Katarzyna Kalita

Estrogen receptor β: Potential functional significance of a variety of mRNA isoforms

Recent cloning of estrogen receptor β (ERβ) was followed by the discovery of a variety of its isoforms. This review describes the complexity of ERβ mRNAs in various species for which most data have been gathered so far. The most surprising finding is the great variation in isoform structure among various mammalian species. This may reflect either the fact that only a very limited number of isoforms have been described so far or between‐species specificity, especially as common elements in closely related species could still be noted. Isoform variations, as detected mainly at the mRNA sequence level, should result in profound functional differences at the level of proteins as already shown in selected cases. Thus, it is proposed that the diversity of ERβ isoforms implies a functional role of this phenomenon in cellular physiology and pathology of estrogen response.

Two subtypes of G protein‐coupled nucleotide receptors, P2Y1 and P2Y2 are involved in calcium signalling in glioma C6 cells

In glioma C6 cells, the stimulation of P2Y receptors by ADP, ATP and UTP initiated an increase in the intracellular Ca2+ concentration, in a process that involved the release of Ca2+ from InsP3‐sensitive store and the capacitative, extracellular Ca2+ entry. The presence of external Ca2+ was not necessary to elevate Ca2+. The rank order of potencies of nucleotide analogues in stimulating [Ca2+]i was: 2MeSADP > ADP > 2MeSATP = 2ClATP > ATP > UTP. α,β‐Methylene ATP, adenosine and AMP were ineffective. ADP and UTP effects were additive, while actions of ATP and UTP were not additive on [Ca2+]i increase. Similarly, cross‐desensitization between ATP and UTP but not between ADP and UTP occurred. Suramin, a non‐specific nucleotide receptors inhibitor, antagonized ATP‐, UTP‐ and ADP‐evoked Ca2+ responses. PPADS, a selective antagonist of the P2Y1 receptor‐generated InsP3 accumulation, decreased ADP‐initiated Ca2+ response with no effect on ATP and UTP. Pertussis toxin (PTX) reduced ADP‐ and ATP‐induced Ca2+ increases. Short‐term treatment with TPA, inhibited both ATP and ADP stimulatory effects on [Ca2+]i. ADP inhibited isoproterenol‐induced cyclic AMP accumulation. PTX blocked this effect, but PPADS did not. RT – PCR analysis revealed the molecular identity of P2Y receptors expressed by glioma C6 cells to be both P2Y1 and P2Y2. It is concluded that both P2Y1 and P2Y2 receptors co‐exist in glioma C6 cells. ADP acts as agonist of the first, and ATP and UTP of the second one. Both receptors are linked to phospholipase C (PLC).

Role for MMP-9 in stress-induced downregulation of nectin-3 in hippocampal CA1 and associated behavioural alterations

Chronic stress is a risk factor for the development of psychopathologies characterized by cognitive dysfunction and deregulated social behaviours. Emerging evidence suggests a role for cell adhesion molecules, including nectin-3, in the mechanisms that underlie the behavioural effects of stress. We tested the hypothesis that proteolytic processing of nectins by matrix metalloproteinases (MMPs), an enzyme family that degrades numerous substrates, including cell adhesion molecules, is involved in hippocampal effects induced by chronic restraint stress. A reduction in nectin-3 in the perisynaptic CA1, but not in the CA3, compartment is observed following chronic stress and is implicated in the effects of stress in social exploration, social recognition and a CA1-dependent cognitive task. Increased MMP-9-related gelatinase activity, involving N-methyl-D-aspartate receptor, is specifically found in the CA1 and involved in nectin-3 cleavage and chronic stress-induced social and cognitive alterations. Thus, MMP-9 proteolytic processing emerges as an important mediator of stress effects in brain function and behaviour.

Brain-Derived Neurotrophic Factor Induces Matrix Metalloproteinase 9 Expression in Neurons via the Serum Response Factor/c-Fos Pathway

ABSTRACT Brain-derived neurotrophic factor (BDNF) plays a pivotal role in the regulation of the transcription of genes that encode proplasticity proteins. In the present study, we provide evidence that stimulation of rat primary cortical neurons with BDNF upregulates matrix metalloproteinase 9 (MMP-9) mRNA and protein levels and increases enzymatic activity. The BDNF-induced MMP-9 transcription was dependent on extracellular signal-regulated kinase 1/2 (ERK1/2) pathway and c-Fos expression. Overexpression of AP-1 dimers in neurons led to MMP-9 promoter activation, with the most potent being those that contained c-Fos, whereas knockdown of endogenous c-Fos by small hairpin RNA (shRNA) reduced BDNF-mediated MMP-9 transcription. Additionally, mutation of the proximal AP-1 binding site in the MMP-9 promoter inhibited the activation of MMP-9 transcription. BDNF stimulation of neurons induced binding of endogenous c-Fos to the proximal MMP-9 promoter region. Furthermore, as the c-Fos gene is a known target of serum response factor (SRF), we investigated whether SRF contributes to MMP-9 transcription. Inhibition of SRF and its cofactors by either overexpression of dominant negative mutants or shRNA decreased MMP-9 promoter activation. In contrast, MMP-9 transcription was not dependent on CREB activity. Finally, we showed that neuronal activity stimulates MMP-9 transcription in a tyrosine kinase receptor B (TrkB)-dependent manner.

Sampling issues in quantitative analysis of dendritic spines morphology

BackgroundQuantitative analysis of changes in dendritic spine morphology has become an interesting issue in contemporary neuroscience. However, the diversity in dendritic spine population might seriously influence the result of measurements in which their morphology is studied. The detection of differences in spine morphology between control and test group is often compromised by the number of dendritic spines taken for analysis. In order to estimate the impact of dendritic spine diversity we performed Monte Carlo simulations examining various experimental setups and statistical approaches. The confocal images of dendritic spines from hippocampal dissociated cultures have been used to create a set of variables exploited as the simulation resources.ResultsThe tabulated results of simulations given in this article, provide the number of dendritic spines required for the detection of hidden morphological differences between control and test groups in terms of spine head-width, length and area. It turns out that this is the head-width among these three variables, where the changes are most easily detected. Simulation of changes occurring in a subpopulation of spines reveal the strong dependence of detectability on the statistical approach applied. The analysis based on comparison of percentage of spines in subclasses is less sensitive than the direct comparison of relevant variables describing spines morphology.ConclusionsWe evaluated the sampling aspect and effect of systematic morphological variation on detecting the differences in spine morphology. The results provided here may serve as a guideline in selecting the number of samples to be studied in a planned experiment. Our simulations might be a step towards the development of a standardized method of quantitative comparison of dendritic spines morphology, in which different sources of errors are considered.

MKLs: co-factors of serum response factor (SRF) in neuronal responses.

Serum response factor (SRF) is a major transcription factor that regulates activity-driven gene expression in neurons. Activation of SRF-driven transcription occurs through its interaction with two families of co-factors: ternary complex factor (TCF) and myocardin-related transcription factors (MRTFs). This review focuses on the MRTF family members MKL1 (MAL/MRTF-A/BSAC) and MKL2 (MRTF-B/MAL16). MKLs share several high-homology domains but a low level of sequence identity in the transactivation domain. Both co-activators are expressed in the brain and regulate SRF-dependent gene expression. MKL1 and MKL2 function as major co-activators of SRF function in the developing mouse brain. MKLs inactivation causes ineffective neuronal migration and aberrant neurite outgrowth during development. Moreover, inhibition of MKL1 or MKL2 by short-hairpin RNAs results in a decreased number of dendritic processes and dendritic length. Altogether, MKLs appear to regulate plasticity-related structural changes in neurons.

Activation function 1 domain plays a negative role in dimerization of estrogen receptor beta.

Transcriptional potential of estrogen receptor beta (ERbeta) depends on the ligand binding and subsequent dimerization of the receptor protein. In order to examine the role of N-terminally located activation function 1 (AF-1) protein domain in the dimerization process of ERbeta, we used yeast SOS-Recruitment System (SRS). Two variants of ERbeta protein were expressed in the yeast cells: full length receptor and a truncated form, lacking AF-1. We observed that upon 17beta-estradiol treatment only the shorter form of the receptor dimerized, whereas the full-length one did not. This result suggests an inhibitory function of AF-1 in dimer formation and supports previous studies showing that N-terminal domain of ERbeta suppresses transcriptional activity.

Synaptic localisation of SRF coactivators, MKL1 and MKL2, and their role in dendritic spine morphology

The megakaryoblastic leukaemia (MKL) family are serum response factor (SRF) coactivators, which are highly expressed in the brain. Accordingly, MKL plays important roles in dendritic morphology, neuronal migration, and brain development. Further, nucleotide substitutions in the MKL1 and MKL2 genes are found in patients with schizophrenia and autism spectrum disorder, respectively. Thus, studies on the precise synaptic localisation and function of MKL in neurons are warranted. In this study, we generated and tested new antibodies that specifically recognise endogenously expressed MKL1 and MKL2 proteins in neurons. Using these reagents, we biochemically and immunocytochemically show that MKL1 and MKL2 are localised at synapses. Furthermore, shRNA experiments revealed that postsynaptic deletion of MKL1 or MKL2 reduced the percentage of mushroom- or stubby-type spines in cultured neurons. Taken together, our findings suggest that MKL1 and MKL2 are present at synapses and involved in dendritic spine maturation. This study may, at least in part, contribute to better understanding of the molecular mechanisms underlying MKL-mediated synaptic plasticity and neurological disorders.