Dopamine D2 Receptor-Deficient Mice Exhibit Decreased Dopamine Transporter Function but No Changes in Dopamine Release in Dorsal Striatum

Abstract : Presynaptic D₂ dopamine (DA) autoreceptors, which are well known to modulate DA release, have recently been shown to regulate DA transporter (DAT) activity. To examine the effects of D₂ DA receptor deficiency on DA release and DAT activity in dorsal striatum, we used mice genetically engineered to have two (D₂^+/+), one (D₂^+/-), or no (D₂^-/-) functional copies of the gene coding for the D₂ DA receptor. In vivo microdialysis studies demonstrated that basal and K⁺-evoked extracellular DA concentrations were similar in all three genotypes. However, using in vivo electrochemistry, the D₂^-/- mice were found to have decreased DAT function, i.e., clearance of locally applied DA was decreased by 50% relative to that in D₂^+/+ mice. In D₂^+/+ mice, but not D₂^-/- mice, local application of the D₂-like receptor antagonist raclopride increased DA signal amplitude, indicating decreased DA clearance. Binding assays with the cocaine analogue [³H]WIN 35,428 showed no genotypic differences in either density or affinity of DAT binding sites in striatum or substantia nigra, indicating that the differences seen in DAT activity were not a result of decreased DAT expression. These results further strengthen the idea that the D₂ DA receptor subtype modulates activity of the striatal DAT.     

Ca2+-Dependent Enhancement of [3H]Dopamine Uptake in Rat Striatum: Possible Involvement of Calmodulin-Dependent Kinases

Abstract: We studied effects of Ca²⁺ in the incubation medium on [³H]dopamine ([³H]DA) uptake by rat striatal synaptosomes. Both the duration of the preincubation period with Ca²⁺ (0–30 min) and Ca²⁺ concentration (0–10 m M ) in Krebs-Ringer medium affected [³H]DA uptake by the synaptosomes. The increase was maximal at a concentration of 1 m M Ca²⁺ after a 10-min preincubation (2.4 times larger than the uptake measured without preincubation), which reflected an increase in V _max of the [³H]DA uptake process. On the other hand, [³H]DA uptake decreased rapidly after addition of ionomycin in the presence of 1 m M Ca²⁺. The Ca²⁺-dependent enhancement of the uptake was still maintained after washing synaptosomes with Ca²⁺-free medium following preincubation with 1 m M Ca²⁺. Protein kinase C inhibitors did not affect apparently Ca²⁺-dependent enhancement of the uptake, whereas 1-[ N,O -bis(1,5-isoquinolinesulfonyl)- N -methyl- l -tyrosyl]-4-phenylpiperazine (KN-62; a Ca²⁺/calmodulin-dependent kinase II inhibitor) and wortmannin (a myosin light chain kinase inhibitor) significantly reduced it. Inhibitory effects of KN-62 and wortmannin appeared to be additive. N -(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7; a calmodulin antagonist) also remarkably inhibited the enhancement. These results suggest that Ca²⁺-dependent enhancement of [³H]DA uptake is mediated by activation of calmodulin-dependent protein kinases.     

Fibroblast growth factor 9 prevents MPP+-induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP⁺ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP⁺ intoxication and melatonin protection. We found that MPP⁺ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP⁺-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP⁺-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP⁺, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP⁺ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.     

A β-N-acetylhexosaminidase from Penicillium oxalicum implicated in its cell-wall degradation

High β- N -acetylhexosaminidase (EC.3.2.1.52) activity was detected during autolysis of Penicillium oxalicum . Purification of the enzyme to homogeneity yielded an enzyme with a molecular weight of 132 000 Da by gel filtration and 71 900 Da by SDS polyacrylamide gel electrophoresis, suggesting a dimeric structure. The enzyme is an acidic protein with a pl of 5.0. Optimal activity was at pH 4.0 and 40°C, with a K _m of 0.80 mmol 1^-1 for p -nitrophenyl-β- N -acetylglucosaminide and 1.03 mmol 1^-1 for p -nitrophenyl-β- N -acetylgalactosaminide. The K _i with the competitive inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino- N -phenylcarbamate was 1 μmol 1^-1. Hg²⁺, Ag⁺ and Fe³⁺ were effective inhibitors. β- N -acetylhexosaminidase hydrolysed chitobiose, chitotriose, chitotetrose and chitopentose to monomer to an extent of 92, 74, 44 and 17% respectively in 40 min. This enzyme, in conjunction with a purified endochitinase from P. oxalicum , hydrolysed a cell-wall chitin fraction isolated from this fungus, with the production of N -acetylglucosamine.     

Isoform-Specific Up-Regulation by Ouabain of Na+,K+-ATPase in Cultured Rat Astrocytes

Abstract: There are two α-subunit isoforms (α1 and α2) and two β-subunit isoforms (β1 and β2) of Na⁺,K⁺-ATPase in astrocytes, but the functional heterodimer composition is not known. Ouabain (0.5–1.0 m M ) increased the levels of α1 and β1 mRNAs, whereas it decreased those of α2 and β2 mRNAs in cultured rat astrocytes. The increases in α1 and β1 mRNAs were observed at 6–48 h after addition of the inhibitor. Immunochemical analyses showed that ouabain increased α1 and β1, but not α2 and β2, proteins, and that the isoforms in control and ouabain-treated cultures were of glial origin. Low extracellular K⁺ and monensin (20 µ M ) mimicked the effect of ouabain on α1 mRNA. The ouabain-induced increase in α1 mRNA was blocked by the protein synthesis inhibitor cycloheximide (10 µ M ), the intracellular Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane- N,N,N',N' -tetraacetic acid tetraacetoxymethyl ester (30 µ M ), and the calcineurin inhibitor FK506 (1 n M ). These findings indicate that chronic inhibition of Na⁺,K⁺-ATPase up-regulates the α1 and β1, but not α2 and β2, isoforms in astrocytes, suggesting a functional coupling of α1β1 complex. They also suggest that intracellular Na⁺, Ca²⁺, and calcineurin may be involved in ouabain-induced up-regulation of the enzyme in astrocytes.     

Uptake of 1-Methyl-4-Phenylpyridinium and Dopamine in the Mouse Brain Cell Nuclei

Abstract: The neurotoxin N -methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes, via its metabolite 1-methyl-4-phenylpyridinium (MPP⁺), parkinsonism in humans, monkeys, and mice but not in rats. When incubated with mouse brain homogenates, [³H]-MPP⁺ is recovered in relatively large concentrations in the brain cell nucleus. Although isolated cell nuclei from rat and mouse brain contain uptake systems for dopamine (DA), only brain cell nuclei from mice avidly take up [³H]MPP⁺. This nuclear uptake is ATP dependent and can be blocked by ouabain and Nethylmaleimide. It is not, however, affected by neuronal and vesicular blockers such as benztropine. mazindol, and reserpine. Selective uptake of MPP⁺ into brain cell nuclei may provide a new avenue for future investigation into the complex modes of action of the neurotoxin MPTP.     

Chronic Dopamine D2 Receptor Activation Does Not Affect Survival and Differentiation of Cultured Dopaminergic Neurons: Morphological and Neurochemical Observations

Abstract: Primary cultures of rat ventral mesencephalon were used to elucidate the role of chronic stimulation of dopamine (DA) D₂ autoreceptors in the development of fetal dopaminergic neurons in vitro. Cultured dopaminergic neurons, as visualized by tyrosine hydroxylase immunocytochemistry, became more differentiated in the course of cultivation time and exhibited specific high-affinity uptake for [³H]DA. In rat striatal tissue, activation of D₂ receptors has been shown to inhibit the release of DA. Previously accumulated [³H]DA was released from the cultures upon depolarization in a Ca²⁺-dependent manner. K⁺-evoked [³H]DA release could be inhibited by the selective D₂ receptor agonists LY 171555 and N0437 in a concentration-dependent manner. The inhibitory effects of LY 171555 and N0437 were antagonized by the selective DA D₂ receptor antagonist sulpiride. These observations are indicative for the expression of functional D₂ receptors in the cultures. Daily treatment of these cultures for 7 days with LY 171555 or sulpiride did not lead to any change in protein content, the number of tyrosine hydroxylase-immunoreactive neurons, or the uptake capacity for [³_H]DA. Our data demonstrate that chronic stimulation of DA D₂ receptors does not impair survival or differentiation of cultured fetal dopaminergic neurons.     

Dopamine Receptor Stimulation Decreases Cytosolic γ Protein Kinase C Immunoreactivity in Rat Hippocampal Slices: Evidence for Increased Ca2+-Dependent Proteolysis

Abstract: The effect of dopamine (DA) receptor stimulation on the distribution of γ protein kinase C (γPKC) in hippocampal slices was assessed. Nanomolar concentrations of DA decreased cytosolic γPKC (56%) without altering membrane γPKC levels, resulting in decreased total γPKC immunoreactivity. The maximal decrease in cytosolic γPKC occurred at 20 min of incubation and was significantly blocked by the D₁ DA antagonist SCH 23390 (10⁻⁶ M ) but not by the D₂ antagonist sulpiride (10⁻⁵ M ). The D₁ agonists SKF 38393 and A 77636 mimicked the effect of DA with similar responses produced at 10 µ M and 1 n M , respectively. The D₂ agonist quinpirole had no effect on γPKC immunoreactivity, thus indicating that this dopaminergic response is mediated through a D₁-like receptor. DA had no effect on α, δ, or ζPKC isozyme immunoreactivity in the same hippocampal preparations. The DA-induced decrease in cytosolic γPKC immunoreactivity was blocked by the Ca²⁺-dependent protease inhibitor N -acetyl-Leu-Leu-norleucinal (100 µ M ) and by the inorganic Ca²⁺ channel blocker Co²⁺. The data suggest that DA stimulates a D₁-like DA receptor, which increases the influx of Ca²⁺ and activates the Ca²⁺-dependent proteolysis of γPKC.     

Cytotoxic Effect of β-Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation

Abstract: The effects of synthetic β-amyloid (Aβ1–42) on cell viability and cellular Ca²⁺ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. Aβ1–42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 µ M , and the strongest effect was produced by 40 µ M . Control NT2N cells produced spontaneous cytosolic Ca²⁺ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4–40 µ M ) by Aβ1–42 that was present in the cell culture for 1 or 4 days. Ca²⁺ wave frequency was decreased from 0.21 ± 0.02 to 0.05 ± 0.02/min, amplitude from 88 ± 8 to 13 ± 4 n M , and average Ca²⁺ level from 130 ± 8 to 58 ± 3 n M . The Ca²⁺ responses to 30 m M K⁺ and 100 µ M glutamate were not different between control and Aβ-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca²⁺ accumulation mediates Aβ-induced NT2N cell death.     

Voltammetric Studies on Mechanisms of Dopamine Efflux in the Presence of Substrates and Cocaine from Cells Expressing Human Norepinephrine Transporter

Abstract: The effects of substrates m -tyramine and β-phenethylamine, as well as cocaine, on the DA efflux from a cell line stably expressing the human norepinephrine transporter (hNET) were investigated by using rotating disk electrode voltammetry. Both the substrates and cocaine induced apparent DA efflux in a concentration-dependent manner. Their EC₅₀ values for inducing DA efflux were similar to their IC₅₀ values for inhibiting DA uptake. The substrate-induced DA efflux was inhibited by various NET blockers, enhanced by raising the internal [Na⁺] with Na⁺,K⁺-ATPase inhibition, but was insensitive to membrane potential-altering agents valinomycin, veratridine, and high [K⁺]. The initial rate of m -tyramine-induced DA efflux was related to preloaded [DA] in a manner defined by a Michaelis-Menten expression. In contrast, DA efflux in the presence of cocaine displayed a much slower efflux rate, lower efficacy, was not stimulated by elevated internal [Na⁺], and was nonsaturable with preloaded [DA]. Single exponential kinetic analysis of the entire time course of the DA efflux showed that the apparent first-order rate constant for m -tyramine-induced DA efflux declined with increased preloaded [DA], whereas that for the DA efflux in the presence of cocaine was unchanged with varying preloaded [DA]. These results suggest that the substrates stimulate the NET-dependent DA efflux by increasing the accessibility of the NET to internal DA, whereas cocaine "uncovers" NET-independent DA efflux by reducing the accessibility of diffused/leaked external DA to the NET.     

Calcium is involved in regulation of the synthesis of HSPs in suspension-cultured sugar beet cells under hyperthermia

Exposure of plants to elevated temperatures induces a complex set of changes that enable plants to adapt following heat stress. In order to test the effect of Ca²⁺ on heat shock-induced changes in cell protein synthesis the incorporation of [ 35 S]methionine into protein was studied in cultured sugar beet ( Beta vulgaris L.) cells incubated in media containing different calcium concentrations. Heat shock inhibited the synthesis of non-heat shock proteins (non-HSPs) and promoted the synthesis of a set of HSPs, typical of plants. The synthesis of non-HSPs was greatly inhibited by external Ca²⁺ removal by treatment of the cells with ethylene glycol-bis( β -aminoethylether)- N,N,N',N'- tetraacetic acid. In contrast, extracellular Ca²⁺ appeared not to be strictly required for the de novo production of HSPs, but this cation exerted different effects on the synthesis of individual HSPs. Cell injury increased if the cells were exposed simultaneously to high temperature and Ca²⁺-deficient medium. Recovery of HSP synthesis and reduced cell injury were observed after addition of exogenous calcium to Ca²⁺-depleted cells. These findings are consistent with a Ca²⁺ requirement for the survival of the cells under heat shock, and likely for the development of cell thermotolerance.     

Neural precursors derived from human embryonic stem cells maintain long-term proliferation without losing the potential to differentiate into all three neural lineages, including dopaminergic neurons

Human embryonic stem (hES) cells have the ability to renew themselves and differentiate into multiple cell types upon exposure to appropriate signals. In particular, the ability of hES cells to differentiate into defined neural lineages, such as neurons, astrocytes, and oligodendrocytes, is fundamental to developing cell-based therapies for neurodegenerative disorders and studying developmental mechanisms. However, the utilization of hES cells for basic and applied research is hampered by the lack of well-defined methods to maintain their self-renewal and direct their differentiation. Recently we reported that neural precursor (NP) cells derived from mouse ES cells maintained their potential to differentiate into dopaminergic (DA) neurons after significant expansion in vitro . We hypothesized that NP cells derived from hES cells (hES-NP) could also undergo the same in vitro expansion and differentiation. To test this hypothesis, we passaged hES-NP cells and analyzed their proliferative and developmental properties. We found that hES-NP cells can proliferate approximately 380 000-fold after in vitro expansion for 12 weeks and maintain their potential to generate Tuj1⁺ neurons, GFAP⁺ astrocytes, and O4⁺ oligodendrocytes as well as tyrosine hydroxylase-positive (TH⁺) DA neurons. Furthermore, TH⁺ neurons originating from hES-NP cells expressed other midbrain DA markers, including Nurr1, Pitx3, Engrail-1, and aromatic l -amino acid decarboxylase, and released significant amounts of DA. In addition, hES-NP cells maintained their developmental potential through long-term storage (over 2 years) in liquid nitrogen and multiple freeze–thaw cycles. These results demonstrate that hES-NP cells have the ability to provide an expandable and unlimited human cell source that can develop into specific neuronal and glial subtypes.     

Aluminum enhancement of plant growth in acid rooting media. A case of reciprocal alleviation of toxicity by two toxic cations

The generally rhizotoxic ion Al³⁺ often enhances root growth at low concentrations. The hypothesis that Al³⁺ enhances growth by relieving H⁺ toxicity was tested with wheat seedlings ( Triticum aestivum L.). Growth enhancement by Al³⁺ only occurred under acidic conditions that reduced root elongation. Al³⁺ increased cell membrane electrical polarity and stimulated H⁺ extrusion. Previous investigations have shown that Al³⁺ decreases solute leakage at low _pH and that the alleviation of H⁺ toxicity by cations appears to be a general phenomenon with effectiveness dependent upon charge (C³⁺>C²⁺>C^l+). Alleviation of one cation toxicity by another toxic cation appears to be reciprocal so that Al³⁺ toxicity is relieved by H⁺. It has been argued previously that this latter phenomenon accounts for the apparent toxicity of ALOH²⁺ and Al(OH)⁺₂. Reduction of cell-surface electrical potential by the ameliorative cation may reduce the cell-surface activity of the toxic cation.     

Change of specific T cells in an emerging neonatal infectious disease induced by a bacterial superantigen

A new epidemic, NTED, has recently occurred in Japan. The cause of NTED is a bacterial superantigen, TSST-1. The aim of the present study was to analyze the change in Vβ2⁺ T cells reactive to TSST-1 in NTED in order to establish T-cell-targeted diagnostic criteria for NTED. Blood samples from 75 patients with clinically diagnosed NTED were collected from 13 neonatal intensive care units throughout Japan. We investigated the percentages of Vβ2⁺, Vβ3⁺ and Vβ12⁺ T cells and their CD45RO expressions in the samples using flow cytometry. In 18 of the 75 patients, we conducted multiple examinations of the T cells and monitored serial changes. The Vβ2⁺ T-cell population rapidly changed over three phases of the disease. Whereas the percentage of Vβ2⁺ T cells was widely distributed over the entire control range, CD45RO expression on Vβ2⁺ T cells in CD4⁺ in all 75 patients was consistently higher than the control range. Patients cannot necessarily be diagnosed as having NTED based on expansion of Vβ2⁺ T cells alone in the early acute phase. Instead, CD45RO expression on specific Vβ2⁺ cells is a potential diagnostic marker for a rapid diagnosis of NTED. We present three diagnostic categories of NTED. Fifty patients (66.7%) were included in the category 'definitive NTED'. It is important to demonstrate an increase of Vβ2⁺ T cells in the following phase in cases of 'probable NTED' or 'possible NTED'.     

The roles of membrane estrogen receptor subtypes in modulating dopamine transporters in PC-12 cells

The effects of 17β-estradiol (E₂) on dopamine (DA) transport could explain gender and life-stage differences in the incidence of some neurological disorders. We tested the effects of E₂ at physiological concentrations on DA efflux in nerve growth factor-differentiated rat pheochromocytoma cells that express estrogen receptors (ER) α, ERβ, and G-protein coupled receptor 30 (GPR30), and DA transporter (DAT). DAT efflux was determined as the transporter-specific loss of ³H-DA from pre-loaded cells; a 9–15 min 10⁻⁹ M E₂ treatment caused maximal DA efflux. Such rapid estrogenic action suggests a non-genomic response, and an E₂-dendrimer conjugate (limited to non-nuclear actions) caused DA efflux within 5 min. Efflux dose–responses for E₂ were non-monotonic, also characteristic of non-genomic estrogenic actions. ERα siRNA knockdown abolished E₂-mediated DA efflux, while ERβ knockdown did not, and GPR30 knockdown increased E₂-mediated DA efflux (suggesting GPR30 is inhibitory). Use of ER-selective agonists/antagonists demonstrated that ERα is the predominant mediator of E₂-mediated DA efflux, with inhibitory contributions from GPR30 and ERβ. E₂ also caused trafficking of ERα to the plasma membrane, trafficking of ERβ away from the plasma membrane, and unchanged membrane GPR30 levels. Therefore, ERα is largely responsible for non-genomic estrogenic effects on DAT activity.     

Resuscitation of Brain Neurons in the Presence of Ca2+ After Toxic NMDA-Receptor Activity

Abstract: Cultured cerebellar granule cells were subjected to toxic activation of the NMDA receptor that was terminated by MK-801. Subsequent resuscitation experiments were mostly conducted in the presence of a physiological concentration of Ca²⁺. Addition of pyruvate and inorganic phosphate, in addition to glucose, which was always present, rescued ∼40% of the dying neurons. La³⁺ and ruthenium red were also effective resuscitating agents. The combination of pyruvate, inorganic phosphate, and ruthenium red rescued 65% of the dying neurons. Parallel studies with ⁴⁵Ca indicated that La³⁺ and ruthenium red facilitated the decrease of ⁴⁵Ca in the neurons, whereas inorganic phosphate, supported by energy-yielding pyruvate, formed perhaps, a less harmful Ca complex inside the neurons.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Defining pre-synaptic nicotinic receptors regulated by beta amyloid in mouse cortex and hippocampus with receptor null mutants

Disruption of neuronal signaling by soluble β-amyloid has been implicated in deficits in short-term recall in the early stages of Alzheimer's disease. One potential target for β-amyloid is the synapse, with evidence for differential interaction with both pre- and post-synaptic elements. Our previous work revealed an agonist-like action of soluble β-amyloid (pM to nM) on isolated pre-synaptic terminals to increase [Ca²⁺]i, with apparent involvement of pre-synaptic nicotinic receptors. To directly establish the role of nicotinic receptors in pre-synaptic Ca²⁺ regulation, we investigated the pre-synaptic action of β-amyloid on terminals isolated from mice harboring either β2 or α7 nicotinic receptor null mutants (knockouts). Average pre-synaptic responses to β-amyloid in hippocampal terminals of α7 knockout mice were unchanged, whereas responses in hippocampal terminals from β2 knockout mice were strongly attenuated. In contrast, pre-synaptic responses to soluble β-amyloid were strongly attenuated in cortical terminals from α7 knockout mice but were moderately attenuated in cortical terminals from β2 knockout mice. The latter responses, having distinct kinetics, were completely blocked by α-bungarotoxin. The use of receptor null mutants thus permitted direct demonstration of the involvement of specific nicotinic receptors in pre-synaptic Ca²⁺ regulation by soluble β-amyloid, and also indicated differential neuromodulation by β-amyloid of synapses in hippocampus and cortex.     

Axonal Contact Regulates Expression of α2 and β2 Isoforms of Na+,K+-ATPase in Schwann Cells: Adhesion Molecules and Nerve Regeneration

Abstract: Three isoforms of catalytic α subunits and two isoforms of β subunits of Na⁺,K⁺-ATPase were detected in rat sciatic nerves by western blotting. Unlike the enzyme in brain, sciatic nerve Na⁺,K⁺-ATPase was highly resistant to ouabain. The ouabain-resistant α1 isoform was demonstrated to be the predominant form in rat intact sciatic nerve by quantitative densitometric analysis and is mainly responsible for sciatic nerve Na⁺,K⁺-ATPase activity. After sciatic nerve injury, the α3 and β1 isoforms completely disappeared from the distal segment owing to Wallerian degeneration. In contrast, α2 and β2 isoform expression and Na⁺,K⁺-ATPase activity sensitive to pyrithiamine (a specific inhibitor of the α2 isoform) were markedly increased in Schwann cells in the distal segment of the injured sciatic nerve. These latter levels returned to baseline with nerve regeneration. Our results suggest that α3 and β1 isoforms are exclusive for the axon and α2 and β2 isoforms are exclusive for the Schwann cell, although axonal contact regulates α2 and β2 isoform expressions. Because the β2 isoform of Na⁺,K⁺-ATPase is known as an adhesion molecule on glia (AMOG), increased expression of AMOG/β2 on Schwann cells in the segment distal to sciatic nerve injury suggests that AMOG/β2 may act as an adhesion molecule in peripheral nerve regeneration.     

Brain-derived neurotrophic factor facilitates maturation of mesenchymal stem cell-derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson's disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain-derived neurotrophic factor (BDNF) in the maturation of the MSC-derived DA progenitors. 9-day induced MSCs show significant tropomyosin-receptor-kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca²⁺ imaging analyses and electrophysiology. BDNF-treated cells showed the following: increases in intracellular Ca²⁺ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post-synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC-derived DA progenitors.