Marcin Rylski

Matrix Metalloproteinase-9 Undergoes Expression and Activation during Dendritic Remodeling in Adult Hippocampus

Neurons of adult brain are able to remodel their synaptic connections in response to various stimuli. Modifications of the peridendritic environment, including the extracellular matrix, are likely to play a role during synapse remodeling. Proteolytic disassembly of ECM is a complex process using the regulated actions of specific extracellular proteinases. One of best-characterized families of matrix-modifying enzymes is the matrix metalloproteinase (MMP) family. Here, we describe changes in the expression and function of two well known MMPs, MMP-9 and MMP-2, in adult rat brain before and after systemic administration of the glutamate receptor agonist kainate. Kainate application results in enhanced synaptic transmission and seizures followed by selective tissue remodeling, primarily in hippocampal dentate gyrus. MMP-9 but not MMP-2 was highly expressed by neurons in normal adult rat brain. MMP-9 protein was localized in neuronal cell bodies and dendrites. Kainate upregulated the level of MMP-9 mRNA and protein within hours after drug administration. This was followed several hours later by MMP-9 enzymatic activation. Within hippocampus, MMP-9 mRNA and activity were increased selectively in dentate gyrus, including its dendritic layer. In addition, MMP-9 mRNA levels decreased in areas undergoing neuronal cell loss. This unique spatiotemporal pattern of MMP-9 expression suggests its involvement in activity-dependent remodeling of dendritic architecture with possible effects on synaptic physiology.

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.

The critical role of cyclin D2 in adult neurogenesis

Adult neurogenesis (i.e., proliferation and differentiation of neuronal precursors in the adult brain) is responsible for adding new neurons in the dentate gyrus of the hippocampus and in the olfactory bulb. We describe herein that adult mice mutated in the cell cycle regulatory gene Ccnd2, encoding cyclin D2, lack newly born neurons in both of these brain structures. In contrast, genetic ablation of cyclin D1 does not affect adult neurogenesis. Furthermore, we show that cyclin D2 is the only D-type cyclin (out of D1, D2, and D3) expressed in dividing cells derived from neuronal precursors present in the adult hippocampus. In contrast, all three cyclin D mRNAs are present in the cultures derived from 5-day-old hippocampi, when developmental neurogenesis in the dentate gyrus takes place. Thus, our results reveal the existence of molecular mechanisms discriminating adult versus developmental neurogeneses.

SYNAPTIC LOCALIZATION OF SEIZURE-INDUCED MATRIX METALLOPROTEINASE-9 mRNA

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.

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.

JunB is a repressor of MMP-9 transcription in depolarized rat brain neurons.

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.

Yin Yang 1 is a critical repressor of matrix metalloproteinase-9 expression in brain neurons.

Membrane depolarization controls long lasting adaptive neuronal changes in brain physiology and pathology. Such responses are believed to be gene expression-dependent. Notably, however, only a couple of gene repressors active in nondepolarized neurons have been described. In this study, we show that in the unstimulated rat hippocampus in vivo, as well as in the nondepolarized brain neurons in primary culture, the transcriptional regulator Yin Yang 1 (YY1) is bound to the proximal Mmp-9 promoter and strongly represses Mmp-9 transcription. Furthermore, we demonstrate that monoubiquitinated and CtBP1 (C-terminal binding protein 1)-bound YY1 regulates Mmp-9 mRNA synthesis in rat brain neurons controlling its transcription apparently via HDAC3-dependent histone deacetylation. In conclusion, our data suggest that YY1 exerts, via epigenetic mechanisms, a control over neuronal expression of MMP-9. Because MMP-9 has recently been shown to play a pivotal role in physiological and pathological neuronal plasticity, YY1 may be implicated in these phenomena as well.

Yin Yang 1 expression in the adult rodent brain.

Yin Yang 1 (YY1) is a ubiquitous transcription factor belonging to Polycomb group proteins. Its expression patterns in the adult brain have not been before clearly elucidated. Using immunohistochemical stainings, we show a distribution of YY1 protein throughout the adult rodent brain. Furthermore, we characterize a cellular localization of YY1 protein and mRNA in the adult rat hippocampus. We have found that YY1 is expressed in all major brain regions, although not ubiquitously in all cells, and its expression levels vary significantly depending on the brain structure. In most of the regions YY1 is not very abundant, but in the olfactory bulb, cerebellar cortex, hippocampus, cerebral cortex, wall of the lateral ventricle and rostral migratory stream intense YY1 staining is observed. In the rat hippocampus, YY1 protein and mRNA are very strongly expressed in neurons, and to a lesser extent in oligodendroglia and microglia. In contrast, we have not detected YY1 protein in astrocytes, which are the most abundant component of hippocampal glia. Moreover, we show that in the adult rodent brain, YY1 is expressed exclusively in the cell nuclei, except of a molecular layer of cerebellar cortex, where it is also present in the cytoplasm. Interestingly, YY1 staining is accumulated in a form of granules in cell nuclei of different types of brain cells. Thus, our data demonstrate that in the adult rodent brain YY1 is predominantly localized to neurons.

Epigenetics of Epileptogenesis-Evoked Upregulation of Matrix Metalloproteinase-9 in Hippocampus

Enhanced levels of Matrix Metalloproteinase-9 (MMP-9) have been implicated in the pathogenesis of epilepsy in humans and rodents. Lack of Mmp-9 impoverishes, whereas excess of Mmp-9 facilitates epileptogenesis. Epigenetic mechanisms driving the epileptogenesis-related upregulation of MMP-9 expression are virtually unknown. The aim of this study was to reveal these mechanisms. We analyzed hippocampi extracted from adult and pediatric patients with temporal lobe epilepsy as well as from partially and fully pentylenetetrazole kindled rats. We used a unique approach to the analysis of the kindling model results (inclusion in the analysis of rats being during kindling, and not only a group of fully kindled animals), which allowed us to separate the molecular effects exerted by the epileptogenesis from those related to epilepsy and epileptic activity. Consequently, it allowed for a disclosure of molecular mechanisms underlying causes, and not consequences, of epilepsy. Our data show that the epileptogenesis-evoked upregulation of Mmp-9 expression is regulated by removal from Mmp-9 gene proximal promoter of the two, interweaved potent silencing mechanisms–DNA methylation and Polycomb Repressive Complex 2 (PRC2)-related repression. Demethylation depends on a gradual dissociation of the DNA methyltransferases, Dnmt3a and Dnmt3b, and on progressive association of the DNA demethylation promoting protein Gadd45β to Mmp-9 proximal gene promoter in vivo. The PRC2-related mechanism relies on dissociation of the repressive transcription factor YY1 and the dissipation of the PRC2-evoked trimethylation on Lys27 of the histone H3 from the proximal Mmp-9 promoter chromatin in vivo. Moreover, we show that the DNA hydroxymethylation, a new epigenetic DNA modification, which is localized predominantly in the gene promoters and is particularly abundant in the brain, is not involved in a regulation of MMP-9 expression during the epileptogenesis in the rat hippocampus as well as in the hippocampi of pediatric and adult epileptic patients. Additionally, we have also found that despite of its transient nature, the histone modification H3S10ph is strongly and gradually accumulated during epileptogenesis in the cell nuclei and in the proximal Mmp-9 gene promoter in the hippocampus, which suggests that H3S10ph can be involved in DNA demethylation in mammals, and not only in Neurospora. The study identifies MMP-9 as the first protein coding gene which expression is regulated by DNA methylation in human epilepsy. We present a detailed epigenetic model of the epileptogenesis-evoked upregulation of MMP-9 expression in the hippocampus. To our knowledge, it is the most complex and most detailed mechanism of epigenetic regulation of gene expression ever revealed for a particular gene in epileptogenesis. Our results also suggest for the first time that dysregulation of DNA methylation found in epilepsy is a cause rather than a consequence of this condition.

Localization and regulation of PML bodies in the adult mouse brain

PML is a tumor suppressor protein involved in the pathogenesis of promyelocytic leukemia. In non-neuronal cells, PML is a principal component of characteristic nuclear bodies. In the brain, PML has been implicated in the control of embryonic neurogenesis, and in certain physiological and pathological phenomena in the adult brain. Yet, the cellular and subcellular localization of the PML protein in the brain, including its presence in the nuclear bodies, has not been investigated comprehensively. Because the formation of PML bodies appears to be a key aspect in the function of the PML protein, we investigated the presence of these structures and their anatomical distribution, throughout the adult mouse brain. We found that PML is broadly expressed across the gray matter, with the highest levels in the cerebral and cerebellar cortices. In the cerebral cortex PML is present exclusively in neurons, in which it forms well-defined nuclear inclusions containing SUMO-1, SUMO 2/3, but not Daxx. At the ultrastructural level, the appearance of neuronal PML bodies differs from the classic one, i.e., the solitary structure with more or less distinctive capsule. Rather, neuronal PML bodies have the form of small PML protein aggregates located in the close vicinity of chromatin threads. The number, size, and signal intensity of neuronal PML bodies are dynamically influenced by immobilization stress and seizures. Our study indicates that PML bodies are broadly involved in activity-dependent nuclear phenomena in adult neurons.