Lotfi Ferhat

Synaptic Regulation of Microtubule Dynamics in Dendritic Spines by Calcium, F-Actin, and Drebrin

Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.

Drebrin A regulates dendritic spine plasticity and synaptic function in mature cultured hippocampal neurons

Drebrin A, one of the most abundant neuron-specific F-actin-binding proteins, is found exclusively in dendrites and is particularly concentrated in dendritic spines receiving excitatory inputs. We investigated the role of drebrin A in synaptic transmission and found that overexpression of drebrin A augmented the glutamatergic synaptic transmission, probably through an increase of active synaptic site density. Interestingly, overexpression of drebrin A also affected the frequency, amplitude and kinetics of miniature inhibitory postsynaptic currents (mIPSCs), despite the fact that GABAergic synapse density and transmission efficacy were not modified. Downregulation of drebrin A led to a decrease of both glutamatergic and GABAergic synaptic activity. In heterologous cells, drebrin A reorganized and stabilized F-actin and these effects were mediated by its actin-binding domain. Thus, drebrin A might regulate dendritic spine morphology via regulation of actin cytoskeleton remodeling and dynamics. Our data demonstrate for the first time that drebrin A modulates glutamatergic and GABAergic synaptic activities.

Changes in vesicular transporters for γ-aminobutyric acid and glutamate reveal vulnerability and reorganization of hippocampal neurons following pilocarpine-induced seizures

The reorganizations of the overall intrinsic glutamatergic and γ‐aminobutyric acid (GABA)‐ergic hippocampal networks as well as the time course of these reorganizations during development of pilocarpine‐induced temporal lobe epilepsy were studied with in situ hybridization and immunohistochemistry experiments for the vesicular glutamate transporter 1 (VGLUT1) and the vesicular GABA transporter (VGAT). These transporters are particularly interesting as specific markers for glutamatergic and GABAergic neurons, respectively, whose expression levels could reflect the demand for synaptic transmission and their average activity. We report that 1) concomitantly with the loss of some subpopulations of VGAT‐containing neurons, there was an up‐regulation of VGAT synthesis in all remaining GABA neurons as early as 1 week after pilocarpine injection. This enhanced synthesis is characterized by marked increases in the relative amount of VGAT mRNAs in interneurons associated with increased intensity of axon terminal labeling for VGAT in all hippocampal layers. 2) There was a striking loss of mossy cells during the latent period, demonstrated by a long‐term decrease of VGLUT1 mRNA‐containing hilar neurons and associated loss of VGLUT1‐containing terminals in the dentate gyrus inner molecular layer. 3) There were aberrant VGLUT1‐containing terminals at the chronic stage resulting from axonal sprouting of granule and pyramidal cells. This is illustrated by a recovery of VGLUT1 immunoreactivity in the inner molecular layer and an increased VGLUT1 immunolabeling in the CA1–CA3 dendritic layers. These data indicate that an increased activity of remaining GABAergic interneurons occurs during the latent period, in parallel with the loss of vulnerable glutamatergic and GABAergic neurons preceding the reorganization of glutamatergic networks. J. Comp. Neurol. 503:466–485, 2007.

Basic Fibroblast Growth Factor-Induced Increase in zif/268 and c-fos mRNA Levels Is Ca2+ Dependent in Primary Cultures of Hippocampal Neurons

Abstract: Basic fibroblast growth factor (bFGF) is present in the developing rat brain and has been shown to provide critical trophic support for hippocampal neurons in culture. The influence of bFGF on the expression of mRNAs encoding the transcription factors zif/268 and c‐fos was studied in primary cultures of hippocampal neurons (derived from rat embryos) using reverse transcription‐coupled PCR. In these cultures grown for 3 days in the absence of serum, bFGF causes a dramatic and transient increase in the levels of zif/268 and c‐fos, within 15 and 30 min, respectively. A similar induction of these two early genes occurs following activation of protein kinase C (PKC). The bFGF‐induced activation persists after PKC desensitization but is inhibited by chelation of intracellular Ca2+. These results suggest that in primary cultures of hippocampal neurons, bFGF induces the expression of immediate early genes through a pathway that requires Ca2+ mobilization.

TRANSIENT INCREASE OF TENASCIN-C IN IMMATURE HIPPOCAMPUS : ASTROGLIAL AND NEURONAL EXPRESSION

SummaryIn the present report we describe the anatomical localization of cells expressing tenascin-C, an extracellular matrix glycoprotein, in the hippocampal complex of developing rats. We report a development-dependent down regulation of both tenascin-C protein and mRNA. The highest levels of expression of tenascin-C was observed in rat pups from embryonic day 18 to postnatal day 7. Double labelling experiments performed with a tenascin-C antibody or tenascin-C probes combined with specific markers of astrocytes (GFAP) or neurons (MAP2 and Tau) allowed us to demonstrate that tenascin-C is expressed by both immature astrocytes and neurons in immature hippocampus. The temporal and topographic distribution of cells expressing of post-mitotic cells. In view of these data we discuss the hypothesis that tenascin-C, as a mediator of neuron-glia interaction, may contribute to the development of hippocampal cells.

Acidic Calponin Cloned from Neural Cells is Differentially Expressed During Rat Brain Development

Calponin is an actin‐, tropomyosin‐ and Ca2+ calmodulin‐binding protein that inhibits in vitro the actomyosin MgATPase. Basic and acidic variants of calponin have been described to date. Although the cerebral expression of calponin remained controversial for some time, transcripts encoding acidic calponin in the adult rat brain and calponin immunoreactivity in rat and pig brain and in cultured cerebellar cells have been reported. In the present work, we report the expression of acidic calponin mRNAs and the isolation of cDNAs encoding the full‐length acidic calponin in cultured neuronal and glial cells and in adult rat brain. Sequence analysis reveals that acidic calponin in the brain is identical to that previously described in rat aortic vascular smooth muscle. In situ hybridization shows that calponin is highly expressed during ontogenesis in granule cells of the dentate gyrus of the hippocampus, in all layers of the olfactory bulb and in cerebellar granule neurons of the external and internal layers. In the adult rat brain, calponin expression decreased in these fields, but increased in choroid plexus cells. Bergmann glial cells were also labelled by a calponin probe. The reverse transcription‐coupled polymerase chain reaction confirms that calponin mRNA levels are highest in the early stages of hippocampal development and that expression levels are low in adult hippocampi. The developmental expression pattern of brain acidic calponin suggests that calponin could be involved in contractile activity associated with neural cell proliferation or neuronal migration.

Seizures induce tenascin-C mRNA expression in neurons.

SummaryTenascin-C, an extracellular matrix glycoprotein that exhibits both growth-promoting and growth-inhibiting properties, is produced in the CNS mainly by astrocytes. In the present study we show that kainate-induced seizures result in an increased expression of tenascin-C in rat brain. Tenascin-C mRNA was increased mainly in the granule cell layer of the hippocampal complex, but tenascin-C mRNA expression was also observed in the pyriform cortex and amygdalo-cortical nucleus. Double labelling experiments using tenascin-C probes and MAP2 (a neuronal microtubule associated protein) antibodies revealed many neurons in these layers that express tenascin-C mRNA. These results support our previous findings of an increased tenascin-C immunoreactivity associated with the axons of granule cells. Tenascin-C expression is rapidly induced by seizures (6h), preceding any lesion and glial reaction. In this pathological condition tenascin-C appears to be produced by both glia and neurons. The functional repercussions on the scarring and remodelling processes are also discussed.

Structure, regional and developmental expression of rat MAP2d, a MAP2 splice variant encoding four microtubule-binding domains

MAP2, a major component of microtubule polymers in neurons consists of high molecular weight (HMW) proteins MAP2a, MAP2b and a low molecular weight (LMW) MAP2c, expressed in the developing brain. These isoforms are produced from a single gene by alternative splicing and share identical C-termini encompassing 3 tandem repeats, critical in microtubule binding. We describe the structure, regional and developmental expression of a novel MAP2 splice variant, MAP2d, containing an insertion whose sequence is homologous to the three and four repeats of MAP2 and Tau respectively. This insertion is absent from the mRNAs encoding HMW MAP2. MAP2d mRNAs are expressed at higher levels than MAP2c in all adult nervous tissues of the rat, and are found at low levels in glial cell cultures when compared to primary cultures of cerebellar neurons. Splicing of the fourth repeat in mature Tau precedes that in MAP2d during rat brain development. The tardive expression of a four microtubule-binding domain LMW MAP2 suggests it could play in extended neurites a similar role as mature Tau in axons.

Trafficking and secretion of matrix metalloproteinase-2 in olfactory ensheathing glial cells: A role in cell migration?

Olfactory ensheathing cells (OECs) are unique glia found only in the olfactory system. They retain exceptional plasticity and support olfactory neurogenesis and retargeting across the PNS:CNS boundary in the olfactory system. OECs have been shown to improve functional outcome when transplanted into rodents with spinal cord injury. The growth‐promoting properties of implanted OECs encompass their ability to migrate through the scar tissue and render it more permissive for axonal outgrowth, but the underlying molecular mechanisms remain poorly understood. OECs appear to regulate molecules of the extracellular matrix (ECM) that inhibit axonal growth. Among the proteins that have the potential to promote cell migration, axonal regeneration and remodeling of the ECM are matrix metalloproteinases (MMPs), a family of endopeptidases that cleave matrix, soluble, and membrane‐bound proteins and that are regulated by their endogenous inhibitors, the tissue inhibitors of MMPs (TIMPs). Little is known about MMP/TIMP trafficking, secretion, and role in OECs. Using a combination of cell biology, biochemistry, pharmacology, and imaging techniques, we show that MMP‐2 and MMP‐9 are expressed and proteolytically active in the olfactory epithelium and in particular in the OECs of the lamina propria. These proteinases and regulatory proteins such as MT1‐MMP and TIMP‐2 are expressed in cultured OECs. MMPs exhibit nuclear localization and vesicular trafficking and secretion, with distribution along microtubules and microfilaments and co‐localization with the molecular motor protein kinesin. Finally, we show that MMPs are involved in migration of OECs in vitro on different ECM substrates.

Role of drebrin A in dendritic spine plasticity and synaptic function Implications in neurological disorders

Drebrin A is one of the most abundant neuron-specific binding proteins of F-actin and its expression is increased in parallel with synapse formation. Drebrin A is particularly concentrated in dendritic spines, postsynaptic sides of excitatory glutamatergic synapses. More recently, Ferhat and colleagues reported the functional role of drebrin A in regulating synaptic transmission. Indeed, our study showed that overexpression of drebrin A induced an increase of glutamatergic but not GABAergic synapses and resulted in the alteration of the normal excitatory-inhibitory ratio in favour of excitation in mature hippocampal neurons. Downregulation of drebrin A expression by antisense oligonucleotides resulted in the decrease of both miniature- glutamatergic and GABAergic synaptic activities without affecting the excitatory-inhibitory ratio. Studies performed in heterologous cells revealed that drebrin A reorganized the actin filaments and stabilized them and that these effects are depend upon its actin-binding domain. These results suggest that drebrin A regulates dendritic spine morphology, size and density, presumably via regulation of actin cytoskeleton remodeling and dynamics. These data demonstrate for the first time that an actin-binding protein such as drebrin A regulates both glutamatergic and GABAergic synaptic transmissions, probably through an increase of active synaptic site density for glutamatergic transmission and through homeostatic mechanisms for the GABAergic one.