Distinct Cellular Tools of Mild Hyperthermia-Induced Acquired Stress Tolerance in Chinese Hamster Ovary Cells.

Mild stress could help cells to survive more severe environmental or pathophysiological conditions. In the current study, we investigated the cellular mechanisms which contribute to the development of stress tolerance upon a prolonged (0-12 h) fever-like (40 °C) or a moderate (42.5 °C) hyperthermia in mammalian Chinese Hamster Ovary (CHO) cells. Our results indicate that mild heat triggers a distinct, dose-dependent remodeling of the cellular lipidome followed by the expression of heat shock proteins only at higher heat dosages. A significant elevation in the relative concentration of saturated membrane lipid species and specific lysophosphatidylinositol and sphingolipid species suggests prompt membrane microdomain reorganization and an overall membrane rigidification in response to the fluidizing heat in a time-dependent manner. RNAseq experiments reveal that mild heat initiates endoplasmic reticulum stress-related signaling cascades resulting in lipid rearrangement and ultimately in an elevated resistance against membrane fluidization by benzyl alcohol. To protect cells against lethal, protein-denaturing high temperatures, the classical heat shock protein response was required. The different layers of stress response elicited by different heat dosages highlight the capability of cells to utilize multiple tools to gain resistance against or to survive lethal stress conditions.

Alzheimer risk factors age and female sex induce cortical Aß aggregation by raising extracellular zinc.

Aging and female sex are the major risk factors for Alzheimer's disease and its associated brain amyloid-ß (Aß) neuropathology, but the mechanisms mediating these risk factors remain uncertain. Evidence indicates that Aß aggregation by Zn2+ released from glutamatergic neurons contributes to amyloid neuropathology, so we tested whether aging and sex adversely influences this neurophysiology. Using acute hippocampal slices, we found that extracellular Zn2+-elevation induced by high K+ stimulation was significantly greater with older (65 weeks vs 10 weeks old) rats, and was exaggerated in females. This was driven by slower reuptake of extracellular Zn2+, which could be recapitulated by mitochondrial intoxication. Zn2+:Aß aggregates were toxic to the slices, but Aß alone was not. Accordingly, high K+ caused synthetic human Aß added to the slices to form soluble oligomers as detected by bis-ANS, attaching to neurons and inducing toxicity, with older slices being more vulnerable. Age-dependent energy failure impairing Zn2+ reuptake, and a higher maximal capacity for Zn2+ release by females, could contribute to age and sex being major risk factors for Alzheimer's disease.

The impact of dihydropyridine derivatives on the cerebral blood flow response to somatosensory stimulation and spreading depolarization.

BACKGROUND AND PURPOSE: A new class of dihydropyridine derivatives, which act as co-inducers of heat shock protein but are devoid of calcium channel antagonist and vasodilator effects, has recently been developed with the purpose of selectively targeting neurodegeneration. Here, we evaluated the action of one of these novel compounds LA1011 on neurovascular coupling in the ischaemic rat cerebral cortex. As a reference, we applied nimodipine, a vasodilator dihydropyridine and well-known calcium channel antagonist. EXPERIMENTAL APPROACH: Rats were treated with LA1011 or nimodipine, either by chronic, systemic (LA1011), or acute, local administration (LA1011 and nimodipine). In the latter treatment group, global forebrain ischaemia was induced in half of the animals by bilateral common carotid artery occlusion under isoflurane anaesthesia. Functional hyperaemia in the somatosensory cortex was created by mechanical stimulation of the contralateral whisker pad under α-chloralose anaesthesia. Spreading depolarization (SD) events were elicited subsequently by 1 M KCl. Local field potential and cerebral blood flow (CBF) in the parietal somatosensory cortex were monitored by electrophysiology and laser Doppler flowmetry. KEY RESULTS: LA1011 did not alter CBF, but intensified SD, presumably indicating the co-induction of heat shock proteins, and, perhaps an anti-inflammatory effect. Nimodipine attenuated evoked potentials and SD. In addition to the elevation of baseline CBF, nimodipine augmented hyperaemia in response to both somatosensory stimulation and SD, particularly under ischaemia. CONCLUSIONS AND IMPLICATIONS: In contrast to the CBF improvement achieved with nimodipine, LA1011 seems not to have discernible cerebrovascular effects but may up-regulate the stress response.

Studies for Improving a Rat Model of Alzheimer's Disease: Icv Administration of Well-Characterized ß-Amyloid 1-42 Oligomers Induce Dysfunction in Spatial Memory.

During the past 15 years, several genetically altered mouse models of human Alzheimer's disease (AD) have been developed. These costly models have greatly facilitated the evaluation of novel therapeutic approaches. Injecting synthetic ß-amyloid (Aß) 1-42 species into different parts of the brain of non-transgenic rodents frequently provided unreliable results, owing to a lack of a genuine characterization of the administered Aß aggregates. Previously, we have published a new rat AD-model in which protofibrillar-fibrillar Aß1-42 was administered into rat entorhinal cortex (Sipos 2007). In order to develop a more reliable model, we have injected well-characterized toxic soluble Aß1-42 species (oligomers, protofibrils and fibrils) intracerebroventricularly (icv) into rat brain. Studies of the distribution of fluorescent-labeled Aß1-42 in the brain showed that soluble Aß-species diffused into all parts of the rat brain. After seven days, the Aß-treated animals showed a significant decrease of spatial memory in Morris water maze test and impairment of synaptic plasticity (LTP) measured in acute hippocampal slices. The results of histological studies (decreased number of viable neurons, increased tau levels and decreased number of dendritic spines) also supported that icv administration of well-characterized toxic soluble Aß species into rat brain provides a reliable rat AD-model.

Dihydropyridine Derivatives Modulate Heat Shock Responses and have a Neuroprotective Effect in a Transgenic Mouse Model of Alzheimer's Disease.

Heat shock proteins (Hsps) have chaperone activity and play a pivotal role in the homeostasis of proteins by preventing misfolding, by clearing aggregated and damaged proteins from cells, and by maintaining proteins in an active state. Alzheimer's disease (AD) is thought to be caused by amyloid-ß peptide that triggers tau hyperphosphorylation, which is neurotoxic. Although proteostasis capacity declines with age and facilitates the manifestation of neurodegenerative diseases such as AD, the upregulation of chaperones improves prognosis. Our research goal is to identify potent Hsp co-inducers that enhance protein homeostasis for the treatment of AD, especially 1,4-dihydropyridine derivatives optimized for their ability to modulate cellular stress responses. Based on favorable toxicological data and Hsp co-inducing activity, LA1011 was selected for the in vivo analysis of its neuroprotective effect in the APPxPS1 mouse model of AD. Here, we report that 6 months of LA1011 administration effectively improved the spatial learning and memory functions in wild type mice and eliminated neurodegeneration in double mutant mice. Furthermore, Hsp co-inducer therapy preserves the number of neurons, increases dendritic spine density, and reduces tau pathology and amyloid plaque formation in transgenic AD mice. In conclusion, the Hsp co-inducer LA1011 is neuroprotective and therefore is a potential pharmaceutical candidate for the therapy of neurodegenerative diseases, particularly AD.

Involvement of small heat shock proteins, trehalose, and lipids in the thermal stress management in Schizosaccharomyces pombe.

Changes in the levels of three structurally and functionally different important thermoprotectant molecules, namely small heat shock proteins (sHsps), trehalose, and lipids, have been investigated upon heat shock in Schizosaccharomyces pombe. Both α-crystallin-type sHsps (Hsp15.8 and Hsp16) were induced after prolonged high-temperature treatment but with different kinetic profiles. The shsp null mutants display a weak, but significant, heat sensitivity indicating their importance in the thermal stress management. The heat induction of sHsps is different in wild type and in highly heat-sensitive trehalose-deficient (tps1Δ) cells; however, trehalose level did not show significant alteration in shsp mutants. The altered timing of trehalose accumulation and induction of sHsps suggest that the disaccharide might provide protection at the early stage of the heat stress while elevated amount of sHsps are required at the later phase. The cellular lipid compositions of two different temperature-adapted wild-type S. pombe cells are also altered according to the rule of homeoviscous adaptation, indicating their crucial role in adapting to the environmental temperature changes. Both Hsp15.8 and Hsp16 are able to bind to different lipids isolated from S. pombe, whose interaction might provide a powerful protection against heat-induced damages of the membranes. Our data suggest that all the three investigated thermoprotectant macromolecules play a pivotal role during the thermal stress management in the fission yeast.

Hydroximic acid derivatives: pleiotropic HSP co-inducers restoring homeostasis and robustness.

According to the "membrane sensor" hypothesis, the membrane's physical properties and microdomain organization play an initiating role in the heat shock response. Clinical conditions such as cancer, diabetes and neurodegenerative diseases are all coupled with specific changes in the physical state and lipid composition of cellular membranes and characterized by altered heat shock protein levels in cells suggesting that these "membrane defects" can cause suboptimal hsp-gene expression. Such observations provide a new rationale for the introduction of novel, heat shock protein modulating drug candidates. Intercalating compounds can be used to alter membrane properties and by doing so normalize dysregulated expression of heat shock proteins, resulting in a beneficial therapeutic effect for reversing the pathological impact of disease. The membrane (and lipid) interacting hydroximic acid (HA) derivatives discussed in this review physiologically restore the heat shock protein stress response, creating a new class of "membrane-lipid therapy" pharmaceuticals. The diseases that HA derivatives potentially target are diverse and include, among others, insulin resistance and diabetes, neuropathy, atrial fibrillation, and amyotrophic lateral sclerosis. At a molecular level HA derivatives are broad spectrum, multi-target compounds as they fluidize yet stabilize membranes and remodel their lipid rafts while otherwise acting as PARP inhibitors. The HA derivatives have the potential to ameliorate disparate conditions, whether of acute or chronic nature. Many of these diseases presently are either untreatable or inadequately treated with currently available pharmaceuticals. Ultimately, the HA derivatives promise to play a major role in future pharmacotherapy.

A novel method for the rapid determination of beta-amyloid toxicity on acute hippocampal slices using MTT and LDH assays.

It is difficult task to measure precisely the toxic effect of beta-amyloid (Aß 1-42) peptides and also the protective effect of novel drug candidates against Aß-peptides. The widely used MTT-assay in cell lines or primary cell cultures could be insensitive against Aß-peptides. We describe here an easy and relevant method for testing Aß 1-42 toxicity on acute hippocampal slices derived from rat. Brain slice viability in different conditions was measured using MTT and LDH assays. The concomitant use of these two assays can give detailed and relevant results on the toxic effect of Aß 1-42 in oxygen-glucose deprived (OGD) acute brain slice model. Both assays are capable of quantifying tissue viability by measuring optical density (OD). We found that simultaneous application of OGD and Aß 1-42 treatment induced a more intensive decrease in hippocampal slice viability than their separate effects. The use of MTT and LDH assay for quantifying brain slice viability proved to be an easy ex vivo method for investigating Aß toxicity. Testing brain slices is more relevant in Alzheimer's Disease research than using in vitro cell cultures, due to maintenance of the three dimensional cellular network, the cell variability and intact cell connections.

Membrane-lipid therapy in operation: the HSP co-inducer BGP-15 activates stress signal transduction pathways by remodeling plasma membrane rafts.

Aging and pathophysiological conditions are linked to membrane changes which modulate membrane-controlled molecular switches, causing dysregulated heat shock protein (HSP) expression. HSP co-inducer hydroxylamines such as BGP-15 provide advanced therapeutic candidates for many diseases since they preferentially affect stressed cells and are unlikely have major side effects. In the present study in vitro molecular dynamic simulation, experiments with lipid monolayers and in vivo ultrasensitive fluorescence microscopy showed that BGP-15 alters the organization of cholesterol-rich membrane domains. Imaging of nanoscopic long-lived platforms using the raft marker glycosylphosphatidylinositol-anchored monomeric green fluorescent protein diffusing in the live Chinese hamster ovary (CHO) cell plasma membrane demonstrated that BGP-15 prevents the transient structural disintegration of rafts induced by fever-type heat stress. Moreover, BGP-15 was able to remodel cholesterol-enriched lipid platforms reminiscent of those observed earlier following non-lethal heat priming or membrane stress, and were shown to be obligate for the generation and transmission of stress signals. BGP-15 activation of HSP expression in B16-F10 mouse melanoma cells involves the Rac1 signaling cascade in accordance with the previous observation that cholesterol affects the targeting of Rac1 to membranes. Finally, in a human embryonic kidney cell line we demonstrate that BGP-15 is able to inhibit the rapid heat shock factor 1 (HSF1) acetylation monitored during the early phase of heat stress, thereby promoting a prolonged duration of HSF1 binding to heat shock elements. Taken together, our results indicate that BGP-15 has the potential to become a new class of pharmaceuticals for use in 'membrane-lipid therapy' to combat many various protein-misfolding diseases associated with aging.

A novel application of the fluorescent dye bis-ANS for labeling neurons in acute brain slices.

The cell-impermeant oligomer-(e.g. beta-amyloid-, or tubulin-) specific fluorescent dye, bis-ANS (4,4'-bis-1-anilinonaphtalene-8-sulfonate), was successfully used for labeling mechanically damaged but still viable neuron bodies, neurites and neurite cross sections in acute brain slices. Acute hippocampal brain slices of rats were co-stained with bis-ANS and the cell-impermeant, DNA-specific dye propidium iodide (PI) and were then analyzed using fluorescence and confocal microscopes. Both the neuron bodies and the neurites were found to exhibit increased fluorescence intensities, suggesting that using this method they can be detected more easily. In addition, bis-ANS showed good region - but not cell specific co-localization with the neuron-specific fluorescent dye Dil (1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate). These two dyes label different neuronal structures: Dil binds specifically to intact cell membranes while bis-ANS can enter cells with compromised cell membranes and then stain the microtubules in the cytoplasm. For a quick (10min) staining of acute brain slices with bis-ANS both HEPES and NaHCO(3) were needed in order to achieve high signal intensity. Labeling with bis-ANS fluorescent dye is an easy method for imaging the neuronal structures on the surface of acute brain slices.

Fibrillar Abeta (1-42) enhances NMDA receptor sensitivity via the integrin signaling pathway.

The aggregated form of amyloid-beta (Abeta) (1-42) has been shown to increase N-methyl-D-aspartic acid (NMDA) evoked neuronal activity in vivo. Here we further characterized this phenomenon by investigating the role of integrin activation and downstream Src kinase activity using in vivo electrophysiology and in vitro intracellular Ca (2+) measurements. Pretreatment of differentiated SH-SY5Y cells with fibrillar Abeta (1-42) markedly enhanced the intracellular calcium increases caused by NMDA receptor (NMDA-R) stimulation. Function blocking antibody against beta1 integrin depressed the facilitatory effects of Abeta (1-42). Similarly, Abeta (1-42) facilitated NMDA-R driven firing of hippocampal neurons in vivo, and this effect was reduced by neutralizing antibody against beta1 integrins. The positive action of Abeta (1-42) on NMDA-R dependent responses was also depressed by an inhibitor known to block Src kinase. These results support the hypothesis that aggregated Abeta (1-42) is recognized by the beta1 subunit containing integrins and may induce a Src kinase dependent NMDA receptor phosphorylation.

Differences between normal and alpha-synuclein overexpressing SH-SY5Y neuroblastoma cells after Abeta(1-42) and NAC treatment.

Alpha-synuclein (alphaSN) plays a major role in numerous neurodegenerative disorders, such as Alzheimer's disease and Parkinson's disease. Intracellular inclusions containing aggregated alphaSN have been reported in Alzheimer's and Parkinson's affected brains. Moreover, a proteolytic fragment of alphaSN, the so-called non-amyloid component of Alzheimer's disease amyloid (NAC) was found to be an integral part of Alzheimer's dementia related plaques. Despite the extensive research on this topic, the exact toxic mechanism of alphaSN remains elusive. We have taken the advantage of an alphaSN overexpressing SH-SY5Y cell line and investigated the effects of classical apoptotic factors (e.g. H(2)O(2), amphotericin B and ruthenium red) and aggregated disease-related peptides on cell viability compared to wild type neuroblastoma cells. It was found that alphaSN overexpressing cells are more sensitive to aggregated peptides treatment than normal expressing counterparts. In contrast, cells containing elevated amount of alphaSN were less vulnerable to classical apoptotic stressors than wild type cells. In addition, alphaSN overexpression is accompanied by altered phenotype, attenuated proliferation kinetics, increased neurite arborisation and decreased cell motility. Based on these results, the alphaSN overexpressing cell lines may represent a good and effective in vitro model for Alzheimer's and Parkinson's disease.

A novel and simple fluorescence method for the measurement of presynaptic vesicular zinc release in acute hippocampal slices with a fluorescence plate reader.

The synaptic vesicles in the hippocampal neuronal terminals are abundantly supplied with zinc ions (Zn2+), which can be released into the synaptic cleft. In the glutamatergic systems (e.g. the hippocampus and the amygdala), the vesicular Zn2+ is co-localized with glutamate (Glu). Glu functions as a neurotransmitter, and Zn2+ as a neuromodulator (effecting basic synaptic functions). Electrical stimulation or chemical treatment (e.g. KCl) of hippocampal slices evokes the release of presynaptic vesicular Zn2+ into the synapse, together with Glu. This paper reports on the development of a rapid and simple method with which to assess the vesicular Zn2+ release and the effects of Zn2+-binding chelators in rat acute hippocampal slices. This method uses a 96-well fluorescence plate reader and the well-known zinc-sensitive fluorescence dye, FluoZin-3, which is cell-impermeable. This dye forms a stable complex with Zn2+ (Kd = 15 nM at pH 7.4); the amount of Zn2+ can be measured by fluorometry (lambda ex. 480-485 nm, em. 520-535 nm). Using 96-well plates, we could measure the Zn2+ release with high sensitivity, in at most 10 slices with a 15-s cycle time. This novel method can readily be used for the ex vivo modelling of the stress-evoked neuronal presynaptic Zn2+ release characteristic of neurodegenerative processes (e.g. Alzheimer's disease), or for the testing of Zn2+ chelators including putative drug candidates. This novel fluorescence plate reader method offers a simple, rapid and cost-effective technique for the measurement of vesicular Zn2+ release. It permits the simultaneous measurement of all mechanically undamaged hippocampal slices, regardless of size, thereby reducing the number of rats required experimentally.