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Kainate receptors (KARs) are key regulators of synaptic circuits by acting at pre- and postsynaptic sites through either ionotropic or metabotropic actions. KARs can be activated by kainate, a potent neurotoxin, which induces acute convulsions. Here, we report that the acute convulsive effect of kainate mostly depends on GluK2/GluK5 containing KARs. By contrast, the acute convulsive activity of pilocarpine and pentylenetetrazol is not alleviated in the absence of KARs. Unexpectedly, the genetic inactivation of GluK2 rather confers increased susceptibility to acute pilocarpine-induced seizures. The mechanism involves an enhanced excitability of GluK2−/− CA3 pyramidal cells compared with controls upon pilocarpine application. Finally, we uncover that the absence of GluK2 increases pilocarpine modulation of Kv7/M currents. Taken together, our findings reveal that GluK2-containing KARs can control the excitability of hippocampal circuits through interaction with the neuromodulatory cholinergic system.

The lysine acetyltransferases type 3 (KAT3) family members CBP and p300 are important transcriptional co-activators, but their specific functions in adult post-mitotic neurons remain unclear. Here, we show that the combined elimination of both proteins in forebrain excitatory neurons of adult mice resulted in a rapidly progressing neurological phenotype associated with severe ataxia, dendritic retraction and reduced electrical activity. At the molecular level, we observed the downregulation of neuronal genes, as well as decreased H3K27 acetylation and pro-neural transcription factor binding at the promoters and enhancers of canonical neuronal genes. The combined deletion of CBP and p300 in hippocampal neurons resulted in the rapid loss of neuronal molecular identity without de- or transdifferentiation. Restoring CBP expression or lysine acetylation rescued neuronal-specific transcription in cultured neurons. Together, these experiments show that KAT3 proteins maintain the excitatory neuron identity through the regulation of histone acetylation at cell type-specific promoter and enhancer regions., M.L. is recipient of a Santiago Grisolia fellowship given by the Generalitat Valenciana, J.M.C. is recipient of a fellowship from the Spanish Ministry of Education, Culture and Sport (MECD), J.F.-A. and C.M.N. are recipients of fellowships from the Spanish Ministry of Science and Innovation (MICINN). The ultrastructure research was supported by the Polish National Science Center Grant UMO-2014/15/N/NZ3/04468 and by the European Regional Development Fund POIG 01.01.02-00-008/08. J.P.L.-A. research is supported by Grants RYC-2015-18056 and RTI2018-102260-B-I00 from MICINN co-financed by ERDF. A.B. research is supported by Grants SAF2017-87928-R, PCIN-2015-192-C02-01, and SEV-2017-0723 from MICINN co-financed by ERDF, PROMETEO/2016/026 from the Generalitat Valenciana, and RGP0039/2017 from the Human Frontiers Science Program Organization (HFSPO). The Instituto de Neurociencias is a “Centre of Excellence Severo Ochoa”.

To date, neurons have been the primary focus of research on the role of glucocorticoids in the regulation of brain function and pathological behaviors, such as addiction. Astrocytes, which are also glucocorticoid-responsive, have been recently implicated in the development of drug abuse, albeit through as yet undefined mechanisms. Here, using a spectrum of tools (whole-transcriptome profiling, viral-mediated RNA interference in vitro and in vivo, behavioral pharmacology and electrophysiology), we demonstrate that astrocytes in the nucleus accumbens (NAc) are an important locus of glucocorticoid receptor (GR)-dependent transcriptional changes that regulate rewarding effects of morphine. Specifically, we show that targeted knockdown of the GR in the NAc astrocytes enhanced conditioned responses to morphine, with a concomitant inhibition of morphine-induced neuronal excitability and plasticity. Interestingly, GR knockdown did not influence sensitivity to cocaine. Further analyses revealed GR-dependent regulation of astroglial metabolism. Notably, GR knockdown inhibited induced by glucocorticoids lactate release in astrocytes. Finally, lactate administration outbalanced conditioned responses to morphine in astroglial GR knockdown mice. These findings demonstrate a role of GR-dependent regulation of astrocytic metabolism in the NAc and a key role of GR-expressing astrocytes in opioid reward processing.

The Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity. In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting—an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning occurring after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

Spatial chromatin organization is crucial for transcriptional regulation and might therefore be particularly dynamic in neurons since these terminally differentiated cells dramatically change their transcriptome in response to external stimuli. Here, we show that stimulation of neurons causes condensation of large chromatin domains. We find that this phenomenon is not only induced in rat hippocampal neurons cultured in vitro, but is also present in vivo in amygdala neurons of rats subjected to fear conditioning, and hippocampal neurons of animals subjected to kainate evoked seizures or High-Frequency Stimulation (HFS). The activity-induced chromatin condensation is an active, very rapid, and reversible process, that is independent of transcription and precedes the expression of Immediate Early Genes (IEG). It is accompanied by the redistribution of posttranslational modifications of histones, and rearrangements in the spatial organization of chromosome territories. Moreover, it leads to the reorganization of nuclear speckles and active domains located in their proximity. Finally, we find that neurons depleted of the histone deacetylase HDAC1 fail to condense chromatin upon stimulation, a phenomenon that can be fully reversed by the introduction of human HDAC1. Taken together, our results suggest that the HDAC1-dependent chromatin reorganization might constitute an important level of fine-tuning of transcriptional regulation in stimulated neurons.

Brain-Derived Neurotrophic Factor is one of the most important trophic proteins in the brain. The role of this growth factor in neuronal plasticity, in health and disease, has been extensively studied. However, mechanisms of epigenetic regulation of Bdnf gene expression in epilepsy are still elusive. In our previous work, using a rat model of neuronal activation upon kainate-induced seizures, we observed a repositioning of Bdnf alleles from the nuclear periphery towards the nuclear center. This change of Bdnf intranuclear position was associated with transcriptional gene activity.In the present study, using the same neuronal activation model, we analyzed the relation between the percentage of the Bdnf allele at the nuclear periphery and clinical and morphological traits of epilepsy. We observed that the decrease of the percentage of the Bdnf allele at the nuclear periphery correlates with stronger mossy fiber sprouting - an aberrant form of excitatory circuits formation. Moreover, using in vitro hippocampal cultures we showed that Bdnf repositioning is a consequence of the transcriptional activity. Inhibition of RNA polymerase II activity in primary cultured neurons with Actinomycin D completely blocked Bdnf gene transcription and repositioning observed after neuronal excitation. Interestingly, we observed that histone deacetylases inhibition with Trichostatin A induced a slight increase of Bdnf gene transcription and its repositioning even in the absence of neuronal excitation. Presented results provide novel insight into the role of BDNF in epileptogenesis. Moreover, they strengthen the statement that this particular gene is a good candidate to search for a new generation of antiepileptic therapies.

The human genome is extensively folded into 3-dimensional organization, yet the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra- resolution imaging capability. Here, we report the development of a novel electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5×5×30 nm in xyz dimensions, respectively). We applied 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualized a large number of distinctive 3D chromatin folding structures in high ultra-resolution. We further quantitatively characterized the reconstituted chromatin folding structures by identifying sub-domains, and uncovered a high level of heterogeneity in chromatin folding ultrastructures, suggestive of extensive dynamic fluidity in 3D chromatin states.

Simple Summary Multiple myeloma (MM) is a still uncurable tumor of mainly elderly patients originating from the terminally differentiated B cells. Introduction to the treatment of MM patients of a new class of drugs called proteasome inhibitors (bortezomib followed by carfilzomib and ixazomib) significantly improved disease control. Proteasome inhibitors interfere with the major mechanism of protein degradation in a cell leading to the severe imbalance in the protein turnover that is deadly to MM cells. Currently, these drugs are the mainstream of MM therapy but are also associated with an increased rate of the injuries to multiple organs and tissues. In this review, we summarize the current knowledge on the molecular mechanisms of the first-in-class proteasome inhibitor bortezomib-induced disturbances in the function of peripheral nerves and cardiac and skeletal muscle. Abstract The overall approach to the treatment of multiple myeloma (MM) has undergone several changes during the past decade. and proteasome inhibitors (PIs) including bortezomib, carfilzomib, and ixazomib have considerably improved the outcomes in affected patients. The first-in-class selective PI bortezomib has been initially approved for the refractory forms of the disease but has now become, in combination with other drugs, the backbone of the frontline therapy for newly diagnosed MM patients, as well as in the maintenance therapy and relapsed/refractory setting. Despite being among the most widely used and highly effective agents for MM, bortezomib can induce adverse events that potentially lead to early discontinuation of the therapy with negative effects on the quality of life and outcome of the patients. Although peripheral neuropathy and myelosuppression have been recognized as the most relevant bortezomib-related adverse effects, cardiac and skeletal muscle toxicities are relatively common in MM treated patients, but they have received much less attention. Here we review the neuromuscular and cardiovascular side effects of bortezomib. focusing on the molecular mechanisms underlying its toxicity. We also discuss our preliminary data on the effects of bortezomib on skeletal muscle tissue in mice receiving the drug.

Rewiring of synaptic circuitry pertinent to memory formation has been associated with morphological changes in dendritic spines and with extracellular matrix (ECM) remodeling. Here, we mechanistically link these processes by uncovering a signaling pathway involving the serotonin 5-HT7 receptor (5-HT7R), matrix metalloproteinase 9 (MMP-9), the hyaluronan receptor CD44, and the small GTPase Cdc42. We highlight a physical interaction between 5-HT7R and CD44 (identified as an MMP-9 substrate in neurons) and find that 5-HT7R stimulation increases local MMP-9 activity, triggering dendritic spine remodeling, synaptic pruning, and impairment of long-term potentiation (LTP). The underlying molecular machinery involves 5-HT7R-mediated activation of MMP-9, which leads to CD44 cleavage followed by Cdc42 activation. One important physiological consequence of this interaction includes an increase in neuronal outgrowth and elongation of dendritic spines, which might have a positive effect on complex neuronal processes (e.g., reversal learning and neuronal regeneration).

Water-soluble upconversion nanoparticles (UCNPs), based on polyvinylpyrrolidone (PVP)-coated NaYF4:Er3+,Yb3+,Gd3+, with various concentrations of Gd3+ ions and relatively high upconversion efficiencies, were synthesized. The internalization and cytotoxicity of the thus obtained UCNPs were evaluated in three cell lines (HeLa, HEK293 and astrocytes). No cytotoxicity was observed even at concentrations of UCNPs up to 50 μg ml-1. The fate of the UCNPs within the cells was studied by examining their upconversion emission spectra with confocal microscopy and confirming these observations with transmission electron microscopy. It was found that the cellular uptake of the UCNPs occurred primarily by clathrin-mediated endocytosis, whereas they were secreted from the cells via lysosomal exocytosis. The results of this study, focused on the mechanisms of the cellular uptake, localization and secretion of UCNPs, demonstrate, for the first time, the co-localization of UCNPs within discrete cell organelles.

Very little is known about the mechanisms responsible for maintaining cell identity in mature tissues. The paralogous type 3 lysine acetyltransferases (KAT3) CBP and p300 are both essential during development, but their specific functions in nondividing differentiated cells remains unclear. Here, we show that when both proteins are simultaneously knocked-out in excitatory neurons of the adult brain, the mice express a rapidly progressing neurological phenotype associated with reduced dendritic complexity and electrical activity, the transcriptional shutdown of neuronal genes, and a dramatic loss of H3K27 acetylation and pro-neural transcription factor binding at neuronal enhancers. The neurons lacking both KAT3 rapidly acquire a molecularly undefined fate with no sign of dedifferentiation, transdifferentiation or death. Restoring CBP expression or lysine acetylation reestablished neuronal-specific transcription. Our experiments demonstrate that KAT3 proteins act as fate-keepers in excitatory neurons and other cell types by jointly safeguarding chromatin acetylation levels at cell type-specific enhancers throughout life.

The human genome is extensively folded into 3-dimensional organization. However, the detailed 3D chromatin folding structures have not been fully visualized due to the lack of robust and ultra-resolution imaging capability. Here, we report the development of an electron microscopy method that combines serial block-face scanning electron microscopy with in situ hybridization (3D-EMISH) to visualize 3D chromatin folding at targeted genomic regions with ultra-resolution (5 × 5 × 30 nm in xyz dimensions) that is superior to the current super-resolution by fluorescence light microscopy. We apply 3D-EMISH to human lymphoblastoid cells at a 1.7 Mb segment of the genome and visualize a large number of distinctive 3D chromatin folding structures in ultra-resolution. We further quantitatively characterize the reconstituted chromatin folding structures by identifying sub-domains, and uncover a high level heterogeneity of chromatin folding ultrastructures in individual nuclei, suggestive of extensive dynamic fluidity in 3D chromatin states., The genome is folded in 3-dimensions, though the lack of robust ultra-resolution imaging makes this difficult to visualise. Here, the authors present 3D-EMISH that combines serial block-face scanning electron microscopy with in situ hybridization.

The detailed architectural examination of the neuronal nuclei in any brain region, using confocal microscopy, requires quantification of fluorescent signals in three-dimensional stacks of confocal images. An essential prerequisite to any quantification is the segmentation of the nuclei which are typically tightly packed in the tissue, the extreme being the hippocampal dentate gyrus (DG), in which nuclei frequently appear to overlap due to limitations in microscope resolution. Segmentation in DG is a challenging task due to the presence of a significant amount of image artifacts and densely packed nuclei. Accordingly, we established an algorithm based on continuous boundary tracing criterion aiming to reconstruct the nucleus surface and to separate the adjacent nuclei. The presented algorithm neither uses a pre-built nucleus model, nor performs image thresholding, which makes it robust against variations in image intensity and poor contrast. Further, the reconstructed surface is used to study morphology and spatial arrangement of the nuclear interior. The presented method is generally dedicated to segmentation of crowded, overlapping objects in 3D space. In particular, it allows us to study quantitatively the architecture of the neuronal nucleus using confocal-microscopic approach.

CD44 is a novel molecular player that regulates structure and function of the synapse. It affects excitatory synaptic transmission, dendritic spine shape, number of functional synapses, and activity-dependent neuronal plasticity. These functions are exerted via the regulation of small Rho GTPases., Synaptic cell adhesion molecules regulate signal transduction, synaptic function, and plasticity. However, their role in neuronal interactions with the extracellular matrix (ECM) is not well understood. Here we report that the CD44, a transmembrane receptor for hyaluronan, modulates synaptic plasticity. High-resolution ultrastructural analysis showed that CD44 was localized at mature synapses in the adult brain. The reduced expression of CD44 affected the synaptic excitatory transmission of primary hippocampal neurons, simultaneously modifying dendritic spine shape. The frequency of miniature excitatory postsynaptic currents decreased, accompanied by dendritic spine elongation and thinning. These structural and functional alterations went along with a decrease in the number of presynaptic Bassoon puncta, together with a reduction of PSD-95 levels at dendritic spines, suggesting a reduced number of functional synapses. Lack of CD44 also abrogated spine head enlargement upon neuronal stimulation. Moreover, our results indicate that CD44 contributes to proper dendritic spine shape and function by modulating the activity of actin cytoskeleton regulators, that is, Rho GTPases (RhoA, Rac1, and Cdc42). Thus CD44 appears to be a novel molecular player regulating functional and structural plasticity of dendritic spines.

Simple Summary We review published clinico-histopathological studies establishing entosis an important prognostic and predictor factor in various cancer types. We also propose a new model to study this phenomenon, which involves active entry of one cell into another one. The internalized cell can remain viable and leave the host cell after a long time, potentially leading to cancer recurrence. Entotic figures are cell in cell structures, in which the nucleus of external cell is crescent-shaped, and the inner cell is surrounded by the extensive space entotic vacuole, distinguishing entosis from cell cannibalism. Entosis correlates with cancer worse prognosis in head and neck squamous cell carcinoma, anal carcinoma, lung adenocarcinoma, pancreatic ductal carcinoma, and some breast ductal carcinoma. The BxPC-3 pancreatic cancer cells provide a new, more convenient model for entosis research in comparison to the previously described semidherent MCF7 model. BxPC-3 cells undergo and survive spontaneous entosis in normal adherent culture conditions. Abstract Entosis is a phenomenon, in which one cell enters a second one. New clinico-histopathological studies of entosis prompted us to summarize its significance in cancer. It appears that entosis might be a novel, independent prognostic predictor factor in cancer histopathology. We briefly discuss the biological basis of entosis, followed by a summary of published clinico-histopathological studies on entosis significance in cancer prognosis. The correlation of entosis with cancer prognosis in head and neck squamous cell carcinoma, anal carcinoma, lung adenocarcinoma, pancreatic ductal carcinoma and breast ductal carcinoma, is shown. Numerous entotic figures are associated with a more malignant cancer phenotype and poor prognosis in many cancers. We also showed that some anticancer drugs could induce entosis in cell culture, even as an escape mechanism. Thus, entosis is likely beneficial for survival of malignant cells, i.e., an entotic cell can hide from unfavourable factors in another cell and subsequently leave the host cell remaining intact, leading to failure in therapy or cancer recurrence. Finally, we highlight the potential relationship of cell adhesion with entosis in vitro, based on the model of the BxPc3 cells cultured in full adhesive conditions, comparing them to a commonly used MCF7 semiadhesive model of entosis.

Introduction. The main component of extralysosomal proteolysis is the ubiquitin-proteasome system (UPS), which is supplemented by tripeptidyl peptidase II (TPPII). That system is a target for anticancer strategies by using proteasome inhibitors. Data from several studies on leukemic cells share evidence for the beneficial and potential role of TPPII in cell survivability. Therefore, the aim of this work was to analyze the effect of AAF-cmk, a membrane permeable semi-specific TPPII inhibitor, on human monocytic leukemic cells U937 for translational research. Material and methods. We studied the viability of U937 cells incubated with AAF-cmk using tetrazolium salt reduction assay (MTT) and apoptosis induction by assessing caspase activation by Western blotting and Annexin V binding assays. Transmission electron microscopy (TEM), a gold standard for apoptosis and autophagy detection, was used to assess the ultrastructure of U937 cells. Results. Incubation of cells with AAF-cmk reduced their viability and induced apoptosis by intrinsic pathway. In groups treated with AAF-cmk, activation of caspases 9 and 3 was observed and caspase inhibition by zVDA restored cell viability. TEM revealed the presence of ultrastructural features of apoptosis and authophagy. Moreover, we identified two types of protein aggregates. The first one was found in close proximity to the endoplasmic reticulum (ER) and corresponds to Aggresome-Like Structure (ALIS); however, the second novel type of aggregate was not related to ER elements, but rather to free cytosolic ribosomes. This type did not correspond to the aggresome neither in localization nor the structure, thus we referred these aggregates as ALiSNER (Aggresome-Like Structure Not Associated With the ER). Conclusions. Our results provide novel and important findings about the role of TPPII in protein homeostasis and cell survival. Since semispecific TPPII inhibitor AAF-cmk displays cytotoxic activity against leukemic U937 cells in vitro it can be considered as a potential anticancer agent.

The expression level of complement regulators in ovarian and corpus uteri tumors was not fully established so far. In current manuscript we performed gene expression analysis by the real-time PCR approach to investigate both membrane bound - CD55 and CD59 and fluid phase - factor H and factor H-like 1 complement regulators. We found increased CD55 expression in corpus uteri tumors when compared to control tissues, whereas in ovarian cancer CD55 expression was lower than in control sections. Additionally we found CD59 expression to be more prominent in ovarian cancer than in corpus uteri tumor samples. We observed also the strong positive correlation between the level of expression of the whole group of regulators, which was particularly significant between the expression of factor H and factor H- like 1. In conclusion we present novel results which implicates different role of particular complement inhibitors in the regulation of the complement system in two cancer types examined. Strong positive correlation between examined proteins implicates similar pattern of the regulation which should be taken into consideration with regards to the possible immunotherapy applied as adjuvant therapeutic approach in these two indications. The inhibition of complement regulation may serve as a strategy to potentiate the efficacy of such treatment.

Differentiation of progenitor cells into adipocytes is accompanied by remarkable changes in cell morphology, cytoskeletal organization, and gene expression profile. Mature adipocytes are filled with a large lipid droplet and the nucleus tends to move to the cell periphery. It was hypothesized that the differentiation process is also associated with changes of nuclear organization. The aim of this study was to determine the number and distribution of selected components of nuclear architecture during porcine in vitro adipogenesis. The pig is an important animal model sharing many similarities to humans at the anatomical, physiological, and genetic levels and has been recognized as a good model for human obesity. Thus, understanding how cellular structures important for fundamental nuclear processes may be altered during adipocyte differentiation is of great importance. Mesenchymal stem cells (MSCs) were derived from bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) and were cultured for 7 days in the adipogenic medium. A variable differentiation potential of these cell populations towards adipogenic lineage was observed, and for further study, a comparative characteristic of the nuclear organization in BM-MSCs and AD-MSCs was performed. Nuclear substructures were visualized by indirect immunofluorescence (nucleoli, nuclear speckles, PML bodies, lamins, and HP1α) or fluorescence in situ hybridization (telomeres) on fixed cells at 0, 3, 5, and 7 days of differentiation. Comprehensive characterization of these structures, in terms of their number, size, dynamics, and arrangement in three-dimensional space of the nucleus, was performed. It was found that during differentiation of porcine MSCs into adipocytes, changes of nuclear organization occurred and concerned: (1) the nuclear size and shape; (2) reduced lamin A/C expression; and (3) reorganization of chromocenters. Other elements of nuclear architecture such as nucleoli, SC-35 nuclear speckles, and telomeres showed no significant changes when compared to undifferentiated and mature fat cells. In addition, the presence of a low number of PML bodies was characteristic of the studied porcine mesenchymal stem cell adipogenesis system. It has been shown that the arrangement of selected components of nuclear architecture was very similar in MSCs derived from different sources, whereas adipocyte differentiation involves nuclear reorganization. This study adds new data on nuclear organization during adipogenesis using the pig as a model organism.

We present the in-vivo imaging of the global mouse brain ischemia using Bessel beam optical coherence microscopy. This method allows to monitor changes in brain structure with extra control of blood flow during the process of artery occlusion. The results show the capability and sensitivity of OCM system with Bessel beam to analyze brain plasticity after severe injury within a period of 8 days.

The importance of the extracellular matrix (ECM) glycoprotein tenascin-C (TnC) and the ECM degrading enzymes, matrix metalloproteinases (MMPs) -2 and -9, in cerebellar histogenesis is well established. This study aimed to examine whether there is a functional relationship between these molecules in regulating structural plasticity of the lateral deep cerebellar nucleus. To this end, starting from postnatal day 21, TnC- or MMP-9-deficient mice were exposed to an enriched environment (EE). We show that 8 weeks of exposure to EE leads to reduced lectin-based staining of perineuronal nets (PNNs), reduction in the size of GABAergic and increase in the number and size of glutamatergic synaptic terminals in wild-type mice. Conversely, TnC-deficient mice showed reduced staining of PNNs compared to wild-type mice maintained under standard conditions, and exposure to EE did not further reduce, but even slightly increased PNN staining. EE did not affect the densities of the two types of synaptic terminals in TnC-deficient mice, while the size of inhibitory, but not excitatory synaptic terminals was increased. In the time frame of 4-8 weeks, MMP-9, but not MMP-2, was observed to influence PNN remodeling and cerebellar synaptic plasticity as revealed by measurement of MMP-9 activity and colocalization with PNNs and synaptic markers. These findings were supported by observations on MMP-9-deficient mice. The present study suggests that TnC contributes to the regulation of structural plasticity in the cerebellum and that interactions between TnC and MMP-9 are likely to be important for these processes to occur.

Nanostructures as color-tunable luminescent markers have become major, promising tools for bioimaging and biosensing. In this paper separated molybdate/Gd2O3 doped rare earth ions (erbium, Er3+ and ytterbium, Yb3+) core–shell nanoparticles (NPs), were fabricated by a one-step homogeneous precipitation process. Emission properties were studied by cathodo- and photoluminescence. Scanning electron and transmission electron microscopes were used to visualize and determine the size and shape of the NPs. Spherical NPs were obtained. Their core–shell structures were confirmed by x-ray diffraction and energy-dispersive x-ray spectroscopy measurements. We postulated that the molybdate rich core is formed due to high segregation coefficient of the Mo ion during the precipitation. The calcination process resulted in crystallization of δ/ξ (core/shell) NP doped Er and Yb ions, where δ—gadolinium molybdates and ξ—molybdates or gadolinium oxide. We confirmed two different upconversion mechanisms. In the presence of molybdenum ions, in the core of the NPs, Yb3+–${{{\rm{MoO}}}_{4}}^{2-}$ (mid2F7/2, 3T2〉) dimers were formed. As a result of a two 980 nm photon absorption by the dimer, we observed enhanced green luminescence in the upconversion process. However, for the shell formed by the Gd2O3:Er, Yb NPs (without the Mo ions), the typical energy transfer upconversion takes place, which results in red luminescence. We demonstrated that the NPs were transported into cytosol of the HeLa and astrocytes cells by endocytosis. The core–shell NPs are sensitive sensors for the environment prevailing inside (shorter luminescence decay) and outside (longer luminescence decay) of the tested cells. The toxicity of the NPs was examined using MTT assay., The research was partially supported by the European Union within European Regional Development Fund, through grant Innovative Economy (POIG.01.01.02-00-008/08) and was partially supported by a grant from the Polish National Science Center 2013/11/B/N21/00089 and partially supported by the grant DEC-2012/07/B/ST5/02080 of the National Science Center of Poland and Center of Excellence. This work has been done in the NanoFun laboratories co-financed by the European Regional Development Fund within the Innovation Economy Operational Program, the Project no. POIG.02.02.00-00-025/09/. This research has been co-finananced with the European Union funds by the European Social Fund and was partially supported by the cluster of Biomedical Engineering Center co-financed by European Union funds under the Operational Programme Innovative Economy (project number UDA-POIG.05.01.00-00). The research was partially financed by the project Sonata from the National Science Centre, UMO-2014/15/D/ST5/02604. The research was partially supported by the Foundation for Polish Science through the International Research Agenda Programme co-financed by the European Union within Smart Growth Operational Program. The research was supported by the National Science Centre (Poland) through Grants No. DEC-2014/14/M/ST3/00484.

Activity-regulatedcytoskeleton-associated protein (Arc) protein is implicated as a master regulator of long-term forms of synaptic plasticity and memory formation, but the mechanisms controlling Arc protein function are little known. Post-translation modification by small ubiquitin-like modifier (SUMO) proteins has emerged as a major mechanism for regulating protein-protein interactions and function. We first show in cell lines that ectopically expressed Arc undergoes mono-SUMOylation. The covalent addition of a single SUMO1 protein was confirmed by in vitro SUMOylation of immunoprecipitated Arc. To explore regulation of endogenous Arc during synaptic plasticity, we induced long-term potentiation (LTP) in the dentate gyrus of live anesthetized rats. Using coimmunoprecipitation of native proteins, we show that Arc synthesized during the maintenance phase of LTP undergoes dynamic mono-SUMO1-ylation. Levels of unmodified Arc increase in multiple subcellular fractions (cytosol, membrane, nuclear and cytoskeletal), whereas enhanced Arc SUMOylation was specific to the synaptoneurosomal and the cytoskeletal fractions. Dentate gyrus LTP consolidation requires a period of sustained Arc synthesis driven by brain-derived neurotrophic factor (BDNF) signaling. Local infusion of the BDNF scavenger, TrkB-Fc, during LTP maintenance resulted in rapid reversion of LTP, inhibition of Arc synthesis and loss of enhanced Arc SUMO1ylation. Furthermore, coimmunoprecipitation analysis showed that SUMO1-ylated Arc forms a complex with the F-actin-binding protein drebrin A, a major regulator of cytoskeletal dynamics in dendritic spines. Although Arc also interacted with dynamin 2, calcium/calmodulindependentprotein kinase II-beta (CaMKIIβ), and postsynaptic density protein-95 (PSD-95), these complexes lacked SUMOylated Arc. The results support a model in which newly synthesized Arc is SUMOylated and targeted for actin cytoskeletal regulation during in vivo LTP. publishedVersion

Multiple myeloma (MM) accounts for ∼13% of all hematologic malignancies. Bortezomib treatment is effective in MM, but can be complicated with neurological side effects. We describe a patient with symptomatic MM who had a reversible metabolic myopathy associated with bortezomib administration and pathologically characterized by excessive storage of lipid droplets together with mitochondrial abnormalities. In a single-center prospective study, 14 out of 24 patients with symptomatic MM were treated with bortezomib and, among these, 7 developed muscular signs and/or symptoms. The myopathy was characterized by a proximal muscle weakness involving lower limbs and was an early complication. Complete resolution of muscle weakness occurred after treatment discontinuation. Conversely, none of the patients who received a treatment without bortezomib developed muscular symptoms. Experimental studies demonstrate that in primary human myoblasts bortezomib at low concentrations leads to excessive storage of lipid droplets together with structural mitochondrial abnormalities, recapitulating the pathologic findings observed in patient's muscle. Our data suggest that patients treated with bortezomib should be monitored for muscular signs and/or symptoms and muscle weakness should alert the clinician to the possibility of myopathy. Bortezomib-induced metabolic myopathy is a potentially reversible entity with important implications for management and treatment of patients with MM.

Inhibition of the proteasome offers many therapeutic possibilities in inflammation as well as in neoplastic diseases. However, clinical use of proteasome inhibitors is limited by the development of resistance or severe side effects. In our study we characterized the anti-tumor properties of the novel proteasome inhibitor BSc2118. The sensitivity of tumor lines to BSc2118 was analyzed in comparison to bortezomib using crystal violet staining in order to assess cell viability. The In Vivo distribution of BSc2118 in mouse tissues was tracked by a fluorescent-modified form of BSc2118 (BSc2118-FL) and visualized by confocal microscopy. Inhibition of the 20S proteasome was monitored both in cultured cell lines and in mice, respectively. Finally, safety and efficacy of BSc2118 was evaluated in a mouse melanoma model. BSc2118 inhibits proliferation of different tumor cell lines with a similar potency as compared with bortezomib. Systemic administration of BSc2118 in mice is well tolerated, even when given in a dose of 60 mg/kg body weight. After systemic injection of BSc2118 or bortezomib similar proteasome inhibition patterns are observed within the murine organs. Detection of BSc2118-FL revealed correlation of distribution pattern of BSc2118 with inhibition of proteasomal activity in cells or mouse tissues. Finally, administration of BSc2118 in a mouse melanoma model shows significant local anti-tumor effects. Concluding, BSc2118 represents a novel low-toxic agent that might be alternatively used for known proteasome inhibitors in anti-cancer treatment. peerReviewed

The gelatinases MMP-9 and MMP-2 have been implicated in skeletal muscle adaptation to training; however, their specific role(s) in the different muscle types are only beginning to be unraveled. Recently, we found that treadmill running increased the activity and/or expression of these enzymes in myonuclei and in activated satellite cells of the soleus (Sol), but not extensor digitorum longus (EDL) muscles on the fifth day of training of adult rats. Here, we asked whether the gelatinases can be involved in physical exercise-induced adaptation of the neuromuscular compartment. To determine the subcellular localization of the gelatinolytic activity, we used high-resolution in situ zymography and immunofluorescence techniques. In both control and trained muscles, strong gelatinolytic activity was associated with myelin sheaths within intramuscular nerve twigs. In EDL, but not Sol, there was an increase in the gelatinolytic activity at the postsynaptic domain of the neuromuscular junction (NMJ). The increased activity was found within punctate structures situated in the vicinity of synaptic cleft of the NMJ, colocalizing with a marker of endoplasmic reticulum. Our results support the hypothesis that the gelatinolytic activity at the NMJ may be involved in NMJ plasticity.

The deleted in liver cancer (DLC) protein family is composed of proteins that are hypothesized to function predominantly by regulating the activity of the small GTPases. The aim of the present study was to determine the expression and exact localization of DLC1 in hepatocellular carcinoma (HCC) tissue sections. In two types of HCC tissues, typical and fibrolamellar, immunohistochemical and immunofluorescent analysis were performed to assess DLC1 immunoreactivity. Additionally, the DLC1 gene status was determined by the fluorescence in situ hybridization. According to the observations, DLC1 is often lost in cancer cells; however, it can remain within the stromal component of tumor sections. The DLC1 immunoreactivity was particularly noticeable within the capsules surrounding the tumor masses. It was found that the DLC1 gene was deleted in 52% of HCC cases. In addition, the hemizygous deletion was observed to be independent of the HCC subtype. The results indicate that although the loss of DLC1 is a common step during hepatocarcinogenesis, this protein may be present in the tumor microenvironment.

Studies in cultured cells have demonstrated the existence of higher-order epigenetic mechanisms, determining the relationship between expression of the gene and its position within the cell nucleus. It is unknown, whether such mechanisms operate in postmitotic, highly differentiated cell types, such as neuronsin vivo. Accordingly, we examined whether the intranuclear positions ofBdnfandTrkbgenes, encoding the major neurotrophin and its receptor respectively, change as a result of neuronal activity, and what functional consequences such movements may have. In a rat model of massive neuronal activation upon kainate-induced seizures we found that elevated neuronal expression ofBdnfis associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressedTrkbremains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleusin vivocontributes to activity-dependent changes in gene expression in the brain.

Membrane-bound complement regulatory proteins protect cells from the complement-based destruction and affects many vital immunological functions. as was shown by many studies, overexpression of these proteins on cancer cells may have a negative influence on the therapeutic effect of monoclonal antibodies and immunotherapy. the most commonly observed complement inhibitors on cancer cells are Cd46, Cd55 and Cd59. in the presented work we evaluated the expression and immunoreactivity pattern of these three regulators in two colon cancer cell lines, one derived from the primary tumor (sw480), and the other from the lymph node metastasis (sw620). Both cell lines were derived from the same patient. we found that in the sw480 cells the expression of all inhibitors was heterogeneous, within this cell line many subpopulations of cells existed, which displayed different levels of complement inhibitors. on the contrary, on sw620 cells the immunoreactivity of examined inhibitors was more homogeneous, virtually all examined cells displayed complement inhibitors immunoreactivity on the same level. the most prominent in both cell lines was the Cd46 expression on the cell surface. our results indicate that during carcinogenesis progression only these cancer cells may spread, migrate and form metastatic foci, which possess more complement inhibitors on their cell surface. Modulation of the function of membrane-bound complement inhibitors may have beneficial influence on such strategies as immunotherapy with monoclonal antibodies.

although recent years have brought many advantages in clinical oncology, colon cancer is still a serious problem. surgical resection remains the most efficient treatment, but its results are not always satisfactory. to improve disease-free survival, overall survival, and to prevent metastases, new strategies for effective treatment are searched for. immunotherapy using monoclonal antibodies was shown to be effective in some types of malignancies, however one of the most important obstacles to this type of treatment is attributed to the presence of the complement inhibitors on cancer cells. the three main membrane-bound complement inhibitors that limit the efficacy of the complement-mediated cytotoxicity on cancer cells are Cd46, Cd55 and Cd59. in the current study, we evaluated the expression of these three complement inhibitors on colon cancer tumor samples. additionally we compared, in samples obtained from the same patients, the immunoreactivities of these inhibitors in the primary and metastatic sites of the tumor. we found that in colon cancer cells the most common is Cd46, whereas Cd55 and Cd59 are present in a small percentage of tumor samples. although the staining intensity varied between primary and metastatic foci, these results, regarding all examined complement inhibitors were not statistically relevant. it appears that Cd46 plays the most significant role among complement inhibitors in colon cancer. thus, attempts should be made to inhibit this regulator when complement-mediated cytotoxicity is taken into consideration as an option for control of cancer cells remaining after surgical resection of the tumor.

We used Optical Coherence Microscopy (OCM) to monitor structural and functional changes due to ischemic stroke in small animals brains in vivo. To obtain lateral resolution of 2.2 μm over the range of 600 μm we used extended focus configuration of OCM instrument involving Bessel beam. It provided access to detailed 3D information about the changes in brain vascular system up to the level of capillaries across I and II/III layers of neocortex. We used photothrombotic stroke model involving photoactive application of rose bengal to assure minimal invasiveness of the procedure and precise localization of the clot distribution center. We present the comparative analysis involving structural and angiographic maps of the stroke-affected brain enabling in-depth insight to the process of development of the disorder.

Communication of cells with their extracellular environment is crucial to fulfill their function in physiological and pathophysiological conditions. The literature data provide evidence that such a communication is also important in case of astrocytes. Mechanisms that contribute to the interaction between astrocytes and extracellular matrix (ECM) proteins are still poorly understood. Hyaluronan is the main component of ECM in the brain, where its major receptor protein CD44 is expressed by a subset of astrocytes. Considering the fact that functions of astrocytes are tightly coupled with changes in their morphology (e.g.: glutamate clearance in the synaptic cleft, migration, astrogliosis), we investigated the influence of hyaluronan cleavage by hyaluronidase, knockdown of CD44 by specific shRNA and CD44 overexpression on astrocyte morphology. Our results show that hyaluronidase treatment, as well as knockdown of CD44, in astrocytes result in a “stellate”-like morphology, whereas overexpression of CD44 causes an increase in cell body size and changes the shape of astrocytes into flattened cells. Moreover, as a dynamic reorganization of the actin cytoskeleton is supposed to be responsible for morphological changes of cells, and this reorganization is controlled by small GTPases of the Rho family, we hypothesized that GTPase Rac1 acts as a downstream effector for hyaluronan and CD44 in astrocytes. We used FRET-based biosensor and a dominant negative mutant of Rac1 to investigate the involvement of Rac1 activity in hyaluronidase- and CD44-dependent morphological changes of astrocytes. Both, hyaluronidase treatment and knockdown of CD44, enhances Rac1 activity while overexpression of CD44 reduces the activity state in astrocytes. Furthermore, morphological changes were blocked by specific inhibition of Rac1 activity. These findings indicate for the first time that regulation of Rac1 activity is responsible for hyaluronidase and CD44-driven morphological changes of astrocytes.

Matrix metalloproteinases (MMPs) are involved in tissue repair, cell death and morphogenesis. We investigated the role of the gelatinases MMP-2 and MMP-9 in the pathogenesis of neuronal death induced by prolonged seizures in the developing brain. Seven-day-old rats, MMP-9 knockout mice and transgenic rats overexpressing MMP-9 received intraperitoneal injections of pilocarpine, 250 mg/kg, to induce seizures. After 6-72 h pups were sacrificed, tissue from different brain regions was isolated and expression of MMP-9 mRNA and protein was analyzed by real-time PCR or Western blot. Additionally, brains were fixed and processed for TUNEL-staining, immunohistochemistry and in situ zymography. We found increased numbers of TUNEL-positive cells 24 h after pilocarpine-induced seizures, most pronounced in cortical areas and the dentate gyrus, and less pronounced in thalamus. At 6-24 h, MMP-9 mRNA levels showed significant elevation compared to sham-treated controls; this effect resolved by 48 h, whereas MMP-2 mRNA levels remained stable. Cortical gelatinolytic activity, monitored by in situ zymography, was enhanced following pilocarpine-induced seizures. The MMP inhibitor GM 6001 ameliorated cell death following pilocarpine-induced seizures in infant rats. MMP-9 knockout mice were less susceptible to seizure-induced brain injury. Transgenic rats overexpressing MMP-9 were equally susceptible to seizure-induced brain injury as wild type rats. Our results suggest a significant contribution of MMP-9 to cell death after pilocarpine-induced seizures in the developing brain. As indicated by Western blot analysis, MMP-9 activation may be linked to activation of the Erk/CREB-pathway. The findings implicate involvement of MMP-9 in the pathophysiology of brain injury following seizures in the developing brain.

The extracellular matrix (ECM) of the central nervous system has a specific structure and protein composition that are different from those in other organs. Today we know that the ECM not only provides physical scaffolding for the neurons and glia, but also actively modifies their functions. Over the last two decades, a growing body of research evidence has been collected, suggesting an important role of ECM proteolysis in synaptic plasticity of the brain. So far the majority of data concern two large families of proteases: the serine proteases and the matrix metalloproteinases. The members of these families are localized at the synapses, and are secreted into the extracellular space in an activity-dependent manner. The proteases remodel the local environment as well as influencing synapse structure and function. The structural modifications induced by proteases include shape and size changes, as well as synapse elimination, and synaptogenesis. The functional changes include modifications of receptor function in the postsynaptic part of the synapse, as well as the potentiation or depression of neurotransmitter secretion by the presynaptic site. The present review summarizes the current view on the role of extracellular proteolysis in the physiological synaptic plasticity underlying the phenomena of learning and memory, as well as in the pathological plasticity occurring during epileptogenesis or development of drug addiction.

Recent case reports provided alarming signals that treatment with bortezomib might be associated with cardiac events. In all reported cases, patients experiencing cardiac problems were previously or concomitantly treated with other chemotherapeutics including cardiotoxic anthracyclines. Therefore, it is difficult to distinguish which components of the therapeutic regimens contribute to cardiotoxicity. Here, we addressed the influence of bortezomib on cardiac function in rats that were not treated with other drugs. Rats were treated with bortezomib at a dose of 0.2 mg/kg thrice weekly. Echocardiography, histopathology, and electron microscopy were used to evaluate cardiac function and structural changes. Respiration of the rat heart mitochondria was measured polarographically. Cell culture experiments were used to determine the influence of bortezomib on cardiomyocyte survival, contractility, Ca(2+) fluxes, induction of endoplasmic reticulum stress, and autophagy. Our findings indicate that bortezomib treatment leads to left ventricular contractile dysfunction manifested by a significant drop in left ventricle ejection fraction. Dramatic ultrastructural abnormalities of cardiomyocytes, especially within mitochondria, were accompanied by decreased ATP synthesis and decreased cardiomyocyte contractility. Monitoring of cardiac function in bortezomib-treated patients should be implemented to evaluate how frequently cardiotoxicity develops especially in patients with pre-existing cardiac conditions, as well as when using additional cardiotoxic drugs.

A proper cooperation between the plasma membrane, the endoplasmic reticulum and the mitochondria seems to be essential for numerous cellular processes involved in Ca 2+ signalling and maintenance of Ca 2+

CD44 is a multifunctional cell surface glycoprotein which regulates cell–cell and cell–matrix interactions in a variety of tissues. In particular, the protein was found to be expressed in glial cells of developing, but not adult, peripheral nerves, where it takes part in signaling mediated by ErbB class of receptors for neuregulins. Here, we demonstrate, using high resolution morphological methods, tissue fractionation and RT-PCR, that CD44 is strongly expressed in terminal Schwann cell (TSC) at the neuromuscular junction (NMJ) of the adult rat skeletal muscle. As CD44 is also expressed by Schwann cells of the non-myelinated Remak bundles of the proximal peripheral nerves, it appears to be a marker of non-myelinating Schwann cell subpopulation. The analysis of transgenic rats bearing a mutated superoxide-dismutase gene (SOD1G93A) causing familial amyotrophic lateral sclerosis (ALS) revealed that TSC activation and morphological plasticity at the NMJ, caused by ongoing denervation–reinnervation is associated with a strong increase in CD44 expression therein. Notably, CD44 immunoreactivity is present in fine axon-escheating processes of the glial cells that guide reinnervation. In addition, we found that both in normal and SOD1G93A muscle, CD44 expressed in TSC partially colocalizes with immunoreactivities of neuregulin receptors ErbB2 and ErbB3. The colocalization appears to reflect a physical interaction, as evidenced by co-immunoprecipitation and fluorescence resonance energy transfer (FRET) analysis between CD44 and ErbB3. Importantly, TSC activation upon ALS-like neurodegeneration results in significant increase in molecular proximity of CD44 and ErbB3, which may have an impact on glial plasticity at the NMJ.

The mitochondrial response to changes of cytosolic calcium concentration has a strong impact on neuronal cell metabolism and viability. We observed that Ca(2+) additions to isolated rat brain mitochondria induced in potassium ion containing media a mitochondrial membrane potential depolarization and an accompanying increase of mitochondrial respiration. These Ca(2+) effects can be blocked by iberiotoxin and charybdotoxin, well known inhibitors of large conductance potassium channel (BK(Ca) channel). Furthermore, NS1619 - a BK(Ca) channel opener - induced potassium ion-specific effects on brain mitochondria similar to those induced by Ca(2+). These findings suggest the presence of a calcium-activated, large conductance potassium channel (sensitive to charybdotoxin and NS1619), which was confirmed by reconstitution of the mitochondrial inner membrane into planar lipid bilayers. The conductance of the reconstituted channel was 265 pS under gradient (50/450 mM KCl) conditions. Its reversal potential was equal to 50 mV, which proved that the examined channel was cation-selective. We also observed immunoreactivity of anti-beta(4) subunit (of the BK(Ca) channel) antibodies with ~26 kDa proteins of rat brain mitochondria. Immunohistochemical analysis confirmed the predominant occurrence of beta(4) subunit in neuronal mitochondria. We hypothesize that the mitochondrial BK(Ca) channel represents a calcium sensor, which can contribute to neuronal signal transduction and survival.

Matrix Metalloproteinase-9 (MMP-9) is an extracellularly operating enzyme involved in the synaptic plasticity, hippocampal-dependent long term memory and neurodegeneration. Previous studies have shown its upregulation following seizure-evoking stimuli. Herein, we show that in the rat brain, MMP-9 mRNA expression in response to pentylenetetrazole-evoked neuronal depolarization is transient. Furthermore, we demonstrate that in the rat hippocampus neuronal activation strongly induces JunB expression, simultaneously leading to an accumulation of JunB/FosB complexes onto the − 88/− 80 bp site of the rat MMP-9 gene promoter in vivo. Surprisingly, manipulations with JunB expression levels in activated neurons revealed its moderate repressive action onto MMP-9 gene expression. Therefore, our study documents the active repressive influence of AP-1 onto MMP-9 transcriptional regulation by the engagement of JunB.

Synaptic plasticity involves remodeling of extracellular matrix. This is mediated, in part, by enzymes of the matrix metalloproteinase (MMP) family, in particular by gelatinase MMP-9. Accordingly, there is a need of developing methods to visualize gelatinolytic activity at the level of individual synapses, especially in the context of neurotransmitters receptors. Here we present a high-resolution fluorescent in situ zymography (ISZ), performed in thin sections of the alcohol-fixed and polyester wax-embedded brain tissue of the rat (Rattus norvegicus), which is superior to the current ISZ protocols. The method allows visualization of structural details up to the resolution-limit of light microscopy, in conjunction with immunofluorescent labeling. We used this technique to visualize and quantify gelatinolytic activity at the synapses in control and seizure-affected rat brain. In particular, we demonstrated, for the first time, frequent colocalization of gelatinase(s) with synaptic N-methyl-D-aspartic acid (NMDA)- and AMPA-type glutamate receptors. We believe that our method represents a valuable tool to study extracellular proteolytic processes at the synapses, it could be used, as well, to investigate proteinase involvement in a range of physiological and pathological phenomena in the nervous system.

In this work we provide evidence for the potential presence of a potassium channel in skeletal muscle mitochondria. In isolated rat skeletal muscle mitochondria, Ca(2+) was able to depolarize the mitochondrial inner membrane and stimulate respiration in a strictly potassium-dependent manner. These potassium-specific effects of Ca(2+) were completely abolished by 200 nM charybdotoxin or 50 nM iberiotoxin, which are well-known inhibitors of large conductance, calcium-activated potassium channels (BK(Ca) channel). Furthermore, NS1619, a BK(Ca)-channel opener, mimicked the potassium-specific effects of calcium on respiration and mitochondrial membrane potential. In agreement with these functional data, light and electron microscopy, planar lipid bilayer reconstruction and immunological studies identified the BK(Ca) channel to be preferentially located in the inner mitochondrial membrane of rat skeletal muscle fibers. We propose that activation of mitochondrial K(+) transport by opening of the BK(Ca) channel may be important for myoprotection since the channel opener NS1619 protected the myoblast cell line C2C12 against oxidative injury.

Temporal lobe epilepsy (TLE) is a devastating disease in which aberrant synaptic plasticity plays a major role. We identify matrix metalloproteinase (MMP) 9 as a novel synaptic enzyme and a key pathogenic factor in two animal models of TLE: kainate-evoked epilepsy and pentylenetetrazole (PTZ) kindling–induced epilepsy. Notably, we show that the sensitivity to PTZ epileptogenesis is decreased in MMP-9 knockout mice but is increased in a novel line of transgenic rats overexpressing MMP-9. Immunoelectron microscopy reveals that MMP-9 associates with hippocampal dendritic spines bearing asymmetrical (excitatory) synapses, where both the MMP-9 protein levels and enzymatic activity become strongly increased upon seizures. Further, we find that MMP-9 deficiency diminishes seizure-evoked pruning of dendritic spines and decreases aberrant synaptogenesis after mossy fiber sprouting. The latter observation provides a possible mechanistic basis for the effect of MMP-9 on epileptogenesis. Our work suggests that a synaptic pool of MMP-9 is critical for the sequence of events that underlie the development of seizures in animal models of TLE.

The phenomenon of dendritic transport and local translation of mRNA is considered to be one of the most fundamental mechanisms underlying long-term synaptic plasticity. Matrix metalloproteinase 9 (gelatinase B) (MMP-9) is a matrix metalloproteinase implicated in synaptic long-term potentiation and hippocampus-dependent memory. It was recently shown to be prominently up-regulated in the hippocampal dentate gyrus (DG) upon kainate-mediated seizures. Here, using a high resolution nonradioactive in situ hybridization at the light- and electron-microscopic levels, as well as subcellular fractionation, we provide evidence that in the rat hippocampus, MMP-9 mRNA is associated with dendrites and dendritic spines bearing asymmetric (excitatory) synapses. Moreover we observe that after kainate treatment the number of dendrites and synapses containing MMP-9 mRNA increases markedly. Our results indicate that we are observing the phenomenon of dendritic transport of seizure-induced MMP-9 mRNA.

Background Understanding of the molecular mechanisms of prefrontal cortex (PFC) plasticity is important for developing new treatment strategies for mental disorders such as depression and schizophrenia. Long-term potentiation (LTP) is a valid model for synaptic plasticity. The extracellular proteolytic system composed of matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs) has recently been shown to play major role in the hippocampal plasticity. Methods We tested whether induction of hippocampal-prefrontal LTP results in accumulation of tissue inhibitor of MMP-1, TIMP-1 mRNA, in the PFC of rats and whether adenovirally driven overexpression of TIMP-1 affects LTP. Additional study of slices was done with a specific MMP-9 inhibitor. Results The TIMP-1 is induced in the rat medial PFC by stimuli evoking late LTP; its overexpression blocks the gelatinolytic activity of the MMP family; its overexpression before tetanization blocks late LTP in vivo; and MMP-9 inhibitor prevents late LTP in vitro. Conclusions We suggest a novel extracellular mechanism of late LTP in the PFC, engaging TIMP-1-controlled proteolysis as an element of information integration. Our results may also be meaningful to an understanding of mental diseases and development of new treatment strategies that are based on extracellular mechanisms of synaptic plasticity.