The enantiomer pair of 24S- and 24R-hydroxycholesterol differentially alter activity of large-conductance Ca2+ -dependent K+ (slo1 BK) channel.

24S-hydroxycholesterol (HC) is most abundant oxysterols in the brain, passes through blood brain barrier, and is therefore regarded as an intermediary for brain cholesterol elimination. We reported that large-conductance Ca2+ - and voltage-activated K+ (slo1 BK) channels are suppressed by this oxysterol, which is presumably intercalated into cell membrane to access the outer surface of the channel. Such an outer approach would make it difficult to interact with the inner, ion-conducting part of the channel. The present findings showed that 24R-HC, the racemic counterpart of 24S-HC, also suppressed slo1 BK channel but in a different voltage-dependent manner. There was a difference between the effects of the two enantiomers on activation kinetics but not on deactivation kinetics. It is suggested that the chirality contributes to the efficacy of channel blockers that act from outer lipophilic parts of channels, as with those which act on the inner, ion-permeable surface.

24S-hydroxycholesterol alters activity of large-conductance Ca2+-dependent K+ (slo1 BK) channel through intercalation into plasma membrane.

Oxysterols, oxidization products of cholesterol, are regarded as bioactive lipids affecting various physiological functions. However, little is known of their effects on ion channels. Using inside-out patch clamp recording, we found that naturally occurring side-chain oxidized oxysterols, 20S­hydroxycholesterol, 22R­hydroxycholesterol, 24S­hydroxycholestero, 25­hydroxycholesterol, and 27­hydroxycholesterol, induced current reduction of large-conductance Ca2+- and voltage-activated K+ (slo1 BK) channels heterologously expressed in HEK293T cells. In contrast with side-chain oxidized oxysterols, naturally occurring ring oxidized ones, 7α­hydroxycholesterol and 7­ketocholesterol were without effect. By using 24S­hydroxycholesterol (24S­HC), the major brain oxysterol, we explored the inhibition mechanism. 24S­HC inhibited Slo1 BK channels with an IC50 of ~2 µM, and decreased macroscopic current by ~60%. This marked current decrease was accompanied by a rightward shift in the conductance-voltage relationship and a slowed activation kinetics, with the deactivation kinetics unaltered. Furthermore, the membrane sterol scavenger γ­cyclodextrin was found to rescue slo1 BK channels from the inhibition, implicating that 24S-HC may be intercalated into the plasma membrane to affect the channel. These findings unveil a novel physiological importance of oxysterols from a new angle that involves ion channel regulation.

A2B5+/GFAP+ Cells of Rat Spinal Cord Share a Similar Lipid Profile with Progenitor Cells: A Comparative Lipidomic Study.

The central nervous system (CNS) harbors multiple glial fibrillary acidic protein (GFAP) expressing cell types. In addition to the most abundant cell type of the CNS, the astrocytes, various stem cells and progenitor cells also contain GFAP+ populations. Here, in order to distinguish between two types of GFAP expressing cells with or without the expression of the A2B5 antigens, we performed lipidomic analyses on A2B5+/GFAP+ and A2B5-/GFAP+ cells from rat spinal cord. First, A2B5+/GFAP- progenitors were exposed to the leukemia inhibitory factor (LIF) or bone morphogenetic protein (BMP) to induce their differentiation to A2B5+/GFAP+ cells or A2B5-/GFAP+ astrocytes, respectively. The cells were then analyzed for changes in their phospholipid, sphingolipid or acyl chain profiles by mass spectrometry and gas chromatography. Compared to A2B5+/GFAP- progenitors, A2B5-/GFAP+ astrocytes contained higher amounts of ether phospholipids (especially the species containing arachidonic acid) and sphingomyelin, which may indicate characteristics of cellular differentiation and inability for multipotency. In comparison, principal component analyses revealed that the lipid composition of A2B5+/GFAP+ cells retained many of the characteristics of A2B5+/GFAP- progenitors, but their lipid profile was different from that of A2B5-/GFAP+ astrocytes. Thus, our study demonstrated that two GFAP+ cell populations have distinct lipid profiles with the A2B5+/GFAP+ cells sharing a phospholipid profile with progenitors rather than astrocytes. The progenitor cells may require regulated low levels of lipids known to mediate signaling functions in differentiated cells, and the precursor lipid profiles may serve as one measure of the differentiation capacity of a cell population.

Learning impairment by minimal cortical injury in a mouse model of Alzheimer's disease.

Brain injury accelerates amyloid-ß (Aß) deposits and exacerbates Alzheimer's disease (AD). Accumulation of intracellular soluble Aß impairs cognition prior to emergence of Aß plaques. However, it is not known whether brain injury affects learning impairment attributable to intracellular soluble Aß. We made a small injury by injecting glutamate into the parietal cortex in 3xTg AD mice of 4-5 months old, at which age soluble Aß is accumulated without Aß deposits. The size of glutamate-induced lesion was significantly larger than that of saline-injected control lesion. We reduced the relative difficulty of Morris water maze (MWM) task by repeating it twice, so that saline-injected 3xTg mice could perform as well as wild-type control mice. Under this condition, glutamate-injected 3xTg mice exhibited learning deficits. DNA microarray analysis revealed that 3 genes are upregulated, with one gene downregulated, more than 2 folds in the hippocampus. These 4 genes do not appear to be involved directly in learning but may be a part of signal cascade triggered by glutamate-induced small injury. Hippocampal content of soluble Aß1-42 was increased in the glutamate 3xTg group. Facilitation of large-conductance calcium-activated potassium (BK) channel accompanied learning recovery in the saline-control 3xTg group in agreement with our previous reports, in which learning deficits attributable to intracellular Aß were alleviated by facilitating BK channels. However, BK channel remained suppressed in the glutamate 3xTg group. It is suggested that glutamate-induced injury worsens learning by enhancing the toxicity of soluble Aß or increasing its content per se.

Omega-3 fatty acids lower blood pressure by directly activating large-conductance Ca²âº-dependent K⁺ channels.

Long-chain polyunsaturated omega-3 fatty acids such as docosahexaenoic acid (DHA), found abundantly in oily fish, may have diverse health-promoting effects, potentially protecting the immune, nervous, and cardiovascular systems. However, the mechanisms underlying the purported health-promoting effects of DHA remain largely unclear, in part because molecular signaling pathways and effectors of DHA are only beginning to be revealed. In vascular smooth muscle cells, large-conductance Ca(2+)- and voltage-activated K(+) (BK) channels provide a critical vasodilatory influence. We report here that DHA with an EC50 of ∼500 nM rapidly and reversibly activates BK channels composed of the pore-forming Slo1 subunit and the auxiliary subunit ß1, increasing currents by up to ∼20-fold. The DHA action is observed in cell-free patches and does not require voltage-sensor activation or Ca(2+) binding but involves destabilization of the closed conformation of the ion conduction gate. DHA lowers blood pressure in anesthetized wild-type but not in Slo1 knockout mice. DHA ethyl ester, contained in dietary supplements, fails to activate BK channels and antagonizes the stimulatory effect of DHA. Slo1 BK channels are thus receptors for long-chain omega-3 fatty acids, and these fatty acids--unlike their ethyl ester derivatives--activate the channels and lower blood pressure. This finding has practical implications for the use of omega-3 fatty acids as nutraceuticals for the general public and also for the critically ill receiving omega-3-enriched formulas.

Activity of BK(Ca) channel is modulated by membrane cholesterol content and association with Na+/K+-ATPase in human melanoma IGR39 cells.

Interaction of large conductance Ca(2+)- and voltage-activated K(+) (BK(Ca)) channels with Na(+)/K(+)-ATPase, caveolin-1, and cholesterol was studied in human melanoma IGR39 cells. Functional BK(Ca) channels were enriched in caveolin-rich and detergent-resistant membranes, i.e. rafts, and blocking of the channels by a specific BK(Ca) blocker paxilline reduced proliferation of the cells. Disruption of rafts by selective depletion of cholesterol released BK(Ca) channels from these domains with a consequent increase in their activity. Consistently, cholesterol enrichment of the cells increased the proportion of BK(Ca) channels in rafts and decreased their activity. Immunocytochemical analysis showed that BK(Ca) channels co-localize with Na(+)/K(+)-ATPase in a cholesterol-dependent manner, thus suggesting their co-presence in rafts. Supporting this, ouabain, a specific blocker of Na(+)/K(+)-ATPase, inhibited BK(Ca) whole-cell current markedly in control cells but not in cholesterol-depleted ones. This inhibition required the presence of external Na(+). Collectively, these data indicate that the presence of Na(+)/K(+)-ATPase in rafts is essential for efficient functioning of BK(Ca) channels, presumably because the pump maintains a low intracellular Na(+) proximal to the BK(Ca) channel. In conclusion, cholesterol could play an important role in cellular ion homeostasis and thus modulate many cellular functions and cell proliferation.

TLX activates MASH1 for induction of neuronal lineage commitment of adult hippocampal neuroprogenitors.

The orphan nuclear receptor TLX has been proposed to act as a repressor of cell cycle inhibitors to maintain the neural stem cells in an undifferentiated state, and prevents commitment into astrocyte lineages. However, little is known about the mechanism of TLX in neuronal lineage commitment and differentiation. A majority of adult rat hippocampus-derived progenitors (AHPs) cultured in the presence of FGF express a high level of TLX and a fraction of these cells also express the proneural gene MASH1. Upon FGF withdrawal, TLX rapidly decreased, while MASH1 was intensely expressed within 1h, decreasing gradually to disappear at 24h. Adenoviral transduction of TLX in AHP cells in the absence of FGF transiently increased cell proliferation, however, later resulted in neuronal differentiation by inducing MASH1, Neurogenin1, DCX, and MAP2ab. Furthermore, TLX directly targets and activates the MASH1 promoter through interaction with Sp1, recruiting co-activators whereas dismissing the co-repressor HDAC4. Conversely, silencing of TLX in AHPs decreased beta-III tubulin and DCX expression and promoted glial differentiation. Our results thus suggest that TLX not only acts as a repressor of cell cycle and glial differentiation but also activates neuronal lineage commitment in AHPs.

Neuronal differentiation of neural progenitor cells by intracellular delivery of synthetic oligopeptide derived from Von Hippel-Lindau protein.

Intracellular delivery of synthetic oligopeptides has the potential to promote the occurrence of various cellular events such as cell death, proliferation, growth inhibition, metabolic changes, and morphological changes. However, the regulation of cellular differentiation by intracellular delivery of synthetic oligopeptides has been little studied. Von Hippel-Lindau protein (pVHL) is one of the proteins that functions to induce the differentiation of neural progenitor cells (NPCs). To function in these cells, pVHL forms a complex composed of itself, elongin BC, Clu-2, and Rbx-1. It is suggested that the binding site of elongin BC in pVHL plays a critical role in pVHL function, i.e., ubiquitination, which is related to neuronal differentiation. So, we synthesized an oligopeptide corresponding to the elongin BC binding site, and delivered the oligopeptide into NPCs by using a mixture of trifluoroacetylated lipopolyamine and diloeoyl phosphatidylethanolamine (BioPorter) to form a peptide-lipid complex. After intracellular delivery of the oligopeptide, induction of differentiation of NPCs was shown in terms of neurite outgrowth and by immunocytochemical and electrophysiological means. The intracellular delivery of the synthetic oligopeptide derived from pVHL may provide a safe and valuable approach for the neuronal differentiation of NPCs.

Ca2+-activated K+ channels in human melanoma cells are up-regulated by hypoxia involving hypoxia-inducible factor-1alpha and the von Hippel-Lindau protein.

Under chronic hypoxia, tumour cells undergo adaptive changes involving hypoxia-inducible factors (HIFs). Here we report that ion currents mediated by Ca2+-activated K+ (K(Ca)) channels in human melanoma IGR1 cells are increased by chronic hypoxia (3% O2), as well as by hypoxia mimetics. This increase involves the HIF system as confirmed by overexpression of HIF-1alpha or the von Hippel-Lindau tumour suppressor gene. Under normoxic conditions the K(Ca) channels in IGR1 cells showed pharmacological characteristics of intermediate conductance K(Ca) subtype IK channels, whereas the subtype SK2 channels were up-regulated under hypoxia, shown with pharmacological tools and with mRNA analysis. Hypoxia increased cell proliferation, but the K(Ca) channel blockers apamin and charybdotoxin slowed down cell growth, particularly under hypoxic conditions. Similar results were obtained for the cell line IGR39 and for acutely isolated cells from a biopsy of a melanoma metastasis. Thus, up-regulation of K(Ca) channels may be a novel mechanism by which HIFs can contribute to the malignant phenotype of human tumour cells.

Specific induction of neuronal cells from bone marrow stromal cells and application for autologous transplantation.

Bone marrow stromal cells (MSCs) have the capability under specific conditions of differentiating into various cell types such as osteocytes, chondrocytes, and adipocytes. Here we demonstrate a highly efficient and specific induction of cells with neuronal characteristics, without glial differentiation, from both rat and human MSCs using gene transfection with Notch intracellular domain (NICD) and subsequent treatment with bFGF, forskolin, and ciliary neurotrophic factor. MSCs expressed markers related to neural stem cells after transfection with NICD, and subsequent trophic factor administration induced neuronal cells. Some of them showed voltage-gated fast sodium and delayed rectifier potassium currents and action potentials compatible with characteristics of functional neurons. Further treatment of the induced neuronal cells with glial cell line-derived neurotrophic factor (GDNF) increased the proportion of tyrosine hydroxylase-positive and dopamine-producing cells. Transplantation of these GDNF-treated cells showed improvement in apomorphine-induced rotational behavior and adjusting step and paw-reaching tests following intrastriatal implantation in a 6-hydroxy dopamine rat model of Parkinson disease. This study shows that a population of neuronal cells can be specifically generated from MSCs and that induced cells may allow for a neuroreconstructive approach.

Free-flow electrophoresis in a microfabricated chamber with a micromodule fraction separator. Continuous separation of proteins.

Continuous free flow electrophoresis of proteins was carried out in a microfabricated free flow electrophoresis (mFFE) module with the 30-microm thick slit of the separation. The newly developed micromodule fraction separator (MFS) was attached to the down-stream end site of the separation chamber of mFFE. By using the MFS, electrolyte solution from the separation chamber was introduced to the peristaltic pump without disturbing the electrolyte solution flow at the bottom side of the chamber. The separation of protein mixture samples was achieved by a hydroxypropylmethylcellulose pretreatment coating of the separation chamber. The pretreatment of the sample chamber effectively suppressed electroosmotic flow. All fractionated samples were collected using the MFS after continuous elecrophoresis and analyzed by reversed-phase HPLC. From the results of HPLC analyses none of the cytochrome c fractions at the other ports revealed cross talk phenomena at adjacent ports. A similar result occurred for the myoglobin. This means that these proteins were completely separated from each other by continuous mFFE, and the MFS functioned efficiently during continuous electrophoresis.

Von Hippel-Lindau tumor suppressor protein transforms human neuroblastoma cells into functional neuron-like cells.

Von Hippel-Lindau (VHL) tumor suppressor protein is expressed in neurons of the central nervous system and plays an important role during the neuronal differentiation of central nervous system progenitor cells. To elucidate the neuronal differentiating potential of VHL protein in neuroblastoma cells, we overexpressed or inhibited VHL protein in human neuroblastoma cells (SY-SH5Y), and examined the morphological change, expressions of neuronal markers, and electrophysiological functions. Here we show that with VHL gene transduction SY-SH5Y cells stably expressing the VHL protein had neurite-like processes with varicosities, showed the distinct expression of the neuronal markers neuropeptide Y and neurofilament 200, acquired regulated neurosecretion competence in response to depolarizing and cholinergic stimuli, and had large voltage-gated fast sodium currents and delayed rectifier potassium (Kv) currents compatible with those of functional neurons. In addition, they displayed inactivated ether-á-go-go potassium channels related to the promotion of the cell cycle and to the termination of differentiation. Also, by treatment with retinoic acid, they rapidly underwent cell death related to apoptosis. These findings suggest that the induction of neuronal function by VHL protein is associated with down-regulation of the cell cycle. In contrast, the inhibition of endogenous expression of VHL protein with antisense-orientated VHL gene transduction reduced such neuronal properties inherent to these cells, including the capacity for activation of ether-á-go-go channels. In conclusion, VHL protein has a neuronal differentiating potential to transform neuroblastoma cells into functional neuron-like cells. Our finding of the neuronal differentiation of neuroblastoma cells under the control of the VHL gene may contribute to the development of clinical techniques for neuronal regeneration in the case of intractable neuronal diseases and for differentiation therapy against neuroblastomas.