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 (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.

In post‐mitotic neurons, the mechanisms of the apoptotic checkpoint that is activated by DNA damage remain unclear. Here we show that in cultured cortical neurons, the DNA damaging agent camptothecin (CPT) reduced transcription of rRNA and disrupted nucleolar staining for B23/nucleophosmin suggesting DNA damage‐induced nucleolar stress. Although CPT activated the pro‐apoptotic protein p53, the CPT‐induced nucleolar stress was unaffected by p53 inhibition. In addition, brain‐derived neurotrophic factor‐mediated protection from CPT‐induced apoptosis prevented neither nucleolar stress nor p53 activation. Therefore, inhibition of rRNA transcription might be upstream of the pro‐apoptotic p53 activity. Indeed, short hairpin RNA‐mediated inhibition of a RNA‐Polymerase‐I co‐factor, transcription initiation factor IA, attenuated rRNA transcription causing nucleolar stress and p53‐dependent neuronal apoptosis. The protein synthesis inhibitor cycloheximide blocked apoptosis that was induced by over‐expressed shTIF‐IA or active form of p53. Also, the general transcription inhibitor actinomycin D triggered nucleolar stress and activated p53. However, it did not induce apoptosis except at the low concentration of 0.05 μg/mL with stronger inhibitory activity against nucleolar than extranucleolar transcription. Hence, nucleolar stress‐activated apoptosis requires extranucleolar transcription. This study identifies the nucleoli of post‐mitotic neurons as sensors of DNA damage coupling reduced rRNA transcription to p53‐mediated apoptosis that requires de novo expression of protein‐coding genes. Thus, rDNA selectivity of DNA damage may determine its ability to induce neuronal apoptosis.

Serum response factor (SRF)-mediated transcription contributes to developmental and adult brain plasticity. Therefore, we investigated the role of a newly identified SRF coactivator, MKL1, in the regulation of SRF-driven transcription in rat forebrain neurons. MKL1 expression was found in newborn rat cortical or hippocampal neurons in culture as well as in adult rat forebrain. Immunostaining demonstrated constitutive nuclear localization of MKL1 in the CA1 region of the hippocampus, in the deep layers of the neocortex, and in cultured neurons. Overexpression of MKL1 in primary cortical neurons elevated SRF-driven transcription and enhanced its stimulation by BDNF. In addition, inhibition of endogenous MKL1 by overexpression of a dominant-negative MKL1 mutant or by small interfering RNA reduced BDNF activation of SRF-driven transcription. In neurons, endogenous MKL1 was associated with SRF-regulated chromatin regions of several endogenous genes including c-fos,JunB,Srf, andCyr61. BDNF activation of MKL1/SRF-driven transcription was dependent on the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, which also led to MKL1 phosphorylation. Finally, synaptic activity stimulation of SRF-driven transcription was reduced by inhibition of endogenous MKL1. Conversely, synaptic activity enhanced transcription by overexpressed MKL1. MKL1 regulation by synaptic activity was mediated through the NMDA receptor-activated ERK1/2. These results suggest that neuronal MKL1 contributes to SRF-regulated gene expression induced by BDNF or synaptic activity. In addition, MKL1 appears as a novel mediator of the signaling between ERK1/2 and SRF. Moreover, MKL1 is a likely regulator of SRF-driven transcription programs that underlie neuronal plasticity.

Background: Quantitative 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. Results: The 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. Conclusions: We 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.