The controlled generation of functional basal forebrain cholinergic neurons from human embryonic stem cells.

An early substantial loss of basal forebrain cholinergic neurons (BFCN) is a constant feature of Alzheimer's disease and is associated with deficits in spatial learning and memory. The ability to selectively control the differentiation of human embryonic stem cells (hESCs) into BFCN would be a significant step toward a cell replacement therapy. We demonstrate here a method for the derivation of a predominantly pure population of BFCN from hESC cells using diffusible ligands present in the forebrain at developmentally relevant time periods. Overexpression of two relevant human transcription factors in hESC-derived neural progenitors also generates BFCN. These neurons express only those markers characteristic of BFCN, generate action potentials, and form functional cholinergic synapses in murine hippocampal slice cultures. siRNA-mediated knockdown of the transcription factors blocks BFCN generation by the diffusible ligands, clearly demonstrating the factors both necessary and sufficient for the controlled derivation of this neuronal population. The ability to selectively control the differentiation of hESCs into BFCN is a significant step both for understanding mechanisms regulating BFCN lineage commitment and for the development of both cell transplant-mediated therapeutic interventions for Alzheimer's disease and high-throughput screening for agents that promote BFCN survival.

Modulation of human microglia and THP-1 cell toxicity by cytokines endogenous to the nervous system.

Neuroinflammatory processes are thought to be a significant factor in the pathology of a number of degenerative neurological diseases. A variety of cytokines influence inflammatory levels. Here we show that a cooperative action of two or more cytokines is required to induce significantly human microglial and monocytic THP-1 cell toxicity towards SH-SY5Y neuroblastoma cells. Such toxicity was induced by the following combinations: interferon-gamma (IFN-gamma) with tumor necrosis factor-alpha (TNF-alpha); IFN-gamma with interleukin (IL) 1alpha or IL-1beta in the presence of TNF-alpha; and IL-6 with TNF-alpha. Toxicity induced by the various stimulatory combinations was not accompanied by an increased nitrite production. Of the potential inhibitors tested, IL-4 downregulated the toxic action of microglia when applied to THP-1 cells either before stimulation or 24 h after stimulation. Toxicity was not inhibited by IL-10, and was even enhanced by transforming growth factor-beta1 (TGF-beta1) and basic fibroblast growth factor (bFGF). These data suggest that antagonists of cytokine receptors, as well as inhibitors of their intracellular pathways may be effective anti-inflammatory agents.

Reduction of human monocytic cell neurotoxicity and cytokine secretion by ligands of the cannabinoid-type CB2 receptor.

1 Two cannabinoid receptors, CB1 and CB2, have been identified. The CB1 receptor is preferentially expressed in brain, and the CB2 receptor in cells of leukocyte lineage. We identified the mRNA for the CB1 receptor in human neuroblastoma SH-SY5Y cells, and the mRNA and protein for the CB2 receptor in human microglia and THP-1 cells. 2 Delta(9)-and Delta(8)-tetrahydrocannabinol (THC) were toxic when added directly to SH-SY5Y neuroblastoma cells. The toxicity of Delta(9)- THC was inhibited by the CB1 receptor antagonist SR141716A but not by the CB2 receptor antagonist SR144528. The endogenous ligand anandamide was also toxic, and this toxicity was enhanced by inhibitors of its enzymatic hydrolysis. 3 The selective CB2 receptor ligands JWH-015 and indomethacin morpholinylamide (BML-190), when added to THP-1 cells before stimulation with lipopolysaccharide (LPS) and IFN-gamma, reduced the toxicity of their culture supernatants to SH-SY5Y cells. JWH-015 was more effective against neurotoxicity of human microglia than THP-1 cells. The antineurotoxic activity of JWH-015 was blocked by the selective CB2 receptor antagonist SR144528, but not by the CB1 receptor antagonist SR141716A. This activity of JWH-015 was synergistic with that of the 5-lipoxygenase (5-LOX) inhibitor REV 5901. 4 Cannabinoids inhibited secretion of IL-1beta and tumor necrosis factor-alpha (TNF-alpha) by stimulated THP-1 cells, but these effects could not be directly correlated with their antineurotoxic activity. 5 Specific CB2 receptor ligands could be useful anti-inflammatory agents, while avoiding the neurotoxic and psychoactive effects of CB1 receptor ligands such as Delta(9)-THC.

Interleukin 1alpha and interleukin 6 protect human neuronal SH-SY5Y cells from oxidative damage.

Interleukin (IL)-1alpha and IL-6 are powerful inflammatory cytokines produced in brain primarily by microglia and astrocytes. Here we demonstrate, using an in vitro assay system, that they can have a direct neuroprotective action against oxidative attack. Exposure of retinoic acid-differentiated human SH-SY5Y neuroblastoma cells to 270 microM hydrogen peroxide caused activation of caspase 3 and significant neuronal death. Treatment with IL-1alpha or IL-6 caused a dose-dependent increase in survival as measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. An antibody against single-strand DNA demonstrated many apoptotic neuroblastoma cells following exposure to hydrogen peroxide, with a decrease following cytokine treatment. These data indicate that IL-1alpha and IL-6 can, under appropriate circumstances, protect neurons from oxidative damage in addition to their well-known action of stimulating inflammation.