Characterization of obesity-related diseases and inflammation using single cell immunophenotyping in two different diet-induced obesity models.

BACKGROUND: Obesity is a growing problem worldwide and a major risk factor for many chronic diseases. The accumulation of adipose tissue leads to the release of significant amounts of pro-inflammatory cytokines and adipokines, resulting in a low-grade systemic inflammation. However, the mechanisms behind the development of obesity-related diseases are not fully understood. Therefore, our study aimed to investigate the pathological changes and inflammatory processes at systemic level and in individual organs in two different diet-induced mouse obesity models. METHODS: Male C57BL6/J mice were fed by high-fat diet (HFD), high-fat/high-fructose diet (HFD + FR) or normal chow for 21 weeks starting at 3 months of age (n = 15 animals/group). Insulin resistance was tested by oral glucose tolerance test. Pathological changes were investigated on hematoxylin-eosin-stained liver and brown adipose tissue sections. The gene expression levels of adipokines and cytokines were analyzed by qPCR in adipose tissues, whereas serum protein concentrations were determined by multiplex immunoassays. Immunophenotyping of isolated blood, bone marrow and spleen cells was performed by single-cell mass cytometry. RESULTS: Weight gain, glucose intolerance and hepatic steatosis were more severe in the HFD + FR group than in the control and HFD groups. This was accompanied by a higher level of systemic inflammation, as indicated by increased expression of pro-inflammatory genes in visceral white adipose tissue and by a higher serum TNFα level. In addition, immunophenotyping revealed the increase of the surface expressions of CD44 and CD69 on various cell types, such as CD8+ and CD4 + T-cells, B-cells and macrophages, in animals with obesity. CONCLUSIONS: The combination of HFD with fructose supplementation promotes more properly the symptoms of metabolic syndrome. Therefore, the combined high-fat/high-fructose nutrition can be a more suitable model of the Western diet. However, despite these differences, both models showed immunophenotypic changes that may be associated with increased risk of obesity-related cancer.

Mild Hyperthermia-Induced Thermogenesis in the Endoplasmic Reticulum Defines Stress Response Mechanisms.

Previous studies reported that a mild, non-protein-denaturing, fever-like temperature increase induced the unfolded protein response (UPR) in mammalian cells. Our dSTORM super-resolution microscopy experiments revealed that the master regulator of the UPR, the IRE1 (inositol-requiring enzyme 1) protein, is clustered as a result of UPR activation in a human osteosarcoma cell line (U2OS) upon mild heat stress. Using ER thermo yellow, a temperature-sensitive fluorescent probe targeted to the endoplasmic reticulum (ER), we detected significant intracellular thermogenesis in mouse embryonic fibroblast (MEF) cells. Temperatures reached at least 8 °C higher than the external environment (40 °C), resulting in exceptionally high ER temperatures similar to those previously described for mitochondria. Mild heat-induced thermogenesis in the ER of MEF cells was likely due to the uncoupling of the Ca2+/ATPase (SERCA) pump. The high ER temperatures initiated a pronounced cytosolic heat-shock response in MEF cells, which was significantly lower in U2OS cells in which both the ER thermogenesis and SERCA pump uncoupling were absent. Our results suggest that depending on intrinsic cellular properties, mild hyperthermia-induced intracellular thermogenesis defines the cellular response mechanism and determines the outcome of hyperthermic stress.

The Crystal Structure of the Hsp90-LA1011 Complex and the Mechanism by Which LA1011 May Improve the Prognosis of Alzheimer's Disease.

Functional changes in chaperone systems play a major role in the decline of cognition and contribute to neurological pathologies, such as Alzheimer's disease (AD). While such a decline may occur naturally with age or with stress or trauma, the mechanisms involved have remained elusive. The current models suggest that amyloid-ß (Aß) plaque formation leads to the hyperphosphorylation of tau by a Hsp90-dependent process that triggers tau neurofibrillary tangle formation and neurotoxicity. Several co-chaperones of Hsp90 can influence the phosphorylation of tau, including FKBP51, FKBP52 and PP5. In particular, elevated levels of FKBP51 occur with age and stress and are further elevated in AD. Recently, the dihydropyridine LA1011 was shown to reduce tau pathology and amyloid plaque formation in transgenic AD mice, probably through its interaction with Hsp90, although the precise mode of action is currently unknown. Here, we present a co-crystal structure of LA1011 in complex with a fragment of Hsp90. We show that LA1011 can disrupt the binding of FKBP51, which might help to rebalance the Hsp90-FKBP51 chaperone machinery and provide a favourable prognosis towards AD. However, without direct evidence, we cannot completely rule out effects on other Hsp90-co-chaprone complexes and the mechanisms they are involved in, including effects on Hsp90 client proteins. Nonetheless, it is highly significant that LA1011 showed promise in our previous AD mouse models, as AD is generally a disease affecting older patients, where slowing of disease progression could result in AD no longer being life limiting. The clinical value of LA1011 and its possible derivatives thereof remains to be seen.

Development of a Laser Microdissection-Coupled Quantitative Shotgun Lipidomic Method to Uncover Spatial Heterogeneity.

Lipid metabolic disturbances are associated with several diseases, such as type 2 diabetes or malignancy. In the last two decades, high-performance mass spectrometry-based lipidomics has emerged as a valuable tool in various fields of biology. However, the evaluation of macroscopic tissue homogenates leaves often undiscovered the differences arising from micron-scale heterogeneity. Therefore, in this work, we developed a novel laser microdissection-coupled shotgun lipidomic platform, which combines quantitative and broad-range lipidome analysis with reasonable spatial resolution. The multistep approach involves the preparation of successive cryosections from tissue samples, cross-referencing of native and stained images, laser microdissection of regions of interest, in situ lipid extraction, and quantitative shotgun lipidomics. We used mouse liver and kidney as well as a 2D cell culture model to validate the novel workflow in terms of extraction efficiency, reproducibility, and linearity of quantification. We established that the limit of dissectible sample area corresponds to about ten cells while maintaining good lipidome coverage. We demonstrate the performance of the method in recognizing tissue heterogeneity on the example of a mouse hippocampus. By providing topological mapping of lipid metabolism, the novel platform might help to uncover region-specific lipidomic alterations in complex samples, including tumors.

Effect of HEAT therapy in patiEnts with type 2 Diabetes mellitus (HEATED): protocol for a randomised controlled trial.

INTRODUCTION: The burden of type 2 diabetes mellitus (T2DM) is increasing worldwide. Heat therapy has been found effective in improving glycaemic control. However, to date, there is a lack of randomised controlled studies investigating the efficacy of heat therapy in T2DM. Therefore, we aim to investigate whether heat therapy with natural thermal mineral water can improve glycaemic control in patients with T2DM. METHODS AND ANALYSIS: The HEAT therapy in patiEnts with type 2 Diabetes mellitus (HEATED) Study is a single-centre, two-arm randomised controlled trial being conducted at Harkány Thermal Rehabilitation Centre in Hungary. Patients with T2DM will be randomly assigned to group A (bath sessions in 38°C natural thermal mineral water) and group B (baths in thermoneutral water (30°C-32°C)). Both groups will complete a maximum of 5 weekly visits, averaging 50-60 visits over the 12-week study. Each session will last 30 min, with a physical check-up before the bath. At baseline, patients' T2DM status will be investigated thoroughly. Possible microvascular and macrovascular complications of T2DM will be assessed with physical and laboratory examinations. The short form-36 questionnaire will assess the quality of life. Patients will also be evaluated at weeks 4, 8 and 12. The primary endpoint will be the change of glycated haemoglobin from baseline to week 12. An estimated 65 patients will be enrolled per group, with a sample size re-estimation at the enrolment of 50% of the calculated sample size. ETHICS AND DISSEMINATION: The study has been approved by the Scientific and Research Ethics Committee of the Hungarian Medical Research Council (818-2/2022/EÜIG). Written informed consent is required from all participants. We will disseminate our results to the medical community and will publish our results in peer-reviewed journals. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, NCT05237219.

The Small Heat Shock Protein, HSPB1, Interacts with and Modulates the Physical Structure of Membranes.

Small heat shock proteins (sHSPs) have been demonstrated to interact with lipids and modulate the physical state of membranes across species. Through these interactions, sHSPs contribute to the maintenance of membrane integrity. HSPB1 is a major sHSP in mammals, but its lipid interaction profile has so far been unexplored. In this study, we characterized the interaction between HSPB1 and phospholipids. HSPB1 not only associated with membranes via membrane-forming lipids, but also showed a strong affinity towards highly fluid membranes. It participated in the modulation of the physical properties of the interacting membranes by altering rotational and lateral lipid mobility. In addition, the in vivo expression of HSPB1 greatly affected the phase behavior of the plasma membrane under membrane fluidizing stress conditions. In light of our current findings, we propose a new function for HSPB1 as a membrane chaperone.

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.

Multifaceted Analyses of Isolated Mitochondria Establish the Anticancer Drug 2-Hydroxyoleic Acid as an Inhibitor of Substrate Oxidation and an Activator of Complex IV-Dependent State 3 Respiration.

The synthetic fatty acid 2-hydroxyoleic acid (2OHOA) has been extensively investigated as a cancer therapy mainly based on its regulation of membrane lipid composition and structure, activating various cell fate pathways. We discovered, additionally, that 2OHOA can uncouple oxidative phosphorylation, but this has never been demonstrated mechanistically. Here, we explored the effect of 2OHOA on mitochondria isolated by ultracentrifugation from U118MG glioblastoma cells. Mitochondria were analyzed by shotgun lipidomics, molecular dynamic simulations, spectrophotometric assays for determining respiratory complex activity, mass spectrometry for assessing beta oxidation and Seahorse technology for bioenergetic profiling. We showed that the main impact of 2OHOA on mitochondrial lipids is their hydroxylation, demonstrated by simulations to decrease co-enzyme Q diffusion in the liquid disordered membranes embedding respiratory complexes. This decreased co-enzyme Q diffusion can explain the inhibition of disjointly measured complexes I-III activity. However, it doesn't explain how 2OHOA increases complex IV and state 3 respiration in intact mitochondria. This increased respiration probably allows mitochondrial oxidative phosphorylation to maintain ATP production against the 2OHOA-mediated inhibition of glycolytic ATP production. This work correlates 2OHOA function with its modulation of mitochondrial lipid composition, reflecting both 2OHOA anticancer activity and adaptation to it by enhancement of state 3 respiration.

Lipids and Trehalose Actively Cooperate in Heat Stress Management of Schizosaccharomyces pombe.

Homeostatic maintenance of the physicochemical properties of cellular membranes is essential for life. In yeast, trehalose accumulation and lipid remodeling enable rapid adaptation to perturbations, but their crosstalk was not investigated. Here we report about the first in-depth, mass spectrometry-based lipidomic analysis on heat-stressed Schizosaccharomyces pombe mutants which are unable to synthesize (tps1Δ) or degrade (ntp1Δ) trehalose. Our experiments provide data about the role of trehalose as a membrane protectant in heat stress. We show that under conditions of trehalose deficiency, heat stress induced a comprehensive, distinctively high-degree lipidome reshaping in which structural, signaling and storage lipids acted in concert. In the absence of trehalose, membrane lipid remodeling was more pronounced and increased with increasing stress dose. It could be characterized by decreasing unsaturation and increasing acyl chain length, and required de novo synthesis of stearic acid (18:0) and very long-chain fatty acids to serve membrane rigidification. In addition, we detected enhanced and sustained signaling lipid generation to ensure transient cell cycle arrest as well as more intense triglyceride synthesis to accommodate membrane lipid-derived oleic acid (18:1) and newly synthesized but unused fatty acids. We also demonstrate that these changes were able to partially substitute for the missing role of trehalose and conferred measurable stress tolerance to fission yeast cells.

Heat therapy shows benefit in patients with type 2 diabetes mellitus: a systematic review and meta-analysis.

AIMS: Type-2 diabetes mellitus (T2DM) is a common health condition which prevalence increases with age. Besides lifestyle modifications, passive heating could be a promising intervention to improve glycemic control. This study aimed to assess the efficacy of passive heat therapy on glycemic and cardiovascular parameters, and body weight among patients with T2DM. METHODS: A systematic review and meta-analysis were reported according to PRISMA Statement. We conducted a systematic search in three databases (MEDLINE, Embase, CENTRAL) from inception to 19 August 2021. We included interventional studies reporting on T2DM patients treated with heat therapy. The main outcomes were the changes in pre-and post-treatment cardiometabolic parameters (fasting plasma glucose, glycated plasma hemoglobin, and triglyceride). For these continuous variables, weighted mean differences (WMD) with 95% confidence intervals (CIs) were calculated. Study protocol number: CRD42020221500. RESULTS: Five studies were included in the qualitative and quantitative synthesis, respectively. The results showed a not significant difference in the hemoglobin A1c [WMD -0.549%, 95% CI (-1.262, 0.164), p = 0.131], fasting glucose [WMD -0.290 mmol/l, 95% CI (-0.903, 0.324), p = 0.355]. Triglyceride [WMD 0.035 mmol/l, 95% CI (-0.130, 0.200), p = 0.677] levels were comparable regarding the pre-, and post intervention values. CONCLUSION: Passive heating can be beneficial for patients with T2DM since the slight improvement in certain cardiometabolic parameters support that. However, further randomized controlled trials with longer intervention and follow-up periods are needed to confirm the beneficial effect of passive heat therapy.

Male and Female Animals Respond Differently to High-Fat Diet and Regular Exercise Training in a Mouse Model of Hyperlipidemia.

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.

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.

Poly(ADP-ribose) polymerase-2 is a lipid-modulated modulator of muscular lipid homeostasis.

There is a growing body of evidence that poly(ADP-ribose) polymerase-2 (PARP2), although originally described as a DNA repair protein, has a widespread role as a metabolic regulator. We show that the ablation of PARP2 induced characteristic changes in the lipidome. The silencing of PARP2 induced the expression of sterol regulatory element-binding protein-1 and -2 and initiated de novo cholesterol biosynthesis in skeletal muscle. Increased muscular cholesterol was shunted to muscular biosynthesis of dihydrotestosterone, an anabolic steroid. Thus, skeletal muscle fibers in PARP2-/- mice were stronger compared to those of their wild-type littermates. In addition, we detected changes in the dynamics of the cell membrane, suggesting that lipidome changes also affect the biophysical characteristics of the cell membrane. In in silico and wet chemistry studies, we identified lipid species that can decrease the expression of PARP2 and potentially phenocopy the genetic abruption of PARP2, including artificial steroids. In view of these observations, we propose a new role for PARP2 as a lipid-modulated regulator of lipid metabolism.

Dihydropyridines Allosterically Modulate Hsp90 Providing a Novel Mechanism for Heat Shock Protein Co-induction and Neuroprotection.

Chaperones play a pivotal role in protein homeostasis, but with age their ability to clear aggregated and damaged protein from cells declines. Tau pathology is a driver of a variety of neurodegenerative disease and in Alzheimer's disease (AD) it appears to be precipitated by the formation of amyloid-ß (Aß) aggregates. Aß-peptide appears to trigger Tau hyperphosphorylation, formation of neurofibrillary tangles and neurotoxicity. Recently, dihydropyridine derivatives were shown to upregulate the heat shock response (HSR) and provide a neuroprotective effect in an APPxPS1 AD mouse model. The HSR response was only seen in diseased cells and consequently these compounds were defined as co-inducers since they upregulate chaperones and co-chaperones only when a pathological state is present. We show for compounds tested herein, that they target predominantly the C-terminal domain of Hsp90, but show some requirement for its middle-domain, and that binding stimulates the chaperones ATPase activity. We identify the site for LA1011 binding and confirm its identification by mutagenesis. We conclude, that binding compromises Hsp90's ability to chaperone, by modulating its ATPase activity, which consequently induces the HSR in diseased cells. Collectively, this represents the mechanism by which the normalization of neurofibrillary tangles, preservation of neurons, reduced tau pathology, reduced amyloid plaque, and increased dendritic spine density in the APPxPS1 Alzheimer's mouse model is initiated. Such dihydropyridine derivatives therefore represent potential pharmaceutical candidates for the therapy of neurodegenerative disease, such as AD.

Mild heat induces a distinct "eustress" response in Chinese Hamster Ovary cells but does not induce heat shock protein synthesis.

The current research on cellular heat stress management focuses on the roles of heat shock proteins (HSPs) and the proteostasis network under severe stress conditions. The mild, fever-type stress and the maintenance of membrane homeostasis are less well understood. Herein, we characterized the acute effect of mild, fever-range heat shock on membrane organization, and HSP synthesis and localization in two mammalian cell lines, to delineate the role of membranes in the sensing and adaptation to heat. A multidisciplinary approach combining ultrasensitive fluorescence microscopy and lipidomics revealed the molecular details of novel cellular "eustress", when cells adapt to mild heat by maintaining membrane homeostasis, activating lipid remodeling, and redistributing chaperone proteins. Notably, this leads to acquired thermotolerance in the complete absence of the induction of HSPs. At higher temperatures, additional defense mechanisms are activated, including elevated expression of molecular chaperones, contributing to an extended stress memory and acquired thermotolerance.

Metabolic crosstalk between membrane and storage lipids facilitates heat stress management in Schizosaccharomyces pombe.

Cell membranes actively participate in stress sensing and signalling. Here we present the first in-depth lipidomic analysis to characterize alterations in the fission yeast Schizosaccharomyces pombe in response to mild heat stress (HS). The lipidome was assessed by a simple one-step methanolic extraction. Genetic manipulations that altered triglyceride (TG) content in the absence or presence of HS gave rise to distinct lipidomic fingerprints for S. pombe. Cells unable to produce TG demonstrated long-lasting growth arrest and enhanced signalling lipid generation. Our results reveal that metabolic crosstalk between membrane and storage lipids facilitates homeostatic maintenance of the membrane physical/chemical state that resists negative effects on cell growth and viability in response to HS. We propose a novel stress adaptation mechanism in which heat-induced TG synthesis contributes to membrane rigidization by accommodating unsaturated fatty acids of structural lipids, enabling their replacement by newly synthesized saturated fatty acids.

Seasonal Variation of Eutrophication in Some Lakes of Danube Delta Biosphere Reserve.

To understand the trophic state of lakes, this study aims to determine the dynamics of phytoplankton assemblages and the main factors that influence their seasonal variation. Sampling campaigns were carried out in three lakes from the Danube Delta Biosphere Reserve. Spectral analysis of specific phytoplankton pigments was applied as a diagnostic marker to establish the distribution and composition of phytoplankton taxonomic groups. Fluorescence spectroscopy was used to quantify changes in dissolved organic matter (DOM). The relative contribution of the main phytoplankton groups to the total phytoplankton biomass and the trend of development during succession of the seasons showed that cyanobacteria could raise potential ecological or human health problems. Moreover, fluorescence spectroscopy revealed that Cryptophyta and cyanobacteria were the main contributors to the protein-like components of DOM. It was concluded that fluorescence could be used to provide a qualitative evaluation of the eutrophication degree in Danube Delta lakes.

The central role of heat shock factor 1 in synaptic fidelity and memory consolidation.

Networks of neuronal synapses are the fundamental basis for making and retaining memory. Reduced synapse number and quality correlates with loss of memory in dementia. Heat shock factor 1 (HSF1), the major transcription factor regulating expression of heat shock genes, plays a central role in proteostasis, in establishing and sustaining synaptic fidelity and function, and in memory consolidation. Support for this thesis is based on these observations: (1) heat shock induces improvements in synapse integrity and memory consolidation; (2) synaptic depolarization activates HSF1; (3) activation of HSF1 alone (independent of the canonical heat shock response) augments formation of essential synaptic elements-neuroligands, vesicle transport, synaptic scaffolding proteins, lipid rafts, synaptic spines, and axodendritic synapses; (4) HSF1 coalesces and activates memory receptors in the post-synaptic dendritic spine; (5) huntingtin or α-synuclein accumulation lowers HSF1 while HSF1 lowers huntingtin and α-synuclein aggregation-a potential vicious cycle; and (6) HSF1 agonists (including physical activity) can improve cognitive function in dementia models. Thus, via direct gene expression of synaptic elements, production of HSPs that assure high protein fidelity, and activation of other neuroprotective signaling pathways, HSF1 agonists could provide breakthrough therapy for dementia-associated disease.

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.

Oxidized Phospholipids Inhibit the Formation of Cholesterol-Dependent Plasma Membrane Nanoplatforms.

We previously developed a single-molecule microscopy method termed TOCCSL (thinning out clusters while conserving stoichiometry of labeling), which allows for direct imaging of stable nanoscopic platforms with raft-like properties diffusing in the plasma membrane. As a consensus raft marker, we chose monomeric GFP linked via a glycosylphosphatidylinositol (GPI) anchor to the cell membrane (mGFP-GPI). With this probe, we previously observed cholesterol-dependent homo-association to nanoplatforms diffusing in the plasma membrane of live CHO cells. Here, we report the release of this homo-association upon addition of 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC) or 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine, two oxidized phospholipids (oxPLs) that are typically present in oxidatively modified low-density lipoprotein. We found a dose-response relationship for mGFP-GPI nanoplatform disintegration upon addition of POVPC, correlating with the signal of the apoptosis marker Annexin V-Cy3. Similar concentrations of lysolipid showed no effect, indicating that the observed phenomena were not linked to properties of the lipid bilayer itself. Inhibition of acid sphingomyelinase by NB-19 before addition of POVPC completely abolished nanoplatform disintegration by oxPLs. In conclusion, we were able to determine how oxidized lipid species disrupt mGFP-GPI nanoplatforms in the plasma membrane. Our results favor an indirect mechanism involving acid sphingomyelinase activity rather than a direct interaction of oxPLs with nanoplatform constituents.