Thiopurines impair the apical plasma membrane expression of CFTR in pancreatic ductal cells via RAC1 inhibition.

BACKGROUND AND AIMS: Thiopurine-induced acute pancreatitis (TIP) is one of the most common adverse events among inflammatory bowel disease patients treated with azathioprine (AZA), representing a significant clinical burden. Previous studies focused on immune-mediated processes, however, the exact pathomechanism of TIP is essentially unclear. METHODS: To model TIP in vivo, we triggered cerulein-induced experimental pancreatitis in mice receiving a daily oral dose of 1.5 mg/kg AZA. Also, freshly isolated mouse pancreatic cells were exposed to AZA ex vivo, and acinar cell viability, ductal and acinar Ca2+ signaling, ductal Cl- and HCO3- secretion, as well as cystic fibrosis transmembrane conductance regulator (CFTR) expression were assessed using microscopy techniques. Ras-related C3 botulinum toxin substrate (RAC1) activity was measured with a G-LISA assay. Super-resolution microscopy was used to determine protein colocalization. RESULTS: We demonstrated that AZA treatment increases tissue damage in the early phase of cerulein-induced pancreatitis in vivo. Also, both per os and ex vivo AZA exposure impaired pancreatic fluid and ductal HCO3- and Cl- secretion, but did not affect acinar cells. Furthermore, ex vivo AZA exposure also inhibited RAC1 activity in ductal cells leading to decreased co-localization of CFTR and the anchor protein ezrin, resulting in impaired plasma membrane localization of CFTR. CONCLUSIONS: AZA impaired the ductal HCO3- and Cl- secretion through the inhibition of RAC1 activity leading to diminished ezrin-CFTR interaction and disturbed apical plasma membrane expression of CFTR. We report a novel direct toxic effect of AZA on pancreatic ductal cells and suggest that the restoration of ductal function might help to prevent TIP in the future.

Prevalence and Determinants of Pain in Spinal Cord Injury During Initial Inpatient Rehabilitation: Data From the Dutch Spinal Cord Injury Database.

OBJECTIVE: To describe the prevalence and characteristics of spinal cord injury (SCI)-related pain during initial inpatient rehabilitation and to investigate relationships with demographic and lesion characteristics. DESIGN: Cohort during inpatient rehabilitation. SETTING: Eight specialized SCI rehabilitation centers in the Netherlands. PARTICIPANTS: Patients with newly acquired SCI admitted for inpatient rehabilitation between November 2013 and August 2019 (N=1432). INTERVENTIONS: Not applicable. MAIN OUTCOME MEASURES: Presence of pain at admission and discharge. Logistic regression analyses were used to study the prevalence of pain related to sex, age, etiology, completeness, and level of injury. RESULTS: Data from 1432 patients were available. Of these patients 64.6% were male, mean age was 56.8 years, 59.9% had a nontraumatic SCI, 63.9% were classified as American Spinal Cord Injury Association Impairment Scale (AIS) D and 56.5% had paraplegia. Prevalence of pain was 61.2% at admission (40.6% nociceptive pain [NocP], 30.2% neuropathic pain [NeuP], 5.4% other pain) and 51.5% at discharge (26.0% NocP, 31.4% NeuP, 5.7% other pain). Having NocP at admission was associated with traumatic SCI. AIS B had a lower risk of NocP than AIS D at admission. Having NocP at discharge was associated with female sex and traumatic SCI. AIS C had a lower risk of NocP at discharge than AIS D. Having NeuP at admission was associated with female sex. Having NeuP at discharge was associated with female sex, age younger than 65 years vs age older than 75 years and tetraplegia. CONCLUSIONS: SCI-related pain is highly prevalent during inpatient rehabilitation. Prevalence of NocP decreased during inpatient rehabilitation, and prevalence of NeuP stayed the same. Different patient and lesion characteristics were related to the presence of SCI-related pain. Healthcare professionals should be aware of these differences in screening patients on presence and development of pain during inpatient rehabilitation.

Bile acid- and ethanol-mediated activation of Orai1 damages pancreatic ductal secretion in acute pancreatitis.

Regardless of its aetiology, sustained intracellular Ca2+ overload is a well-known hallmark of acute pancreatitis (AP). Toxic Ca2+ elevation induces pancreatic ductal cell damage characterized by impaired ion and fluid secretion - essential to wash out the protein-rich fluid secreted by acinar cells while maintaining the alkaline intra-ductal pH under physiological conditions - and mitochondrial dysfunction. While prevention of ductal cell injury decreases the severity of AP, no specific drug target has yet been identified in the ductal cells. Although Orai1, a store-operated Ca2+ influx channel, is known to contribute to sustained Ca2+ overload in acinar cells, details concerning its expression and function in ductal cells are currently lacking. In this study, we demonstrate that functionally active Orai1 channels reside predominantly in the apical plasma membrane of pancreatic ductal cells. Selective CM5480-mediated Orai1 inhibition impairs Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. Furthermore, prevention of sustained extracellular Ca2+ influx protects ductal cell secretory function in vitro and decreases pancreatic ductal cell death. Finally, Orai1 inhibition partially restores and maintains proper exocrine pancreatic secretion in in vivo AP models. In conclusion, our results indicate that Orai1 inhibition prevents AP-related ductal cell function impairment and holds the potential of improving disease outcome. KEY POINTS: Sustained intracellular Ca2+ overload in pancreatic acinar and ductal cells is a hallmark of biliary and alcohol-induced acute pancreatitis, which leads to impaired ductal ion and fluid secretion. Orai1 is a plasma membrane Ca2+ channel that mediates extracellular Ca2+ influx upon endoplasmic reticulum Ca2+ depletion. Results showed that Orai1 is expressed on the luminal plasma membrane of the ductal cells and selective Orai1 inhibition impaired Stim1-dependent extracellular Ca2+ influx evoked by bile acids or ethanol combined with non-oxidative ethanol metabolites. The prevention of sustained extracellular Ca2+ influx protected ductal cell secretory functions in in vitro models and maintained exocrine pancreatic secretion in in vivo acute pancreatitis models. Orai1 inhibition prevents the bile acid- and alcohol-induced damage of the pancreatic ductal secretion and holds the potential of improving the outcome of acute pancreatitis.

Endoplasmic Reticulum-Plasma Membrane Contact Sites as an Organizing Principle for Compartmentalized Calcium and cAMP Signaling.

In eukaryotic cells, ultimate specificity in activation and action-for example, by means of second messengers-of the myriad of signaling cascades is primordial. In fact, versatile and ubiquitous second messengers, such as calcium (Ca2+) and cyclic adenosine monophosphate (cAMP), regulate multiple-sometimes opposite-cellular functions in a specific spatiotemporal manner. Cells achieve this through segregation of the initiators and modulators to specific plasma membrane (PM) subdomains, such as lipid rafts and caveolae, as well as by dynamic close contacts between the endoplasmic reticulum (ER) membrane and other intracellular organelles, including the PM. Especially, these membrane contact sites (MCSs) are currently receiving a lot of attention as their large influence on cell signaling regulation and cell physiology is increasingly appreciated. Depletion of ER Ca2+ stores activates ER membrane STIM proteins, which activate PM-residing Orai and TRPC Ca2+ channels at ER-PM contact sites. Within the MCS, Ca2+ fluxes relay to cAMP signaling through highly interconnected networks. However, the precise mechanisms of MCS formation and the influence of their dynamic lipid environment on their functional maintenance are not completely understood. The current review aims to provide an overview of our current understanding and to identify open questions of the field.

Heat Shock Proteins and Autophagy Pathways in Neuroprotection: from Molecular Bases to Pharmacological Interventions.

Neurodegenerative diseases (NDDs) such as Alzheimer's disease, Parkinson's disease and Huntington's disease (HD), amyotrophic lateral sclerosis, and prion diseases are all characterized by the accumulation of protein aggregates (amyloids) into inclusions and/or plaques. The ubiquitous presence of amyloids in NDDs suggests the involvement of disturbed protein homeostasis (proteostasis) in the underlying pathomechanisms. This review summarizes specific mechanisms that maintain proteostasis, including molecular chaperons, the ubiquitin-proteasome system (UPS), endoplasmic reticulum associated degradation (ERAD), and different autophagic pathways (chaperon mediated-, micro-, and macro-autophagy). The role of heat shock proteins (Hsps) in cellular quality control and degradation of pathogenic proteins is reviewed. Finally, putative therapeutic strategies for efficient removal of cytotoxic proteins from neurons and design of new therapeutic targets against the progression of NDDs are discussed.

Associations Between Self-Efficacy and Secondary Health Conditions in People Living With Spinal Cord Injury: A Systematic Review and Meta-Analysis.

OBJECTIVE: To describe the association between self-efficacy and secondary health conditions (SHCs) in people living with spinal cord injury (SCI). DATA SOURCES: PubMed, EMBASE, the Cochrane Library, and CINAHL were systematically searched from database inception to September 2016. STUDY SELECTION: Studies describing patients living with SCI in which self-efficacy was measured by a standardized questionnaire and an association was made with somatic or psychological SHCs. DATA EXTRACTION: An independent extraction by multiple observers was performed based on the Strengthening the Reporting of Observational Studies in Epidemiology statements checklist. A meta-analysis concerning the association between self-efficacy and SHCs in people with SCI was performed if a minimum of 4 comparable studies were available. DATA SYNTHESIS: Of 670 unique articles screened, 22 met the inclusion criteria. Seven of these 22 studies investigated associations between self-efficacy and somatic SHCs. Only a trend toward an association between higher self-efficacy and less pain, fatigue, number of SHCs, and limitations caused by SHCs was found. Twenty-one studies described the association between self-efficacy and psychological SHCs. All correlations of higher self-efficacy with fewer depressive (18 studies) and anxiety symptoms (7 studies) were significant, and meta-analysis showed a strong negative correlation of -.536 (-.584 to -.484) and -.493 (-.577 to -.399), respectively. A small number of studies (2) showed a trend toward a positive correlation between self-efficacy and quality of life. CONCLUSIONS: Self-efficacy is negatively associated with depressive and anxiety symptoms in SCI. Therefore, self-efficacy seems an important target in the rehabilitation of patients living with SCI. More research is necessary to clarify the associations between self-efficacy and somatic SHCs. Future research should also focus on different types of self-efficacy and their association with SHCs.

Plasma membranes as heat stress sensors: from lipid-controlled molecular switches to therapeutic applications.

The classic heat shock (stress) response (HSR) was originally attributed to protein denaturation. However, heat shock protein (Hsp) induction occurs in many circumstances where no protein denaturation is observed. Recently considerable evidence has been accumulated to the favor of the "Membrane Sensor Hypothesis" which predicts that the level of Hsps can be changed as a result of alterations to the plasma membrane. This is especially pertinent to mild heat shock, such as occurs in fever. In this condition the sensitivity of many transient receptor potential (TRP) channels is particularly notable. Small temperature stresses can modulate TRP gating significantly and this is influenced by lipids. In addition, stress hormones often modify plasma membrane structure and function and thus initiate a cascade of events, which may affect HSR. The major transactivator heat shock factor-1 integrates the signals originating from the plasma membrane and orchestrates the expression of individual heat shock genes. We describe how these observations can be tested at the molecular level, for example, with the use of membrane perturbers and through computational calculations. An important fact which now starts to be addressed is that membranes are not homogeneous nor do all cells react identically. Lipidomics and cell profiling are beginning to address the above two points. Finally, we observe that a deregulated HSR is found in a large number of important diseases where more detailed knowledge of the molecular mechanisms involved may offer timely opportunities for clinical interventions and new, innovative drug treatments. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.

Use and experienced effectiveness of non-pharmacological treatments for chronic spinal cord injury related pain in The Netherlands: A cross-sectional survey.

CONTEXT/OBJECTIVE: Chronic pain is a common secondary condition in spinal cord injury (SCI). Pharmacological interventions to reduce pain are associated with side effects. The reported effects of non-pharmacological treatments are unclear. This study aims to examine the self-reported presence and type of pain, and the use, effectiveness and side effects of non-pharmacological treatments for pain. DESIGN: Cross-sectional survey regarding SCI-related pain and non-pharmacological treatments. SETTING: Community, the Netherlands. PARTICIPANTS: Outpatients with SCI from two rehabilitation centers. INTERVENTIONS: Not applicable. OUTCOME MEASURES: Self-reported presence and type of pain, use, effectiveness and side effects of non-pharmacological treatments. RESULTS: A total of 371 patients (41.5%) returned the questionnaire. Median time since onset of SCI was 7 years. Pain following SCI was reported by 262 patients (70.6%). Neuropathic pain was reported most often (74.4%), followed by musculoskeletal pain (51.5%). Of patients with pain, 204 (77.9%) reported past or current use of non-pharmacological treatments. Non-pharmacological treatments used most were physiotherapy (67.6%), physical exercise (44.7%) and massage (22.5%). Of patients using non-pharmacological treatments, 152 patients (74.5%) reported the effect of their treatment. Most treatments for which the effect was reported, were described as moderately effective. Most side effects were reported for cannabis. CONCLUSION: Patients with SCI experiencing pain often use non-pharmacological treatments. Most treatments were described as moderately effective. Research on specific non-pharmacological treatments and different types of pain separately is needed to further determine the effectiveness of non-pharmacological treatments.

Membrane fluidity matters: hyperthermia from the aspects of lipids and membranes.

Hyperthermia is a promising treatment modality for cancer in combination both with radio- and chemotherapy. In spite of its great therapeutic potential, the underlying molecular mechanisms still remain to be clarified. Due to lipid imbalances and 'membrane defects' most of the tumour cells possess elevated membrane fluidity. However, further increasing membrane fluidity to sensitise to chemo- or radiotherapy could have some other effects. In fact, hyperfluidisation of cell membrane induced by membrane fluidiser initiates a stress response as the heat shock protein response, which may modulate positively or negatively apoptotic cell death. Overviewing some recent findings based on a technology allowing direct imaging of lipid rafts in live cells and lipidomics, novel aspects of the intimate relationship between the 'membrane stress' of tumour cells and the cellular heat shock response will be highlighted. Our findings lend support to both the importance of membrane remodelling and the release of lipid signals initiating stress protein response, which can operate in tandem to control the extent of the ultimate cellular thermosensitivity. Overall, we suggest that the fluidity variable of membranes should be used as an independent factor for predicting the efficacy of combinational cancer therapies.

Orai1 calcium channel inhibition prevents progression of chronic pancreatitis.

Patients with recurrent acute pancreatitis (RAP) are at significant risk of developing early chronic pancreatitis (CP), which progresses into irreversible, end-stage CP with severe symptoms. There is no specific therapy in RAP or in early CP that may hinder disease progression. The pathogenesis of CP is complex and involves interactions among multiple cell types, including pancreatic acinar, ductal, and stellate cells (PSC). Therefore, it is pivotal to identify common pathogenic pathways in these cells that could be targeted pharmacologically. The Orai1-mediated store-operated Ca2+ entry (SOCE) is a ubiquitous signaling mechanism that may become overactivated in pathological states resulting in intracellular Ca2+ overload. In this study, we used ex vivo and in vivo preclinical disease models to demonstrate that Orai1 inhibition prevents progression of RAP and early CP. The selective Orai1 inhibitor CM5480 restored the expression of SOCE-associated regulatory factor in acinar cells, prevented uncontrolled Ca2+ elevation, protected acinar and ductal functions, mitigated immune cell infiltration, and diminished PSC activation, proliferation, and migration. We suggest that the overactivation of Orai1 is a crucial pathogenetic event in the progression of early CP and that inhibition of Orai1 could prevent the development of end-stage CP.

Resistance training prevents deterioration in quadriceps muscle function during acute exacerbations of chronic obstructive pulmonary disease.

RATIONALE: Exacerbations of chronic obstructive pulmonary disease (COPD) acutely reduce skeletal muscle strength and result in long-term loss of functional capacity. OBJECTIVES: To investigate whether resistance training is feasible and safe and can prevent deteriorating muscle function during exacerbations of COPD. METHODS: Forty patients (FEV(1) 49 +/- 17% predicted) hospitalized with a severe COPD exacerbation were randomized to receive usual care or an additional resistance training program during the hospital admission. Patients were followed up for 1 month after discharge. Primary outcomes were quadriceps force and systemic inflammation. A muscle biopsy was taken in a subgroup of patients to assess anabolic and catabolic pathways. MEASUREMENTS AND MAIN RESULTS: Resistance training did not yield higher systemic inflammation as indicated by C-reactive protein levels and could be completed uneventfully. Enhanced quadriceps force was seen at discharge (+9.7 +/- 16% in the training group; -1 +/- 13% in control subjects; P = 0.05) and at 1 month follow-up in the patients who trained. The 6-minute walking distance improved after discharge only in the group who received resistance training (median 34; interquartile range, 14-61 m; P = 0.002). In a subgroup of patients a muscle biopsy showed a more anabolic status of skeletal muscle in patients who followed training. Myostatin was lower (P = 0.03) and the myogenin/MyoD ratio tended to be higher (P = 0.08) in the training group compared with control subjects. CONCLUSIONS: Resistance training is safe, successfully counteracts skeletal muscle dysfunction during acute exacerbations of COPD, and may up-regulate the anabolic milieu in the skeletal muscle. Clinical trial registered with www.clinicaltrials.gov (NCT00877084).

Gene expression profiling in vastus lateralis muscle during an acute exacerbation of COPD.

BACKGROUND/AIMS: The molecular mechanisms leading to loss in muscle force during an acute exacerbation in COPD patients are unknown. A cross-sectional study was designed to compare the gene expression profile of the vastus lateralis muscle in patients with an acute COPD exacerbation and in stable COPD patients. METHODS: Muscle biopsies were taken in 9 COPD patients with an exacerbation on day 4 of hospitalization and in 15 stable COPD patients. Micro-array was performed on an UniSet Human 20K Bioarray. RESULTS: Gene Ontology and Gene Set Enrichment Analysis of the microarray data revealed enrichment of 1) the ubiquitin-dependent protein catabolism, the induction of apoptosis and anti-apoptosis and the response to reactive oxygen species in the upregulated transcripts, and 2) the aspartate catabolism and the mitochondrial respiratory chain in the downregulated transcripts. Real Time PCR data confirmed 1) increased expression of MuRF1 and MAFbx, markers of the ubiquitin dependent catabolism pathway, and 2) decreased expression levels of COX6C, a marker of mitochondrial respiration. CONCLUSIONS: The present study suggests that multiple pathways leading to muscle atrophy and mitochondrial dysfunction are altered in the muscle during an acute exacerbation. Strategies limiting the loss of muscle function during an acute exacerbation need to be developed.

Modulation of Plasma Membrane Composition and Microdomain Organization Impairs Heat Shock Protein Expression in B16-F10 Mouse Melanoma Cells.

The heat shock response (HSR) regulates induction of stress/heat shock proteins (HSPs) to preserve proteostasis during cellular stress. Earlier, our group established that the plasma membrane (PM) acts as a sensor and regulator of HSR through changes in its microdomain organization. PM microdomains such as lipid rafts, dynamic nanoscale assemblies enriched in cholesterol and sphingomyelin, and caveolae, cholesterol-rich PM invaginations, constitute clustering platforms for proteins functional in signaling cascades. Here, we aimed to compare the effect of cyclodextrin (MßCD)- and nystatin-induced cholesterol modulations on stress-activated expression of the representative HSPs, HSP70, and HSP25 in mouse B16-F10 melanoma cells. Depletion of cholesterol levels with MßCD impaired the heat-inducibility of both HSP70 and HSP25. Sequestration of cholesterol with nystatin impaired the heat-inducibility of HSP25 but not of HSP70. Imaging fluorescent correlation spectroscopy marked a modulated lateral diffusion constant of fluorescently labelled cholesterol in PM during cholesterol deprived conditions. Lipidomics analysis upon MßCD treatment revealed, next to cholesterol reductions, decreased lysophosphatidylcholine and phosphatidic acid levels. These data not only highlight the involvement of PM integrity in HSR but also suggest that altered dynamics of specific cholesterol pools could represent a mechanism to fine tune HSP expression.

The Role of Lipids and Membranes in the Pathogenesis of Alzheimer's Disease: A Comprehensive View.

Lipids participate in Amyloid Precursor Protein (APP) trafficking and processing - important factors in the initiation of Alzheimer's disease (AD) pathogenesis and influence the formation of neurotoxic ß-amyloid (Aß) peptides. An important risk factor, the presence of ApoE4 protein in AD brain cells binds the lipids to AD. In addition, lipid signaling pathways have a crucial role in the cellular homeostasis and depend on specific protein-lipid interactions. The current review focuses on pathological alterations of membrane lipids (cholesterol, glycerophospholipids, sphingolipids) and lipid metabolism in AD and provides insight in the current understanding of biological membranes, their lipid structures and functions, as well as their role as potential therapeutic targets. Novel methods for studying the membrane structure and lipid composition will be reviewed in a broad sense whereas the use of lipid biomarkers for early diagnosis of AD will be shortly summarized. Interactions of Aß peptides with the cell membrane and different subcellular organelles are reviewed. Next, the details of the most important lipid signaling pathways, including the role of the plasma membrane as stress sensor and its therapeutic applications are given. 4-hydroxy-2-nonenal may play a special role in the initiation of the pathogenesis of AD and thus the "calpain-cathepsin hypothesis" of AD is highlighted. Finally, the most important lipid dietary factors and their possible use and efficacy in the prevention of AD are discussed.

Chaperone co-inducer BGP-15 inhibits histone deacetylases and enhances the heat shock response through increased chromatin accessibility.

Defects in cellular protein homeostasis are associated with many severe and prevalent pathological conditions such as neurodegenerative diseases, muscle dystrophies, and metabolic disorders. One way to counteract these defects is to improve the protein homeostasis capacity through induction of the heat shock response. Despite numerous attempts to develop strategies for chemical activation of the heat shock response by heat shock transcription factor 1 (HSF1), the underlying mechanisms of drug candidates' mode of action are poorly understood. To lower the threshold for the heat shock response activation, we used the chaperone co-inducer BGP-15 that was previously shown to have beneficial effects on several proteinopathic disease models. We found that BGP-15 treatment combined with heat stress caused a substantial increase in HSF1-dependent heat shock protein 70 (HSPA1A/B) expression already at a febrile range of temperatures. Moreover, BGP-15 alone inhibited the activity of histone deacetylases (HDACs), thereby increasing chromatin accessibility at multiple genomic loci including the stress-inducible HSPA1A. Intriguingly, treatment with well-known potent HDAC inhibitors trichostatin A and valproic acid enhanced the heat shock response and improved cytoprotection. These results present a new pharmacological strategy for restoring protein homeostasis by inhibiting HDACs, increasing chromatin accessibility, and lowering the threshold for heat shock response activation.

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.

Rac1 participates in thermally induced alterations of the cytoskeleton, cell morphology and lipid rafts, and regulates the expression of heat shock proteins in B16F10 melanoma cells.

Eukaryotic cells exhibit a characteristic response to hyperthermic treatment, involving morphological and cytoskeletal alterations and the induction of heat shock protein synthesis. Small GTPases of the Ras superfamily are known to serve as molecular switches which mediate responses to extracellular stimuli. We addressed here how small GTPase Rac1 integrates signals from heat stress and simultaneously induces various cellular changes in mammalian cells. As evidence that Rac1 is implicated in the heat shock response, we first demonstrated that both mild (41.5°C) and severe (43°C) heat shock induced membrane translocation of Rac1. Following inhibition of the activation or palmitoylation of Rac1, the size of its plasma membrane-bound pool was significantly decreased while the heat shock-induced alterations in the cytoskeleton and cell morphology were prevented. We earlier documented that the size distribution pattern of cholesterol-rich rafts is temperature dependent and hypothesized that this is coupled to the triggering mechanism of stress sensing and signaling. Interestingly, when plasma membrane localization of Rac1 was inhibited, a different and temperature independent average domain size was detected. In addition, inhibition of the activation or palmitoylation of Rac1 resulted in a strongly decreased expression of the genes of major heat shock proteins hsp25 and hsp70 under both mild and severe heat stress conditions.

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.

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.