Morphological alterations of the neuronal Golgi apparatus upon seizures.

AIMS: Epilepsy is one of the most common chronic neurological disorders, affecting around 50 million people worldwide, but its underlying cellular and molecular events are not fully understood. The Golgi is a highly dynamic cellular organelle and can be fragmented into ministacks under both physiological and pathological conditions. This phenomenon has also been observed in several neurodegenerative disorders; however, the structure of the Golgi apparatus (GA) in human patients suffering from epilepsy has not been described so far. The aim of this study was to assess the changes in GA architecture in epilepsy. METHODS: Golgi visualisation with immunohistochemical staining in the neocortex of adult patients who underwent epilepsy surgery; 3D reconstruction and quantitative morphometric analysis of GA structure in the rat hippocampi upon kainic acid (KA) induced seizures, as well as in vitro studies with the use of Ca2+ chelator BAPTA-AM in primary hippocampal neurons upon activation were performed. RESULTS: We observed GA dispersion in neurons of the human neocortex of patients with epilepsy and hippocampal neurons in rats upon KA-induced seizures. The structural changes of GA were reversible, as GA morphology returned to normal within 24 h of KA treatment. KA-induced Golgi fragmentation observed in primary hippocampal neurons cultured in vitro was largely abolished by the addition of BAPTA-AM. CONCLUSIONS: In our study, we have shown for the first time that the neuronal GA is fragmented in the human brain of patients with epilepsy and rat brain upon seizures. We have shown that seizure-induced GA dispersion can be reversible, suggesting that enhanced neuronal activity induces Golgi reorganisation that is involved in aberrant neuronal plasticity processes that underlie epilepsy. Moreover, our results revealed that elevated cytosolic Ca2+ is indispensable for these KA-induced morphological alterations of GA in vitro.

Astrocytic CD44 Deficiency Reduces the Severity of Kainate-Induced Epilepsy.

BACKGROUND: Epilepsy affects millions of people worldwide, yet we still lack a successful treatment for all epileptic patients. Most of the available drugs modulate neuronal activity. Astrocytes, the most abundant cells in the brain, may constitute alternative drug targets. A robust expansion of astrocytic cell bodies and processes occurs after seizures. Highly expressed in astrocytes, CD44 adhesion protein is upregulated during injury and is suggested to be one of the most important proteins associated with epilepsy. It connects the astrocytic cytoskeleton to hyaluronan in the extracellular matrix, influencing both structural and functional aspects of brain plasticity. METHODS: Herein, we used transgenic mice with an astrocyte CD44 knockout to evaluate the impact of the hippocampal CD44 absence on the development of epileptogenesis and ultrastructural changes at the tripartite synapse. RESULTS: We demonstrated that local, virally-induced CD44 deficiency in hippocampal astrocytes reduces reactive astrogliosis and decreases the progression of kainic acid-induced epileptogenesis. We also observed that CD44 deficiency resulted in structural changes evident in a higher dendritic spine number along with a lower percentage of astrocyte-synapse contacts, and decreased post-synaptic density size in the hippocampal molecular layer of the dentate gyrus. CONCLUSIONS: Overall, our study indicates that CD44 signaling may be important for astrocytic coverage of synapses in the hippocampus and that alterations of astrocytes translate to functional changes in the pathology of epilepsy.

The interplay of seizures-induced axonal sprouting and transcription-dependent Bdnf repositioning in the model of temporal lobe epilepsy.

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.

MMP-9 Signaling Pathways That Engage Rho GTPases in Brain Plasticity.

The extracellular matrix (ECM) has been identified as a critical factor affecting synaptic function. It forms a functional scaffold that provides both the structural support and the reservoir of signaling molecules necessary for communication between cellular constituents of the central nervous system (CNS). Among numerous ECM components and modifiers that play a role in the physiological and pathological synaptic plasticity, matrix metalloproteinase 9 (MMP-9) has recently emerged as a key molecule. MMP-9 may contribute to the dynamic remodeling of structural and functional plasticity by cleaving ECM components and cell adhesion molecules. Notably, MMP-9 signaling was shown to be indispensable for long-term memory formation that requires synaptic remodeling. The core regulators of the dynamic reorganization of the actin cytoskeleton and cell adhesion are the Rho family of GTPases. These proteins have been implicated in the control of a wide range of cellular processes occurring in brain physiology and pathology. Here, we discuss the contribution of Rho GTPases to MMP-9-dependent signaling pathways in the brain. We also describe how the regulation of Rho GTPases by post-translational modifications (PTMs) can influence these processes.

Longitudinal in-vivo OCM imaging of glioblastoma development in the mouse brain.

We present in-vivo imaging of the mouse brain using custom made Gaussian beam optical coherence microscopy (OCM) with 800nm wavelength. We applied new instrumentation to longitudinal imaging of the glioblastoma (GBM) tumor microvasculature in the mouse brain. We have introduced new morphometric biomarkers that enable quantitative analysis of the development of GBM. We confirmed quantitatively an intensive angiogenesis in the tumor area between 3 and 14 days after GBM cells injection confirmed by considerably increased of morphometric parameters. Moreover, the OCM setup revealed heterogeneity and abnormality of newly formed vessels.

Synaptic Remodeling Depends on Signaling between Serotonin Receptors and the Extracellular Matrix.

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).

CD44: a novel synaptic cell adhesion molecule regulating structural and functional plasticity of dendritic spines.

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.

Cleavage of Hyaluronan and CD44 Adhesion Molecule Regulate Astrocyte Morphology via Rac1 Signalling.

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.

CD44: molecular interactions, signaling and functions in the nervous system.

CD44 is the major surface hyaluronan (HA) receptor implicated in intercellular and cell-matrix adhesion, cell migration and signaling. It is a transmembrane, highly glycosylated protein with several isoforms resulting from alternative gene splicing. The CD44 molecule consists of several domains serving different functions: the N-terminal extracellular domain, the stem region, the transmembrane domain and the C-terminal tail. In the nervous system, CD44 expression occurs in both glial and neuronal cells. The role of CD44 in the physiology and pathology of the nervous system is not entirely understood, however, there exists evidence suggesting it might be involved in the axon guidance, cytoplasmic Ca(2+) clearance, dendritic arborization, synaptic transmission, epileptogenesis, oligodendrocyte and astrocyte differentiation, post-traumatic brain repair and brain tumour development.

CD44 regulates dendrite morphogenesis through Src tyrosine kinase-dependent positioning of the Golgi.

The acquisition of proper dendrite morphology is a crucial aspect of neuronal development towards the formation of a functional network. The role of the extracellular matrix and its cellular receptors in this process has remained enigmatic. We report that the CD44 adhesion molecule, the main hyaluronan receptor, is localized in dendrites and plays a crucial inhibitory role in dendritic tree arborization in vitro and in vivo. This novel function is exerted by the activation of Src tyrosine kinase, leading to the alteration of Golgi morphology. The mechanism operates during normal brain development, but its inhibition might have a protective influence on dendritic trees under toxic conditions, during which the silencing of CD44 expression prevents dendritic shortening induced by glutamate exposure. Overall, our results indicate a novel role for CD44 as an essential regulator of dendritic arbor complexity in both health and disease.

Novel higher-order epigenetic regulation of the Bdnf gene upon seizures.

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 neurons in vivo. Accordingly, we examined whether the intranuclear positions of Bdnf and Trkb genes, 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 of Bdnf is associated with its detachment from the nuclear lamina, and translocation toward the nucleus center. In contrast, the position of stably expressed Trkb remains unchanged after seizures. Our study demonstrates that activation-dependent architectural remodeling of the neuronal cell nucleus in vivo contributes to activity-dependent changes in gene expression in the brain.

Matrix metalloproteinase-9 (MMP-9) in human intractable epilepsy caused by focal cortical dysplasia.

Focal cortical dysplasia (FCD) is a developmental brain disorder characterized by localized abnormalities of cortical layering and neuronal morphology. It is associated with pharmacologically intractable forms of epilepsy in both children and adults. The mechanisms that underlie FCD-associated seizures and lead to the progression of the disease are unclear. Matrix metalloproteinases (MMPs) are enzymes that are able to influence neuronal function through extracellular proteolysis in various normal and pathological conditions. The results of experiments that have used rodent models showed that extracellular MMP-9 can play an important role in epileptogenesis. However, no studies have shown that MMP-9 is involved in the pathogenesis of human epilepsy. The aim of the present study was to determine whether MMP-9 plays a role in intractable epilepsy. Using an unbiased antibody microarray approach, we found that up regulation of MMP-9 is prominent and consistent in FCD tissue derived from epilepsy surgery, regardless of the patient's age. Additionally, an up regulation of MMP-1, -2, -8, -10, and -13 was found but was either less pronounced or limited only to adult cases. In the dysplastic cortex, immunohistochemistry revealed that the highest MMP-9 immuno reactivity occurred in the cytoplasm of abnormal neurons and balloon cells. The neuronal over expression of MMP-9 also occurred in sclerotic hippocampi that were excised together with the dysplastic cortex, but sclerotic hippocampi were free of dysplastic features. In both locations, MMP-9 was also found in reactive astrocytes, albeit to a lesser extent. At the subcellular level, increased MMP-9 immunoreactivity was prominently upregulated at synapses. Thus, although upregulation of the enzyme in FCD is not causally linked to the developmental malformation, it may be a result of ongoing abnormal synaptic plasticity. The present findings support the hypothesis of the pathogenic role of MMP-9 in human epilepsy and may stimulate discussions about whether MMPs could be novel therapeutic targets for intractable epilepsy.

MicroRNA loss enhances learning and memory in mice.

Dicer-dependent noncoding RNAs, including microRNAs (miRNAs), play an important role in a modulation of translation of mRNA transcripts necessary for differentiation in many cell types. In vivo experiments using cell type-specific Dicer1 gene inactivation in neurons showed its essential role for neuronal development and survival. However, little is known about the consequences of a loss of miRNAs in adult, fully differentiated neurons. To address this question, we used an inducible variant of the Cre recombinase (tamoxifen-inducible CreERT2) under control of Camk2a gene regulatory elements. After induction of Dicer1 gene deletion in adult mouse forebrain, we observed a progressive loss of a whole set of brain-specific miRNAs. Animals were tested in a battery of both aversively and appetitively motivated cognitive tasks, such as Morris water maze, IntelliCage system, or trace fear conditioning. Compatible with rather long half-life of miRNAs in hippocampal neurons, we observed an enhancement of memory strength of mutant mice 12 weeks after the Dicer1 gene mutation, before the onset of neurodegenerative process. In acute brain slices, immediately after high-frequency stimulation of the Schaffer collaterals, the efficacy at CA3-to-CA1 synapses was higher in mutant than in control mice, whereas long-term potentiation was comparable between genotypes. This phenotype was reflected at the subcellular and molecular level by the elongated filopodia-like shaped dendritic spines and an increased translation of synaptic plasticity-related proteins, such as BDNF and MMP-9 in mutant animals. The presented work shows miRNAs as key players in the learning and memory process of mammals.

Expression of Ttyh1, a member of the Tweety family in neurons in vitro and in vivo and its potential role in brain pathology.

We have previously shown that Ttyh1 mRNA is expressed in neurons and its expression is up-regulated in the brain during epileptogenesis and epilepsy. In this study, we aimed to elucidate the role of Ttyh1 in neurons. We found widespread expression of Ttyh1 protein in neurons in vivo and in vitro. Ttyh1 immunoreactivity in vitro was frequently found in invaginations of dendritic spines; however, Ttyh1, seldom co-localized with synaptic markers in vivo. Silencing Ttyh1 expression with siRNA in hippocampal cultures resulted in alterations of MAP2 distribution along neurites causing it to appear in the form of chains of beads. Over-expression of Ttyh1 caused intense neuritogenesis and the formation of numerous filopodia-like protrusions. Similar protrusions were also produced in SH-SY5Y neuroblastoma cells over-expressing Ttyh1. Using a biotin-streptavidin pull-down assay and mass spectrometry, we identified proteins that can form complexes with Ttyh1 in the brain. Ttyh1 binding proteins are often expressed in the endoplasmic reticulum or the Golgi apparatus or are localized at synapses. Finally, we found increased expression of Ttyh1 in the inner molecular layer of the dentate gyrus in an animal model of epilepsy. On the basis of our findings, we propose Ttyh1 involvement in brain pathology.

Smad3 is a key nonredundant mediator of transforming growth factor beta signaling in Nme mouse mammary epithelial cells.

Smad2 and Smad3 are intracellular mediators of transforming growth factor beta (TGFbeta) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFbeta as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFbeta-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFbeta-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFbeta-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein.

Beta-dystroglycan as a target for MMP-9, in response to enhanced neuronal activity.

Matrix metalloproteinase-9 has recently emerged as an important molecule in control of extracellular proteolysis in the synaptic plasticity. However, no synaptic targets for its enzymatic activity had been identified before. In this report, we show that beta-dystroglycan comprises such a neuronal activity-driven target for matrix metalloproteinase-9. This notion is based on the following observations. (i) Recombinant, autoactivating matrix metalloproteinase-9 produces limited proteolytic cleavage of beta-dystroglycan. (ii) In neuronal cultures, beta-dystroglycan proteolysis occurs in response to stimulation with either glutamate or bicuculline and is blocked by tissue inhibitor of metalloproteinases-1, a metalloproteinase inhibitor. (iii) Beta-dystroglycan degradation is also observed in the hippocampus in vivo in response to seizures but not in the matrix metalloproteinase-9 knock-out mice. (iv) Beta-dystroglycan cleavage correlates in time with increased matrix metalloproteinase-9 activity. (v) Finally, beta-dystroglycan and matrix metalloproteinase-9 colocalize in postsynaptic elements in the hippocampus. In conclusion, our data identify the beta-dystroglycan as a first matrix metalloproteinase-9 substrate digested in response to enhanced synaptic activity. This demonstration may help to understand the possible role of both proteins in neuronal functions, especially in synaptic plasticity, learning, and memory.

TIMP-1 abolishes MMP-9-dependent long-lasting long-term potentiation in the prefrontal cortex.

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.

Matrix metalloproteinase-9 is required for hippocampal late-phase long-term potentiation and memory.

Matrix metalloproteinases (MMPs) are extracellular proteases that have well recognized roles in cell signaling and remodeling in many tissues. In the brain, their activation and function are customarily associated with injury or pathology. Here, we demonstrate a novel role for MMP-9 in hippocampal synaptic physiology, plasticity, and memory. MMP-9 protein levels and proteolytic activity are rapidly increased by stimuli that induce late-phase long-term potentiation (L-LTP) in area CA1. Such regulation requires NMDA receptors and protein synthesis. Blockade of MMP-9 pharmacologically prevents induction of L-LTP selectively; MMP-9 plays no role in, nor is regulated during, other forms of short-term synaptic potentiation or long-lasting synaptic depression. Similarly, in slices from MMP-9 null-mutant mice, hippocampal LTP, but not long-term depression, is impaired in magnitude and duration; adding recombinant active MMP-9 to null-mutant slices restores the magnitude and duration of LTP to wild-type levels. Activated MMP-9 localizes in part to synapses and modulates hippocampal synaptic physiology through integrin receptors, because integrin function-blocking reagents prevent an MMP-9-mediated potentiation of synaptic signal strength. The fundamental importance of MMP-9 function in modulating hippocampal synaptic physiology and plasticity is underscored by behavioral impairments in hippocampal-dependent memory displayed by MMP-9 null-mutant mice. Together, these data reveal new functions for MMPs in synaptic and behavioral plasticity.

Matrix metalloproteinases and their endogenous inhibitors in neuronal physiology of the adult brain.

More than 20 matrix metalloproteinases (MMPs) and four of their endogenous tissue inhibitors (TIMPs) act together to control tightly temporally restricted, focal proteolysis of extracellular matrix. In the neurons of the adult brain several components of the TIMP/MMP system are expressed and are responsive to changes in neuronal activity. Furthermore, functional studies, especially involving blocking of MMP activities, along with the identification of MMP substrates in the brain strongly suggest that this enzymatic system plays an important physiological role in adult brain neurons, possibly being pivotal for neuronal plasticity.

Matrix metalloproteinases in the adult brain physiology: a link between c-Fos, AP-1 and remodeling of neuronal connections?

Matrix metalloproteinases (MMPs), together with their endogenous inhibitors (TIMPs) form an enzymatic system that plays an important role in a variety of physiological and pathological conditions. These proteins are also expressed in the brain, especially under pathological conditions, in which glia as well as invading inflammatory cells provide the major source of the MMP activity. Surprisingly little is known about the MMP function(s) in adult neuronal physiology. This review describes available data on this topic, which is presented in a context of knowledge about the MMP/TIMP system in other organs as well as in brain disorders. An analysis of the MMP and TIMP expression patterns in the brain, along with a consideration of their regulatory mechanisms and substrates, leads to the proposal of possible roles of the MMP system in the brain. This analysis suggests that MMPs may play an important role in the neuronal physiology, especially in neuronal plasticity, including their direct participation in the remodeling of synaptic connections-a mechanism pivotal for learning and memory.