CD8+ T-cells infiltrate Alzheimer's disease brains and regulate neuronal- and synapse-related gene expression in APP-PS1 transgenic mice.

Neuroinflammation is a major contributor to disease progression in Alzheimer's disease (AD) and is characterized by the activity of brain resident glial cells, in particular microglia cells. However, there is increasing evidence that peripheral immune cells infiltrate the brain at certain stages of AD progression and shape disease pathology. We recently identified CD8+ T-cells in the brain parenchyma of APP-PS1 transgenic mice being tightly associated with microglia as well as with neuronal structures. The functional role of CD8+ T-cells in the AD brain is however completely unexplored. Here, we demonstrate increased numbers of intra-parenchymal CD8+ T-cells in human AD post-mortem hippocampus, which was replicated in APP-PS1 mice. Also, aged WT mice show a remarkable infiltration of CD8+ T-cells, which was more pronounced and had an earlier onset in APP-PS1 mice. To address their functional relevance in AD, we successfully ablated the pool of CD8+ T-cells in the blood, spleen and brain from 12 months-old APP-PS1 and WT mice for a total of 4 weeks using an anti-CD8 antibody treatment. While the treatment at this time of disease stage did neither affect the cognitive outcome nor plaque pathology, RNAseq analysis of the hippocampal transcriptome from APP-PS1 mice lacking CD8+ T-cells revealed highly altered neuronal- and synapse-related gene expression including an up-regulation for neuronal immediate early genes (IEGs) such as the Activity Regulated Cytoskeleton Associated Protein (Arc) and the Neuronal PAS Domain Protein 4 (Npas4). Gene ontology enrichment analysis illustrated that the biological processes "regulation of neuronal synaptic plasticity" and the cellular components "postsynapses" were over-represented upon CD8+ T-cell ablation. Additionally, Kegg pathway analysis showed up-regulated pathways for "calcium signaling", "long-term potentiation", "glutamatergic synapse" and "axon guidance". Therefore, we conclude that CD8+ T-cells infiltrate the aged and AD brain and that brain CD8+ T-cells might directly contribute to neuronal dysfunction in modulating synaptic plasticity. Further analysis will be essential to uncover the exact mechanism of how CD8+ T-cells modulate the neuronal landscape and thereby contribute to AD pathology.

A novel nervous system beta subunit that downregulates human large conductance calcium-dependent potassium channels.

The pore-forming alpha subunits of many ion channels are associated with auxiliary subunits that influence channel expression, targeting, and function. Several different auxiliary (beta) subunits for large conductance calcium-dependent potassium channels of the Slowpoke family have been reported, but none of these beta subunits is expressed extensively in the nervous system. We describe here the cloning and functional characterization of a novel Slowpoke beta4 auxiliary subunit in human and mouse, which exhibits only limited sequence homology with other beta subunits. This beta4 subunit coimmunoprecipitates with human and mouse Slowpoke. beta4 is expressed highly in human and monkey brain in a pattern that overlaps strikingly with Slowpoke alpha subunit, but in contrast to other Slowpoke beta subunits, it is expressed little (if at all) outside the nervous system. Also in contrast to other beta subunits, beta4 downregulates Slowpoke channel activity by shifting its activation range to more depolarized voltages and slowing its activation kinetics. beta4 may be important for the critical roles played by Slowpoke channels in the regulation of neuronal excitability and neurotransmitter release.

Detection of a large cation-selective channel in nuclear envelopes of avian erythrocytes.

To determine whether the nuclear envelope of eukaryotic cells has the capability to regulate ion fluxes, we have used the patch-clamp technique to detect ion channels in this membrane system. Since possible sites for ion channels in the nuclear envelope include not only the nuclear pores, but also both the inner and outer nuclear membranes, we have patched giant liposomes composed of phosphatidylcholine and nuclear envelope fragments isolated from mature avian erythrocytes. A large, cation-selective channel with a maximum conductance of approximately 800 pS in symmetrical 100 mM KCl was detected. This channel is a possible candidate for a nuclear pore.

Long chain diamines inhibit growth of C6 glioma cells according to their hydrophobicity. An in vitro and molecular modeling study.

A series of diamines with the general structure NH2(CH2)xNH2, x=2-12, was tested for their potential effects on cell proliferation of cultured rat C6 glioma cells in comparison to natural polyamines. Long chain diamines reduced cell number after 48 h in culture with a sequence of 1,12-diaminododecane (1,12-DD) >1,10-diaminodecane >1,9-diaminononane. Polyamines (putrescine, spermidine and spermine) as well as diamines up to a CH2-chain length of x=8 were found to be ineffective. The spermine analogue 1,12-DD was the most effective molecule in reducing cell number in an irreversible, dose-dependent manner (EC50=3 microM under serum-free conditions). In further experiments we investigated the mechanisms of action of 1,12-DD. The compound had only a minor effect on cell cycle and did not affect free internal calcium concentration. Under physiological conditions 1,12-DD interacts with triplex DNA but not with duplex DNA. Ornithine decarboxylase activity as well as the concentration of internal polyamines were found to be reduced by 1,12-DD. Polyamine application, however, was not able to reverse the effect of 1,12-DD, indicating a polyamine-independent or non-competitive mechanism of action. 1,12-DD reduced cell number by induction of apoptosis as well as necrosis. In molecular modeling studies it was found that a minimal hydrophobic intersegment of at least 4 A was required to make a diamine an effective drug in respect to cellular growth. A hydrophobic gap of this size fits the minimum requirement expected from molecular modeling to provide space for hydrophobic interactions with parts of proteins like a CH3-group. Our results show that 1,12-DD acts as a potent drug, reducing the number of C6 glioma cells, and suggest that its spatial and hydrophobic properties are responsible for its mechanism of action.

Potassium channel blockers quinidine and caesium halt cell proliferation in C6 glioma cells via a polyamine-dependent mechanism.

Potassium channels are ubiquitous in cells and serve essential functions in physiology and pathophysiology. Potassium channel blockers have been shown to block tumour growth by arresting cells at the G(0)/G(1) checkpoint of the cell cycle. We investigated the effect of quinidine and caesium (Cs(+)) on cell proliferation, LDH (lactate dehydrogenase) release, free internal calcium, membrane potential, polyamine concentration, ODC (ornithine decarboxylase) activity and polyamine uptake in C6 glioma cells. The EC(50) for reducing cell proliferation was 112 microM for quinidine, whereas Cs(+) was less effective with an EC(50) of 4.75 mM. KCl or sucrose did not affect proliferation. LDH release was augmented by quinidine. Quinidine caused a transient increase in free internal calcium but decreased calcium after a 48 h incubation period. Further 300 microM quinidine depolarized the cell membrane in a similar range as did 30 mM KCl. Quinidine decreased cellular putrescine beyond detection levels while spermidine and spermine remained unaffected. ODC activity was reduced. Addition of putrescine could not override the antiproliferative effect owing to a reduced activity of the polyamine transporter. Our study indicates that the antiproliferative effect of quinidine is not due to a simple membrane depolarization but is caused by a block of ODC activity.

Ethanol activates maxi Ca2+-activated K+ channels of clonal pituitary (GH3) cells.

The effect of ethanol on maxi Ca2+-activated K+ channels (BK channels) in GH3 pituitary tumor cells was investigated using single-channel recordings and focusing on intracellular signal transduction. In outside-out patches, ethanol caused a transient concentration-dependent increase of BK-channel activity. 30 mm (1.4 per thousand) ethanol significantly increased mean channel open time and channel open probability by 26.3 +/- 9% and 78.8 +/- 10%, respectively; single-channel current amplitude was not affected by ethanol. The augmenting effect of ethanol was blocked in the presence of protein kinase C (PKC) inhibitors staurosporine, bisindolylmaleimide, and PKC (19-31) pseudosubstrate inhibitor as well as by AMP-PNP (5'-adenylylimidodiphosphate), a nonhydrolyzable ATP-analogue, but not by the phospholipase C blocker U-73122. Phosphatase inhibitors microcystin-LR and okadaic acid promoted the ethanol effect. The blocking effect was released at higher concentrations of ethanol (100 mm) suggesting a second site of action or a competition between blockers and ethanol. Our results suggest that the effect of ethanol on BK-channels is mediated by PKC stimulation and phosphorylation of the channels which increases channel activity and hence may influence action potentials duration and hormone secretion.

External action of di- and polyamines on maxi calcium-activated potassium channels: an electrophysiological and molecular modeling study.

In this study we compared polyamines to various diamines, and we modeled flexibility as well as hydrophobicity properties of these molecules to examine possible structural differences that could explain their external effects on the channels. The natural polyamines (putrescine, cadaverine, spermidine, spermine) and diamines increasing in CH2 chain length from C2 to C12 were used to probe maxi calcium-activated potassium (BK) channels in GH3 pituitary tumor cells when applied extracellularly. In single-channel recordings we found polyamines as well as diamines up to 1,10-diaminodecane to be ineffective in altering channel current amplitudes or kinetics. In contrast, 1,12-diamino dodecane (1,12-DD) was found to be a reversible blocker, with a blocking site at an electrical distance (z delta) of 0.72 within the channel. It reduced single-channel current amplitude, mean channel open time, and channel open probability. In computer simulations structural data, such as flexibility, hydration, and log D values, were calculated. 1,12-DD showed the largest flexibility of all diamines (minimum N-N distance 9.9 A) combined with a marked hydrophobicity due to a 4-5 A hydrophobic intersegment between hydrophilic ends in the molecule, as confirmed by GRID water probe maps and a log D value of -1.82 at pH 7.2. We propose that the amount of hydration of the molecule, more than its flexibility, constitutes an essential parameter for its ability to act as a channel blocker.

Astrocytes and neurons express the tight junction-specific protein occludin in vitro.

The expression of occludin, an integral plasma membrane protein specifically located at tight junctions, was studied in various epithelial and nonepithelial tissues by means of RT-PCR, Western blotting, and immunofluorescent staining. Besides detection in epithelial and endothelial tissue, expression of occludin was found in primary and secondary cultures of neurons and astrocytes. Differentiation of astrocytes in vitro led to a marked decrease in occludin expression. Extractability of occludin from plasma membranes differed considerably between epithelial and nonepithelial cells. Following treatment with Triton X-100, occludin was completely extracted from astrocytic membranes but not from membranes derived from MDCK cells, suggesting a difference in the cytoplasmic and/or plasma membrane anchoring of occludin between these cell types.