Filip A. Konopacki

Important role of matrix metalloproteinase 9 in epileptogenesis

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 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.

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.

High resolution in situ zymography reveals matrix metalloproteinase activity at glutamatergic synapses.

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.

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.

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.

The possible role of factor H in colon cancer resistance to complement attack

A soluble complement inhibitor factor H (FH) and its splice variant factor H‐like protein (FHL) have been recently discovered to play a major role in malignant cell escape from complement‐mediated cytotoxicity in lung‐, ovarian‐ and glia‐derived neoplasms. The role of FH in colon cancer has not yet been examined. Here, we studied immunocytochemically FH/FHL expression in tumor samples derived from 40 patients, with both primary colon adenocarcinoma and metastatic foci in the liver. FH/FHL immunoreactivity was present in stroma of both primary and metastatic tumors, in virtually all patients. The cellular immunoreactivity was observed infrequently. Importantly, when analyzed quantitatively, FH/FHL immunoreactivity was significantly increased in liver metastases when compared with the primary sites. In addition, we have analyzed FH and FHL expression in 5 colon cancer cell lines: SW480, SW620, HCT116, HT‐29 and Lovo. FH mRNA and FH secretion were observed in SW620 and HT‐29 cells, whereas FHL was produced only by HT‐29 cell‐line. By confocal and electron microscopy, FH immunoreactivity was associated with the plasma membrane and intracellular vesicular structures. Finally, we have analyzed the role of FH in the susceptibility of SW620 colon cancer cells to complement‐mediated damage. When FH function was blocked, using specific antibody, the cells became more susceptible to lysis. Taken together, our results suggest an important role of FH/FHL in colon cancer cells defense against complement‐mediated cytotoxicity, and in metastatic process.

Protein kinase G is involved in ammonia-induced swelling of astrocytes

Ammonia‐induced swelling of astrocytes is a primary cause of brain edema associated with acute hepatic encephalopathy. Previous studies have shown that ammonia transiently increases cGMP in brain in vivo and in cultured astrocytes in vitro. We hypothesized that protein kinase G (PKG), an enzyme activated by cGMP and implicated in regulation of cell shape, size, and/or volume in peripheral and CNS cells, may play a role in the ammonia‐induced astrocytic volume increase. Treatment of cultured rat cortical astrocytes with 1 or 5 mM NH4Cl (ammonia) for 24 h increased their cell volume by 50% and 80% above control, respectively, as measured by confocal imaging followed by 3D computational analysis. A cGMP analog, 8‐(4‐chlorophenylthio)‐cGMP, increased the cell volume in control cells and potentiated the increase in 1 mM ammonia‐treated cells. A soluble guanylate cyclase inhibitor (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one) abrogated, and a PKG inhibitor [8‐(4‐chlorophenylthio)‐cGMP‐thioate, Rp‐isomer] dose‐dependently reduced the cell volume‐increasing effect of 5 mM ammonia. The results suggest that (i) PKG may play a permissive role in ammonia‐induced astrocytic swelling and (ii) elevation of brain cGMP associated with acute exposure to ammonia in vivo may aggravate the ensuing brain edema.

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.