Monika Bijata

Matrix Metalloproteinases Regulate the Formation of Dendritic Spine Head Protrusions during Chemically Induced Long-Term Potentiation

Dendritic spines are are small membranous protrusions that extend from neuronal dendrites and harbor the majority of excitatory synapses. Increasing evidence has shown that matrix metalloproteinases (MMPs), a family of extracellularly acting and Zn2+-dependent endopeptidases, are able to rapidly modulate dendritic spine morphology. Spine head protrusions (SHPs) are filopodia-like processes that extend from the dendritic spine head, representing a form of postsynaptic structural remodeling in response to altered neuronal activity. Herein, we show that chemically induced long-term potentiation (cLTP) in dissociated hippocampal cultures upregulates MMP-9 activity that controls the formation of SHPs. Blocking of MMPs activity or microtubule dynamics abolishes the emergence of SHPs. In addition, autoactive recombinant MMP-9, promotes the formation of SHPs in organotypic hippocampal slices. Furthermore, spines with SHPs gained postsynaptic α-amino-3-hydroxyl-5-methyl-4-isoxazole propionic acid (AMPA) receptors upon cLTP and the synaptic delivery of AMPA receptors was controlled by MMPs. The present results strongly imply that MMP-9 is functionally involved in the formation of SHPs and the control of postsynaptic receptor distribution upon cLTP.

Synaptically released matrix metalloproteinase activity in control of structural plasticity and the cell surface distribution of GluA1-AMPA receptors.

Synapses are particularly prone to dynamic alterations and thus play a major role in neuronal plasticity. Dynamic excitatory synapses are located at the membranous neuronal protrusions called dendritic spines. The ability to change synaptic connections involves both alterations at the morphological level and changes in postsynaptic receptor composition. We report that endogenous matrix metalloproteinase (MMP) activity promotes the structural and functional plasticity of local synapses by its effect on glutamate receptor mobility and content. We used live imaging of cultured hippocampal neurons and quantitative morphological analysis to show that chemical long-term potentiation (cLTP) induces the permanent enlargement of a subset of small dendritic spines in an MMP-dependent manner. We also used a superresolution microscopy approach and found that spine expansion induced by cLTP was accompanied by MMP-dependent immobilization and synaptic accumulation as well as the clustering of GluA1-containing AMPA receptors. Altogether, our results reveal novel molecular and cellular mechanisms of synaptic plasticity.

Experience-Dependent Plasticity of the Barrel Cortex in Mice Observed with 2-DG Brain Mapping and c-Fos: Effects of MMP-9 KO

Modifications of properties of the adult sensory cortex by elimination of sensory input (deprivation) serves as a model for studying plasticity in the adult brain. We studied the effects of short- and long-term deprivation (sparing one row of vibrissae) upon the barrel cortex. The response to stimulation (exploration of a new environment) of the spared row was examined with [14C]-2-deoxyglucose autoradiography and c-Fos immunohistochemistry. Both methods found large increases of the functional cortical representation of the spared row of vibrissae, extending into parts of the barrel cortex previously activated by the deprived vibrissae. With both methods, the greatest expansion of spared input was observed in cortical layer IV. In this way, we established a model, which was applied for examining involvement of matrix metalloproteinase 9 (MMP-9), upon experience-dependent cortical plasticity. MMP-9 is an enzyme implicated in plastic modification of the neuronal connections. We found that MMP-9 activity was increased in response to stimulation, and furthermore, MMP-9 knockout mice showed a modest but significant decrease of plasticity in layer IV with 2-DG mapping and in layers II/III with c-Fos mapping. Thus, in adult mouse brain experience-dependent plasticity is in part supported by the activity of MMP-9.

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.

Serotonin 5-HT7 receptor increases the density of dendritic spines and facilitates synaptogenesis in forebrain neurons

Precise control of dendritic spine density and synapse formation is critical for normal and pathological brain functions. Therefore, signaling pathways influencing dendrite outgrowth and remodeling remain a subject of extensive investigations. Here, we report that prolonged activation of the serotonin 5‐HT7 receptor (5‐HT7R) with selective agonist LP‐211 promotes formation of dendritic spines and facilitates synaptogenesis in postnatal cortical and striatal neurons. Critical role of 5‐HT7R in neuronal morphogenesis was confirmed by analysis of neurons isolated from 5‐HT7R‐deficient mice and by pharmacological inactivation of the receptor. Acute activation of 5‐HT7R results in pronounced neurite elongation in postnatal striatal and cortical neurons, thus extending previous data on the morphogenic role of 5‐HT7R in embryonic and hippocampal neurons. We also observed decreased number of spines in neurons with either genetically (i.e. 5‐HT7R‐knock‐out) or pharmacologically (i.e. antagonist treatment) blocked 5‐HT7R, suggesting that constitutive 5‐HT7R activity is critically involved in the spinogenesis. Moreover, cyclin‐dependent kinase 5 and small GTPase Cdc42 were identified as important downstream effectors mediating morphogenic effects of 5‐HT7R in neurons. Altogether, our data suggest that the 5‐HT7R‐mediated structural reorganization during the postnatal development might have a crucial role for the development and plasticity of forebrain areas such as cortex and striatum, and thereby can be implicated in regulation of the higher cognitive functions.

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).

Dystroglycan controls dendritic morphogenesis of hippocampal neurons in vitro.

Dendritic outgrowth and arborization are important for establishing neural circuit formation. To date, little information exists about the involvement of the extracellular matrix (ECM) and its cellular receptors in these processes. In our studies, we focus on the role of dystroglycan (DG), a cell adhesion molecule that links ECM components to the actin cytoskeleton, in dendritic development and branching. Using a lentiviral vector to deliver short-hairpin RNA (shRNA) that specifically silences DG in cultured hippocampal neurons, we found that DG knockdown exerted an inhibitory effect on dendritic tree growth and arborization. The structural changes were associated with activation of the guanosine triphosphatase Cdc42. The overexpression of DG promoted dendritic length and branching. Furthermore, exposure of the cultures to autoactivating matrix metalloproteinase-9 (aaMMP-9), a β-DG-cleaving protease, decreased the complexity of dendritic arbors. This effect was abolished in neurons that overexpressed a β-DG mutant that was defective in MMP-9-mediated cleavage. Altogether, our results indicate that DG controls dendritic arborization in vitro in MMP-9-dependent manner.

Stress-induced changes in the S-palmitoylation and S-nitrosylation of synaptic proteins

The precise regulation of synaptic integrity is critical for neuronal network connectivity and proper brain function. Essential aspects of the activity and localization of synaptic proteins are regulated by posttranslational modifications. S-palmitoylation is a reversible covalent modification of the cysteine with palmitate. It modulates affinity of the protein for cell membranes and membranous compartments. Intracellular palmitoylation dynamics are regulated by other posttranslational modifications, such as S-nitrosylation. Still unclear, however, are the ways in which this crosstalk is affected in brain pathology, such as stress-related disorders. Using a newly developed mass spectrometry-based approach (Palmitoylation And Nitrosylation Interplay Monitoring), we analyzed the endogenous S-palmitoylation and S-nitrosylation of postsynaptic density proteins at the level of specific single cysteines in a mouse model of chronic stress. Our results suggest that atypical mechanism of crosstalk between the S-palmitoylation and S-nitrosylation of synaptic proteins might be one of the major events associated with chronic stress disorders.