Preclinical target validation for non-addictive therapeutics development for pain.

Introduction: The Helping to End Addiction Long-term Initiative supports a wide range of programs to develop new or improved prevention and opioid addiction treatment strategies. An essential component of this effort is to accelerate development of non-opioid pain therapeutics. In all fields of medicine, therapeutics development is an arduous process and late-stage translational efforts such as clinical trials to validate targets are particularly complex and costly.

Development of a high-throughput AlphaScreen assay for modulators of synapsin I phosphorylation in primary neurons.

Alterations in synaptic transmission have been implicated in a number of psychiatric and neurological disorders. The discovery of small-molecule modulators of proteins that regulate neurotransmission represents a novel therapeutic strategy for these diseases. However, high-throughput screening (HTS) approaches in primary neurons have been limited by challenges in preparing and applying primary neuronal cultures under conditions required for generating sufficiently robust and sensitive HTS assays.

Working memory impairment in calcineurin knock-out mice is associated with alterations in synaptic vesicle cycling and disruption of high-frequency synaptic and network activity in prefrontal cortex.

Working memory is an essential component of higher cognitive function, and its impairment is a core symptom of multiple CNS disorders, including schizophrenia. Neuronal mechanisms supporting working memory under normal conditions have been described and include persistent, high-frequency activity of prefrontal cortical neurons. However, little is known about the molecular and cellular basis of working memory dysfunction in the context of neuropsychiatric disorders.

Insulin, IGF-1, and muscarinic agonists modulate schizophrenia-associated genes in human neuroblastoma cells.

Background: Genes associated with energy metabolism are decreased in schizophrenia brain and human and rodent diabetic skeletal muscle. These and other similarities between diabetes and schizophrenia suggest that an insulin signaling deficit may underlie schizophrenia. We determined with human SH-SY5Y neuroblastoma and astrocyte cell lines whether insulin or other molecules could modulate genes opposite to their change reported in schizophrenia brain.

The mood stabilizer valproic acid stimulates GABA neurogenesis from rat forebrain stem cells.

Valproate, an anticonvulsant drug used to treat bipolar disorder, was studied for its ability to promote neurogenesis from embryonic rat cortical or striatal primordial stem cells. Six days of valproate exposure increased by up to fivefold the number and percentage of tubulin beta III-immunopositive neurons, increased neurite outgrowth, and decreased by fivefold the number of astrocytes without changing the number of cells. Valproate also promoted neuronal differentiation in human fetal forebrain stem cell cultures.

Comparison of microarray-based mRNA profiling technologies for identification of psychiatric disease and drug signatures.

The gene expression profiles of human postmortem parietal and prefrontal cortex samples of normal controls and patients with bipolar disease, or human neuroblastoma flat (NBFL) cells treated with the mood-stabilizing drug, valproate, were used to compare the performance of Affymetrix oligonucleotide U133A GeneChips and Agilent Human 1 cDNA microarrays. Among those genes represented on both platforms, the oligo array identified 26-53% more differentially expressed genes compared to the cDNA array in the three experiments, when identical fold change and t-test criteria were applied. The increased sensitivity was primarily the result of more robust fold changes measured by the oligonucleotide system.

Electroconvulsive seizures regulate gene expression of distinct neurotrophic signaling pathways.

Electroconvulsive therapy (ECT) remains the treatment of choice for drug-resistant patients with depressive disorders, yet the mechanism for its efficacy remains unknown. Gene transcription changes were measured in the frontal cortex and hippocampus of rats subjected to sham seizures or to 1 or 10 electroconvulsive seizures (ECS), a model of ECT. Among the 3500-4400 RNA sequences detected in each sample, ECS increased by 1.

Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets.

Although the source of embryonic stem (ES) cells presents ethical concerns, their use may lead to many clinical benefits if differentiated cell types can be derived from them and used to assemble functional organs. In pancreas, insulin is produced and secreted by specialized structures, islets of Langerhans. Diabetes, which affects 16 million people in the United States, results from abnormal function of pancreatic islets.

Defasciculation of neurites is mediated by tenascin-R and its neuronal receptor F3/11.

Fasciculation and defasciculation of axons are major morphogenetic events in the formation of neuronal pathways during development. We have identified the extracellular matrix glycoprotein tenascin-R (TN-R) and its neuronal receptor, the immunoglobulin superfamily recognition molecule F3, as promoters of neurite defasciculation in cerebellar explant cultures. Perturbation of the interaction between these two molecules using both antibodies and an antisense oligonucleotide resulted in increased neurite fasciculation.

Transplantation of oligodendrocyte progenitor cells into the rat retina: extensive myelination of retinal ganglion cell axons.

In most mammals, retinal ganglion cell axons are unmyelinated in the retina. The same axons become myelinated in the optic nerve. Various studies suggest that retinal ganglion cell axons are also in principle, myelination competent intraretinally and that non-neuronal factors at the retinal end of the optic nerve prevent the migration of oligodendrocyte progenitor cells into the retina.

Long-term induction of an aldose reductase protein by basic fibroblast growth factor in rat astrocytes in vitro.

Basic fibroblast growth factor (bFGF) is known to elicit various developmental-like effects on astrocytes in vitro, but these effects were studied mainly over short-term periods. In this work we asked the question whether bFGF could induce long-term effects on rat astrocytes in culture. This factor was found to induce only a transient mitogenic effect lasting less than 48 h, even when the treatment was carried on for 4 days.

Retinoic acid regulates the development of oligodendrocyte precursor cells in vitro.

Cultures of oligodendrocyte precursor cells can be grown from brain hemispheres of newborn rats. These cells, also called O-2A progenitor cells, can differentiate in vitro into oligodendrocytes or type 2 astrocytes. Basic FGF and PDGF are known to stimulate their proliferation and delay their differentiation.

Expression of neuromodulin (GAP-43) and its regulation by basic fibroblast growth factor during the differentiation of O-2A progenitor cells.

In a recent work we have shown that neuromodulin (Nm, also known as GAP-43), a protein kinase C substrate, previously believed to be expressed exclusively in neurons, is also present in glial cells. Here we investigated the expression of Nm and its mRNA in O-2A glial progenitor cells (common precursor for oligodendrocytes and type-2 astrocytes) during their development in secondary culture and under the influence of basic fibroblast growth factor (bFGF). The different stages of oligodendrocyte development were characterized by the expression of surface markers: A2B5, which identifies O-2A glial precursor cells, and O4 and galactocerebroside (GC), which characterize later developmental stages.

The proliferation of mature oligodendrocytes in vitro is stimulated by basic fibroblast growth factor and inhibited by oligodendrocyte-type 2 astrocyte precursors.

Basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) are mitogens for bipotential oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells. We investigated the mitogenic effect of these growth factors on quiescent mature oligodendrocytes (OL) expressing myelin basic protein (MBP) in OL cultures that were treated for 3 days with cytosine arabinoside (ARA-C) in order to kill O-2A precursors which divide in chemically defined medium. After treatment with ARA-C proliferation decreased and O-2A precursors identified with A2B5 monoclonal antibody were nearly undetectable.

Phosphorylation of the MARCKS protein (P87), a major protein kinase C substrate, is not an obligatory step in the mitogenic signaling pathway of basic fibroblast growth factor in rat oligodendrocytes.

Basic fibroblast growth factor (bFGF) is a well-characterized peptide hormone that has mitogenic activity for various cell types and elicits a characteristic set of responses on the cell types investigated. In this report we confirmed that bFGF is a potent mitogen for rat brain-derived oligodendrocyte (OL) precursor cells as well as for differentiated OL in secondary culture. bFGF was shown to induce expression of the protooncogene c-fos in OL.

Transforming growth factor type beta 1 modulates the effects of basic fibroblast growth factor on growth and phenotypic expression of rat astroblasts in vitro.

In a search of the growth factors possibly involved in brain ontogenesis we have examined the effects of transforming growth factor beta 1 (TGF-beta 1) on the growth and phenotypic expression of rat astroblasts in primary culture. Along TGF-beta 1 elicited only a slight negative effect on the growth of these cells. However, this factor was found to modulate the mitogenic effects of other growth factors.

Acidic and basic fibroblast growth factors similarly regulate the rate of biosynthesis of rat astroblast proteins.

Quiescent rat astroblasts in culture have been treated for various periods of time with acidic and basic fibroblast growth factors. Both factors elicited similar effects on the cell proliferation and glutamine synthetase activity. The rate of biosynthesis of the proteins analyzed on autoradiograms of polyacrylamide gels after two-dimensional electrophoresis was also similarly modulated by the two growth factors.