Effects of Tolerance-Induced Preconditioning on Mitochondrial Biogenesis in Undifferentiated and Differentiated Neuronal Cells.

Background: Mitochondrial biogenesis occurs in response to chronic stresses as an adaptation to the increased energy demands and often renders cells more refractive to subsequent injuries which is referred to as preconditioning. This phenomenon is observed in several non-neuronal cell types, but it is not yet fully established in neurons, although it is fundamentally important for neuroprotection and could be exploited for therapeutic purposes.

Methods: This study was designed to examine whether the preconditioning treatment with hypoxia or nitric oxide could trigger biogenesis in undifferentiated and differentiated neuronal cells (rat PC12 and human NT2 cells) as well as in primary mouse cortical neurons.

Electrophysiological- and Neuropharmacological-Based Benchmarking of Human Induced Pluripotent Stem Cell-Derived and Primary Rodent Neurons.

Human induced pluripotent stem cell (iPSC)-derived neurons are of interest for studying neurological disease mechanisms, developing potential therapies and deepening our understanding of the human nervous system. However, compared to an extensive history of practice with primary rodent neuron cultures, human iPSC-neurons still require more robust characterization of expression of neuronal receptors and ion channels and functional and predictive pharmacological responses. In this study, we differentiated human amniotic fluid-derived iPSCs into a mixed population of neurons (AF-iNs).

Development of a Blood-Brain Barrier Permeability Assay Using Human Induced Pluripotent Stem Cell Derived Brain Endothelial Cells.

The development of translational and predictive models in vitro for assessing blood-brain barrier (BBB) delivery has become an important requirement in preclinical testing of CNS-targeting therapeutics. Here we describe a directed monolayer differentiation strategy to generate a population of brain endothelial-like cells (BECs) from human induced pluripotent stem cell (iPSC) with robust BBB properties. To generate BBB permeability assays, the BECs are seeded as a monolayer on a semipermeable Transwell insert placed inside a companion plate to generate a two-compartment Transwell model.

Zika virus crosses an in vitro human blood brain barrier model.

Zika virus (ZIKV) is a flavivirus that is highly neurotropic causing congenital abnormalities and neurological damage to the central nervous systems (CNS). In this study, we used a human induced pluripotent stem cell (iPSC)-derived blood brain barrier (BBB) model to demonstrate that ZIKV can infect brain endothelial cells (i-BECs) without compromising the BBB barrier integrity or permeability. Although no disruption to the BBB was observed post-infection, ZIKV particles were released on the abluminal side of the BBB model and infected underlying iPSC-derived neural progenitor cells (i-NPs).

A novel human induced pluripotent stem cell blood-brain barrier model: Applicability to study antibody-triggered receptor-mediated transcytosis.

We have developed a renewable, scalable and transgene free human blood-brain barrier model, composed of brain endothelial cells (BECs), generated from human amniotic fluid derived induced pluripotent stem cells (AF-iPSC), which can also give rise to syngeneic neural cells of the neurovascular unit. These AF-iPSC-derived BECs (i-BEC) exhibited high transendothelial electrical resistance (up to 1500 Ω cm) inducible by astrocyte-derived molecular cues and retinoic acid treatment, polarized expression of functional efflux transporters and receptor mediated transcytosis triggered by antibodies against specific receptors. In vitro human BBB models enable pre-clinical screening of central nervous system (CNS)-targeting drugs and are of particular importance for assessing species-specific/selective transport mechanisms.

Traumatic brain injury: classification, models, and markers.

Traumatic brain injury (TBI) is a leading cause of morbidity and mortality worldwide. Due to its high incidence rate and often long-term sequelae, TBI contributes significantly to increasing costs of health care expenditures annually. Unfortunately, advances in the field have been stifled by patient and injury heterogeneity that pose a major challenge in TBI prevention, diagnosis, and treatment.

Comparison of S-nitrosoglutathione- and staurosporine-induced apoptosis in human neural cells.

S-nitrosoglutathione (GSNO) is an endogenously produced S-nitrosylating compound that controls the function of various proteins. While a number of rodent cell lines have been used to study GSNO-induced apoptosis, the mechanisms of action remain to be evaluated in human cells and in parallel with other common apoptosis-inducing agents. In this study, we compared the pro-apoptotic effects of GSNO and staurosporine (STS) on human neural progenitors (NT2, hNP1) and neuroblasts (SH-SY5Y).

Neuroprotective effects of GDNF-expressing human amniotic fluid cells.

Brain injury continues to be one of the leading causes of disability worldwide. Despite decades of research, there is currently no pharmacologically effective treatment for preventing neuronal loss and repairing the brain. As a result, novel therapeutic approaches, such as cell-based therapies, are being actively pursued to repair tissue damage and restore neurological function after injury.

Therapeutic potential of amniotic fluid-derived cells for treating the injured nervous system.

There is a need for improved therapy for acquired brain injury, which has proven resistant to treatment by numerous drugs in clinical trials and continues to represent one of the leading causes of disability worldwide. Research into cell-based therapies for the treatment of brain injury is growing rapidly, but the ideal cell source has yet to be determined. Subpopulations of cells found in amniotic fluid, which is readily obtained during routine amniocentesis, can be easily expanded in culture, have multipotent differentiation capacity, are non-tumourigenic, and avoid the ethical complications associated with embryonic stem cells, making them a promising cell source for therapeutic purposes.

Development of BMP7-producing human cells, using a third generation lentiviral gene delivery system.

Bone morphogenetic protein 7 (BMP7), a member of the transforming growth factor β (TGF-β) superfamily, plays important roles in the development of various tissues and organs in mouse and human. In particular, BMP7 is critical for the formation of the nervous system and it is considered to have therapeutic potential in brain injury and stroke. One approach to make BMP7 more suitable for therapeutic purposes is the development of efficient vectors that allow the consistent, reliable and cost-effective production of the BMP7 protein.

Differentiation of mouse Neuro 2A cells into dopamine neurons.

Neuro 2A (N2a) is a mouse neural crest-derived cell line that has been extensively used to study neuronal differentiation, axonal growth and signaling pathways. A convenient characteristic of these cells is their ability to differentiate into neurons within a few days. However, most differentiation methods reported for N2a cells do not provide information about the neuronal types obtained after each treatment.

Regulation of MYPT1 stability by the E3 ubiquitin ligase SIAH2.

Myosin phosphatase target subunit 1 (MYPT1), together with catalytic subunit of type1 delta isoform (PP1cdelta) and a small 20-kDa regulatory unit (M20), form a heterotrimeric holoenzyme, myosin phosphatase (MP), which is responsible for regulating the extent of myosin light chain phosphorylation. Here we report the identification and characterization of a molecular interaction between Seven in absentia homolog 2 (SIAH2) and MYPT1 that resulted in the proteasomal degradation of the latter in mammalian cells, including neurons and glia. The interaction involved the substrate binding domain of SIAH2 (aa 116-324) and a central region of MYPT1 (aa 445-632) containing a degenerate consensus Siah-binding motif RLAYVAP (aa 493-499) evolutionally conserved from fish to humans.

Identification of ataxia-associated mtDNA mutations (m.4052T>C and m.9035T>C) and evaluation of their pathogenicity in transmitochondrial cybrids.

The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035T>C and 4452T>C, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452T>C transition in tRNA(Met) represented a polymorphism; however, 9035T>C conversion in the ATP6 gene was responsible for a defective F(0)-ATPase. Accordingly, mutant cybrids had a reduced oligomycin-sensitive ATP hydrolyzing activity.

Astrocyte-secreted GDNF and glutathione antioxidant system protect neurons against 6OHDA cytotoxicity.

In recent years, GDNF has emerged as a protective and restorative agent in several models of neurodegeneration; however, the exact molecular mechanisms responsible for these effects are not yet fully understood. Here we examined the effects of astrocytes secreting GDNF on neurons subjected to 6OHDA toxicity using in vitro neuron-astroglia co-cultures. Astrocytes were transduced with lentiviral vectors carrying the GDNF gene under the control of either human glial fibrillary acidic protein or cytomegalovirus promoters.

Epigenetic modifications of SOX2 enhancers, SRR1 and SRR2, correlate with in vitro neural differentiation.

SOX2 is a key neurodevelopmental gene involved in maintaining the pluripotency of stem cells and proliferation of neural progenitors and astroglia. Two evolutionally conserved enhancers, SRR1 and SRR2, are involved in controlling SOX2 expression during neurodevelopment; however, the molecular mechanisms regulating their activity are not known. We have examined DNA methylation and histone H3 acetylation at both enhancers in NT2-D1 progenitors, neurons and astrocytes, to establish the role of epigenetic mechanisms in cell-type-specific SOX2 expression.

Novel subtractive transcription-based amplification of mRNA (STAR) method and its application in search of rare and differentially expressed genes in AD brains.

Background: Alzheimer's disease (AD) is a complex disorder that involves multiple biological processes. Many genes implicated in these processes may be present in low abundance in the human brain. DNA microarray analysis identifies changed genes that are expressed at high or moderate levels.

Role of Sox2 in the development of the mouse neocortex.

The mammalian neocortex is established from neural stem and progenitor cells that utilize specific transcriptional and environmental factors to create functional neurons and astrocytes. Here, we examined the mechanism of Sox2 action during neocortical neurogenesis and gliogenesis. We established a robust Sox2 expression in neural stem and progenitor cells within the ventricular zone, which persisted until the cells exited the cell cycle.

Molecular mechanisms of glutamate neurotoxicity in mixed cultures of NT2-derived neurons and astrocytes: protective effects of coenzyme Q10.

Although glutamate excitotoxicity has long been implicated in neuronal cell death associated with a variety of neurological disorders, the molecular mechanisms underlying this process are not yet fully understood. In part, this is due to the lack of relevant experimental cell systems recapitulating the in vivo neuronal environment, mainly neuronal-glial interactions. To explore these mechanisms, we have analyzed the cytotoxic effects of glutamate on mixed cultures of NT2/N neurons and NT2/A astrocytes derived from human NT2/D1 cells.

Transcriptional activation of the human brain-derived neurotrophic factor gene promoter III by dopamine signaling in NT2/N neurons.

We have identified a functional cAMP-response element (CRE) in the human brain-derived neurotrophic factor (BDNF) gene promoter III and established that it participated in the modulation of BDNF expression in NT2/N neurons via downstream signaling from the D1 class of dopamine (DA) receptors. The up-regulation of BDNF expression, in turn, produced neuroprotective signals through receptor tyrosine kinase B (TrkB) and promoted cell survival under the conditions of oxygen and glucose deprivation. To our knowledge this is the first evidence showing the presence of a functional CRE in the human BDNF gene and the role of DA signaling in establishing transcriptional competence of CRE in post-mitotic NT2/N neurons.