Single-Step 3D Bioprinting of Alginate-Collagen Type I Hydrogel Fiber Rings to Promote Angiogenic Network Formation.

In the advent of tissue engineering and regenerative medicine, the demand for innovative approaches to biofabricate complex vascular structures is increasing. We describe a single-step 3D bioprinting method leveraging Aspect Biosystems RX1 technology, which integrates the crosslinking step at a flow-focusing junction, to biofabricate immortalized adult rat brain endothelial cell (SV-ARBEC)-encapsulated alginate-collagen type I hydrogel rings. This single-step biofabrication process involves the strategic layer-by-layer assembly of hydrogel rings, encapsulating SV-ARBECs in a spatially controlled manner while optimizing access to media and nutrients.

Differential Expression of Transporter Genes in Brain Vessels vs. Peripheral Tissues and Vessels from Human, Mouse and Rat.

Background: ATP-binding cassette (ABC) transporters comprise a superfamily of genes encoding membrane proteins with nucleotide-binding domains (NBD). These transporters, including drug efflux across the blood-brain barrier (BBB), carry a variety of substrates through plasma membranes against substrate gradients, fueled by hydrolyzing ATP. The expression patterns/enrichment of transporter genes in brain microvessels compared to peripheral vessels and tissues are largely uncharacterized.

Mouse embryonic stem cell-derived blood-brain barrier model: applicability to studying antibody triggered receptor mediated transcytosis.

Blood brain barrier (BBB) models in vitro are an important tool to aid in the pre-clinical evaluation and selection of BBB-crossing therapeutics. Stem cell derived BBB models have recently demonstrated a substantial advantage over primary and immortalized brain endothelial cells (BECs) for BBB modeling. Coupled with recent discoveries highlighting significant species differences in the expression and function of key BBB transporters, the field is in need of robust, species-specific BBB models for improved translational predictability.

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.

Immunoassay for Quantitative Detection of Antibody Transcytosis Across the Blood-Brain Barrier In Vitro.

Automated high-throughput immunoassays are emerging as reliable analytic techniques for the quantitative detection of proteins from a variety of sample types. Herein, we describe a method using the Protein Simple Wes capillary-based automated immunoassays platform for the quantification of His- and HA-tagged antibody transcytosis across an in vitro transwell blood-brain barrier (BBB) model. Compared to conventional ELISA, fluorescence, and Mass Spec-based detection approaches, Wes provides comparable datasets with additional information regarding size, aggregation, and potential degradation of samples before and after BBB transcytosis.

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.

Differential expression of receptors mediating receptor-mediated transcytosis (RMT) in brain microvessels, brain parenchyma and peripheral tissues of the mouse and the human.

Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. Constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB.

Intracellular sorting and transcytosis of the rat transferrin receptor antibody OX26 across the blood-brain barrier in vitro is dependent on its binding affinity.

Unlabelled: The blood-brain barrier (BBB) is a formidable obstacle to the delivery of therapeutics to the brain. Antibodies that bind transferrin receptor (TfR), which is enriched in brain endothelial cells, have been shown to cross the BBB and are being developed as fusion proteins to deliver therapeutic cargos to brain targets. Various antibodies have been developed for this purpose and their in vivo evaluation demonstrated that either low affinity or monovalent receptor binding re-directs their transcellular trafficking away from lysosomal degradation and toward improved exocytosis on the abluminal side of the BBB.

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.

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).

Nanomicellar formulation of coenzyme Q10 (Ubisol-Q10) effectively blocks ongoing neurodegeneration in the mouse 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model: potential use as an adjuvant treatment in Parkinson's disease.

Although the support for the use of antioxidants, such as coenzyme Q(10) (CoQ(10)), to treat Parkinson's disease (PD) comes from the extensive scientific evidence, the results of conducted thus far clinical trials are inconclusive. It is assumed that the efficacy of CoQ(10) is hindered by insolubility, poor bioavailability, and lack of brain penetration. We have developed a nanomicellar formulation of CoQ(10) (Ubisol-Q(10)) with improved properties, including the brain penetration, and tested its effectiveness in mouse MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) model with the objectives to assess its potential use as an adjuvant therapy for PD.

Method for isolation and molecular characterization of extracellular microvesicles released from brain endothelial cells.

Background: In addition to possessing intracellular vesicles, eukaryotic cells also produce extracellular microvesicles, ranging from 50 to 1000 nm in diameter that are released or shed into the microenvironment under physiological and pathological conditions. These membranous extracellular organelles include both exosomes (originating from internal vesicles of endosomes) and ectosomes (originating from direct budding/shedding of plasma membranes). Extracellular microvesicles contain cell-specific collections of proteins, glycoproteins, lipids, nucleic acids and other molecules.

Involvement of NOS3 in RA-Induced neural differentiation of human NT2/D1 cells.

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process.

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.

Effects of nitric oxide donors on cybrids harbouring the mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) A3243G mitochondrial DNA mutation.

Reactive nitrogen and oxygen species (O2*-, H2O2, NO* and ONOO-) have been strongly implicated in the pathophysiology of neurodegenerative and mitochondrial diseases. In the present study, we examined the effects of nitrosative and/or nitrative stress generated by DETA-NO {(Z)-1-[2-aminoethyl-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate}, SIN-1 (3-morpholinosydnonimine hydrochloride) and SNP (sodium nitroprusside) on U87MG glioblastoma cybrids carrying wt (wild-type) and mutant [A3243G (Ala3243-->Gly)] mtDNA (mitochondrial genome) from a patient suffering from MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes). The mutant cybrids had reduced activity of cytochrome c oxidase, significantly lower ATP level and decreased mitochondrial membrane potential.

S/MAR-binding properties of Sox2 and its involvement in apoptosis of human NT2 neural precursors.

DNA fragmentation in apoptosis, especially in lymphocytic cells, is initiated at scaffold/matrix attachment regions (S/MARs) and is preceded by the degradation of nuclear proteins. The present study was performed to establish whether the same mechanism occurred in human NT2 cells subjected to oxygen and glucose deprivation (OGD). We analyzed the integrity of c-myc S/MAR containing a base-unpairing region (BUR)-like element, which we established to be a binding site of the transcription factor Sox2.

Identification of a functional CRE in the promoter of Fukuyama congenital muscular dystrophy gene fukutin.

We isolated a fragment of the fukutin gene promoter from differentiated human NT2 cells using chromatin immunoprecipitation technique with an anti-CREB antibody. This fragment contained a CRE-like sequence and here we describe its functional validation. The results showed that the element was functional in vitro and in vivo and that CREB in neurons was involved in the transcriptional regulation of the fukutin gene.

Unique technology for solubilization and delivery of highly lipophilic bioactive molecules.

We have produced a family of novel carriers enabling water solubilization of highly lipophilic molecules. The compound carriers were synthesized by conjugating polyethylene glycol to alpha-tocopherol, tocotrienols, beta-sitosterol or cholesterol via an alkanedioyl linker. These PEG- conjugates were amphiphilic and formed stable non-covalent complexes (nanomicelles) with a wide range of molecules including vitamins, carotenoids, ubiquinones, poly-unsaturated fatty acids and polyene macrolide antibiotics.

Regulation of DNaseY activity by actinin-alpha4 during apoptosis.

DNaseY, a Ca(2+)- and Mg(2+)-dependent endonuclease, has been implicated in apoptotic DNA degradation; however, the molecular mechanisms controlling its involvement in this process have not been fully elucidated. We have obtained evidence from yeast two-hybrid screening and coimmunoprecipitation experiments that DNaseY interacted physically with actinin-alpha4 and this interaction significantly enhanced its endonuclease activity. Accordingly, simultaneous overexpression of both proteins in PC12 cells dramatically increased the rate of apoptosis in response to teniposide' VM26.

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.