Plectin plays a role in the migration and volume regulation of astrocytes: a potential biomarker of glioblastoma.

BACKGROUND: The expression of aquaporin 4 (AQP4) and intermediate filament (IF) proteins is altered in malignant glioblastoma (GBM), yet the expression of the major IF-based cytolinker, plectin (PLEC), and its contribution to GBM migration and invasiveness, are unknown. Here, we assessed the contribution of plectin in affecting the distribution of plasmalemmal AQP4 aggregates, migratory properties, and regulation of cell volume in astrocytes. METHODS: In human GBM, the expression of glial fibrillary acidic protein (GFAP), AQP4 and PLEC transcripts was analyzed using publicly available datasets, and the colocalization of PLEC with AQP4 and with GFAP was determined by immunohistochemistry. We performed experiments on wild-type and plectin-deficient primary and immortalized mouse astrocytes, human astrocytes and permanent cell lines (U-251 MG and T98G) derived from a human malignant GBM. The expression of plectin isoforms in mouse astrocytes was assessed by quantitative real-time PCR. Transfection, immunolabeling and confocal microscopy were used to assess plectin-induced alterations in the distribution of the cytoskeleton, the influence of plectin and its isoforms on the abundance and size of plasmalemmal AQP4 aggregates, and the presence of plectin at the plasma membrane. The release of plectin from cells was measured by ELISA. The migration and dynamics of cell volume regulation of immortalized astrocytes were assessed by the wound-healing assay and calcein labeling, respectively. RESULTS: A positive correlation was found between plectin and AQP4 at the level of gene expression and protein localization in tumorous brain samples. Deficiency of plectin led to a decrease in the abundance and size of plasmalemmal AQP4 aggregates and altered distribution and bundling of the cytoskeleton. Astrocytes predominantly expressed P1c, P1e, and P1g plectin isoforms. The predominant plectin isoform associated with plasmalemmal AQP4 aggregates was P1c, which also affected the mobility of astrocytes most prominently. In the absence of plectin, the collective migration of astrocytes was impaired and the dynamics of cytoplasmic volume changes in peripheral cell regions decreased. Plectin's abundance on the plasma membrane surface and its release from cells were increased in the GBM cell lines. CONCLUSIONS: Plectin affects cellular properties that contribute to the pathology of GBM. The observed increase in both cell surface and released plectin levels represents a potential biomarker and therapeutic target in the diagnostics and treatment of GBMs.

Immunophenotypes of anti-SARS-CoV-2 responses associated with fatal COVID-19.

Background: The relationship between anti-SARS-CoV-2 humoral immune response, pathogenic inflammation, lymphocytes and fatal COVID-19 is poorly understood. Methods: A longitudinal prospective cohort of hospitalised patients with COVID-19 (n=254) was followed up to 35 days after admission (median, 8 days). We measured early anti-SARS-CoV-2 S1 antibody IgG levels and dynamic (698 samples) of quantitative circulating T-, B- and natural killer lymphocyte subsets and serum interleukin-6 (IL-6) response. We used machine learning to identify patterns of the immune response and related these patterns to the primary outcome of 28-day mortality in analyses adjusted for clinical severity factors. Results: Overall, 45 (18%) patients died within 28 days after hospitalisation. We identified six clusters representing discrete anti-SARS-CoV-2 immunophenotypes. Clusters differed considerably in COVID-19 survival. Two clusters, the anti-S1-IgGlowestTlowestBlowestNKmodIL-6mod, and the anti-S1-IgGhighTlowBmodNKmodIL-6highest had a high risk of fatal COVID-19 (HR 3.36-21.69; 95% CI 1.51-163.61 and HR 8.39-10.79; 95% CI 1.20-82.67; p≤0.03, respectively). The anti-S1-IgGhighestTlowestBmodNKmodIL-6mod and anti-S1-IgGlowThighestBhighestNKhighestIL-6low cluster were associated with moderate risk of mortality. In contrast, two clusters the anti-S1-IgGhighThighBmodNKmodIL-6low and anti-S1-IgGhighestThighestBhighNKhighIL-6lowest clusters were characterised by a very low risk of mortality. Conclusions: By employing unsupervised machine learning we identified multiple anti-SARS-CoV-2 immune response clusters and observed major differences in COVID-19 mortality between these clusters. Two discrete immune pathways may lead to fatal COVID-19. One is driven by impaired or delayed antiviral humoral immunity, independently of hyper-inflammation, and the other may arise through excessive IL-6-mediated host inflammation response, independently of the protective humoral response. Those observations could be explored further for application in clinical practice.

Length of Hospital Stay and Survival of Hospitalized COVID-19 Patients During the Second Wave of the Pandemic: A Single Centre Retrospective Study from Slovenia.

Background: As of writing, there are no publications pertaining to the prediction of COVID-19-related outcomes and length of stay in patients from Slovene hospitals. Objectives: To evaluate the length of regular ward and ICU stays and assess the survival of COVID-19 patients to develop better prediction models to forecast hospital capacity and staffing demands in possible further pandemic peaks. Methods: In this retrospective, single-site study we analysed the length of stay and survival of all patients, hospitalized due to the novel coronavirus (COVID-19) at the peak of the second wave, between November 18th 2020 and January 27th 2021 at the University Clinic Golnik, Slovenia. Results: Out of 407 included patients, 59% were male. The median length of stay on regular wards was 7.5 (IQR 5-13) days, and the median ICU length of stay was 6 (IQR 4-11) days. Age, male sex, and ICU stay were significantly associated with a higher risk of death. The probability of dying in 21 days at the regular ward was 14.4% (95% CI [10.9-18%]) and at the ICU it was 43.6% (95% CI [19.3-51.8%]). Conclusion: The survival of COVID-19 is strongly affected by age, sex, and the fact that a patient had to be admitted to ICU, while the length of hospital bed occupancy is very similar across different demographic groups. Knowing the length of stay and admission rate to ICU is important for proper planning of resources during an epidemic.

Vesicle cholesterol controls exocytotic fusion pore.

In some lysosomal storage diseases (LSD) cholesterol accumulates in vesicles. Whether increased vesicle cholesterol affects vesicle fusion with the plasmalemma, where the fusion pore, a channel between the vesicle lumen and the extracellular space, is formed, is unknown. Super-resolution microscopy revealed that after stimulation of exocytosis, pituitary lactotroph vesicles discharge cholesterol which transfers to the plasmalemma. Cholesterol depletion in lactotrophs and astrocytes, both exhibiting Ca2+-dependent exocytosis regulated by distinct Ca2+sources, evokes vesicle secretion. Although this treatment enhanced cytosolic levels of Ca2+ in lactotrophs but decreased it in astrocytes, this indicates that cholesterol may well directly define the fusion pore. In an attempt to explain this mechanism, a new model of cholesterol-dependent fusion pore regulation is proposed. High-resolution membrane capacitance measurements, used to monitor fusion pore conductance, a parameter related to fusion pore diameter, confirm that at resting conditions reducing cholesterol increases, while enrichment with cholesterol decreases the conductance of the fusion pore. In resting fibroblasts, lacking the Npc1 protein, a cellular model of LSD in which cholesterol accumulates in vesicles, the fusion pore conductance is smaller than in controls, showing that vesicle cholesterol controls fusion pore and is relevant for pathophysiology of LSD.

AQP4e-Based Orthogonal Arrays Regulate Rapid Cell Volume Changes in Astrocytes.

Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. It is predominantly expressed in astrocytes at the blood-brain and blood-liquor interfaces. Although several AQP4 isoforms have been identified in the mammalian brain, two, AQP4a (M1) and AQP4c (M23), have been confirmed to cluster into plasma membrane supramolecular structures, termed orthogonal arrays of particles (OAPs) and to enhance water transport through the plasma membrane. However, the role of the newly described water-conductive mammalian isoform AQP4e is unknown. Here, the dynamics of AQP4e aggregation into OAPs and its role in the regulation of astrocyte water homeostasis have been studied. Using super-resolution structured illumination, atomic force, and confocal microscopies, the results revealed that, in female rat astrocytes, AQP4e isoform colocalizes with OAPs, affecting its structural dynamics. In hypoosmotic conditions, which elicit cell edema, OAP formation was considerably enhanced by overexpressed AQP4e. Moreover, the kinetics of the cell swelling and of the regulatory volume decrease was faster in astrocytes overexpressing AQP4e compared with untransfected controls. Furthermore, the increase in maximal cell volume elicited by hypoosmotic stimulation was significantly smaller in AQP4e-overexpressing astrocytes. For the first time, this study demonstrates an active role of AQP4e in the regulation of OAP structural dynamics and in water homeostasis.SIGNIFICANCE STATEMENT Water channel aquaporin 4 (AQP4) plays a key role in the regulation of water homeostasis in the brain. To date, only AQP4a and AQP4c isoforms have been confirmed to enhance water transport through plasmalemma and to cluster into orthogonal arrays of particles (OAPs). We here studied the dynamics, aggregation, and role in the regulation of astrocyte water homeostasis of the newly described water-conductive mammalian isoform AQP4e. Our main findings are as follows: brain edema mimicking hypoosmotic conditions stimulates the formation of new OAPs with larger diameters, due to the incorporation of additional cytoplasmic AQP4 channels and the redistribution of AQP4 channels of the existing OAPs; and AQP4e affects the dynamics of cell swelling and regulatory volume decrease in astrocytes exposed to hypoosmotic conditions.

Subanesthetic doses of ketamine stabilize the fusion pore in a narrow flickering state in astrocytes.

Ketamine is an anesthetic that exhibits analgesic, psychotomimetic, and rapid antidepressant effects that are of particular neuropharmacological interest. Recent studies revealed astrocytic Ca(2+) signaling and regulated exocytosis as ketamine-targeted processes. Thus high-resolution cell-attached membrane capacitance measurements were performed to examine the influence of ketamine on individual vesicle interactions with the plasma membrane in cultured rat astrocytes. Ketamine evoked long-lasting bursts of repetitive opening and closing of the fusion pore that were both time- and concentration-dependent. Moreover, acute application and subanesthetic doses of ketamine elicited a significant increase in the occurrence of bursts that were characterized by a decreased fusion pore conductance, indicating that the fusion pore was stabilized in a narrow configuration. The time- and concentration-dependent increase in burst occurrence was correlated with a decrease in full fission events. This study has demonstrated a novel effect of ketamine manifested as stabilization of a fusion pore incapable of transiting to full vesicle fission, suggestive of an inhibitory effect on vesicle retrieval. This until now unrecognized effect of ketamine on the vesicle fusion pore might play a role in astroglial release and (re)uptake of molecules, modulating synaptic activity. This study demonstrates a novel effect of ketamine on the fusion pore. High-resolution cell-attached membrane capacitance measurements revealed that ketamine evokes long-lasting flickering of a narrow fusion pore that is incapable of transiting to full fission. Astrocytic vesicle fusion/retrieval modified by subanesthetic ketamine doses most likely affects gliotransmission and indicates a non-neuronal mechanism of ketamine action that may contribute to its behavioral effects.

Local electrostatic interactions determine the diameter of fusion pores.

In regulated exocytosis vesicular and plasma membranes merge to form a fusion pore in response to stimulation. The nonselective cation HCN channels are involved in the regulation of unitary exocytotic events by at least 2 mechanisms. They can affect SNARE-dependent exocytotic activity indirectly, via the modulation of free intracellular calcium; and/or directly, by altering local cation concentration, which affects fusion pore geometry likely via electrostatic interactions. By monitoring membrane capacitance, we investigated how extracellular cation concentration affects fusion pore diameter in pituitary cells and astrocytes. At low extracellular divalent cation levels predominantly transient fusion events with widely open fusion pores were detected. However, fusion events with predominately narrow fusion pores were present at elevated levels of extracellular trivalent cations. These results show that electrostatic interactions likely help determine the stability of discrete fusion pore states by affecting fusion pore membrane composition.

Hyperpolarization-activated cyclic nucleotide-gated channels and cAMP-dependent modulation of exocytosis in cultured rat lactotrophs.

Hormone and neurotransmitter release from vesicles is mediated by regulated exocytosis, where an aqueous channel-like structure, termed a fusion pore, is formed. It was recently shown that second messenger cAMP modulates the fusion pore, but the detailed mechanisms remain elusive. In this study, we asked whether the hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, which are activated by cAMP, are involved in the regulation of unitary exocytic events. By using the Western blot technique, a real-time PCR, immunocytochemistry in combination with confocal microscopy, and voltage-clamp measurements of hyperpolarizing currents, we show that HCN channels are present in the plasma membrane and in the membrane of secretory vesicles of isolated rat lactotrophs. Single vesicle membrane capacitance measurements of lactotrophs, where HCN channels were either augmented by transfection or blocked with an HCN channel blocker (ZD7288), show modulated fusion pore properties. We suggest that the changes in local cation concentration, mediated through HCN channels, which are located on or near secretory vesicles, have an important role in modulating exocytosis.

Cholesterol-mediated membrane surface area dynamics in neuroendocrine cells.

How cholesterol, a key membrane constituent, affects membrane surface area dynamics in secretory cells is unclear. Using methyl-beta-cyclodextrin (MbetaCD) to deplete cholesterol, we imaged melanotrophs from male Wistar rats in real-time and monitored membrane capacitance (C(m)), fluctuations of which reflect exocytosis and endocytosis. Treatment with MbetaCD reduced cellular cholesterol and caused a dose-dependent attenuation of the Ca(2+)-evoked increase in C(m) (IC50 = 5.3 mM) vs. untreated cells. Cytosol dialysis of MbetaCD enhanced the attenuation of C(m) increase (IC50 = 3.3 mM), suggesting cholesterol depletion at intracellular membrane sites was involved in attenuating exocytosis. Acute extracellular application of MbetaCD resulted in an immediate C(m) decline, which correlated well with the cellular surface area decrease, indicating the involvement of cholesterol in the regulation of membrane surface area dynamics. This decline in C(m) was three-fold slower than MbetaCD-mediated fluorescent cholesterol decay, implying that exocytosis is the likely physiological means for plasma membrane cholesterol replenishment. MbetaCD had no effect on the specific C(m) and the blockade of endocytosis by Dyngo 4a, confirmed by inhibition of dextran uptake, also had no effect on the time-course of MbetaCD-induced C(m) decline. Thus acute exposure to MbetaCD evokes a C(m) decline linked to the removal of membrane cholesterol, which cannot be compensated for by exocytosis. We propose that the primary contribution of cholesterol to surface area dynamics is via its role in regulated exocytosis.

High-resolution membrane capacitance measurements for the study of exocytosis and endocytosis.

In order to understand exocytosis and endocytosis, it is necessary to study these processes directly. An elegant way to do this is by measuring plasma membrane capacitance (C(m)), a parameter proportional to cell surface area, the fluctuations of which are due to fusion and fission of secretory and other vesicles. Here we describe protocols that enable high-resolution C(m) measurements in macroscopic and microscopic modes. Macroscopic mode, performed in whole-cell configuration, is used for measuring bulk C(m) changes in the entire membrane area, and it enables the introduction of exocytosis stimulators or inhibitors into the cytosol through the patch pipette. Microscopic mode, performed in cell-attached configuration, enables measurements of C(m) with attofarad resolution and allows characterization of fusion pore properties. Although we usually apply these protocols to primary pituitary cells and astrocytes, they can be adapted and used for other cell types. After initial hardware setup and culture preparation, several C(m) measurements can be performed daily.

Cholesterol and regulated exocytosis: a requirement for unitary exocytotic events.

Since the 1970s, much effort was been expended researching mechanisms of regulated exocytosis. Early work focused mainly on the role of proteins. Most notably the discovery of SNARE proteins in the 1980s and the zippering hypothesis brought us much closer to understanding the complex interactions in membrane fusion between vesicle and plasma membranes, a pivotal component of regulated exocytosis. However, most likely due to the predictions of the Singer-Nicholson fluid mosaic membrane model, the lipid components of the exocytotic machinery remained largely overlooked. Lipids were considered passive constituents of cellular membranes, not contributing much, if anything, to the process of exocytosis and membrane fusion. Since the 1990s, this so-called proteocentric view has been gradually giving way to the new perspective best described with the term proteolipidic. Many lipids were found to be of great importance in the regulation of exocytosis. Here we highlight the role of cholesterol. Furthermore, by using high-resolution cell-attached membrane capacitance measurements, we have monitored unitary exocytotic events in cholesterol-depleted membranes. We show that the frequency of these events is attenuated, providing evidence at the single vesicle level that cholesterol directly influences the merger of the vesicle and the plasma membranes.

Hypotonicity and peptide discharge from a single vesicle.

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perfused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently labeled atrial natriuretic peptide cargo from approximately 2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of approximately 10% of vesicles/cell, with different unloading/loading time course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, transient vesicle fusion mediates hormone release.