite length, which were further significantly enhanced by electrical stimulation by 2-fold and 1.8-fold, respectively. SIGNIFICANCE: Our results indicate that nanocomposites, where carbon nanotubes have been added to hydrogel substrates, in combination with electrical stimulation provided improved conditions for neural growth and regeneration.

Asunto(s)