Annette Koulakoff

Doublecortin is a developmentally regulated, microtubule-associated protein expressed in migrating and differentiating neurons.

Recently, we and others reported that the doublecortin gene is responsible for X-linked lissencephaly and subcortical laminar heterotopia. Here, we show that Doublecortin is expressed in the brain throughout the period of corticogenesis in migrating and differentiating neurons. Immunohistochemical studies show its localization in the soma and leading processes of tangentially migrating neurons, and a strong axonal labeling is observed in differentiating neurons. In cultured neurons, Doublecortin expression is highest in the distal parts of developing processes. We demonstrate by sedimentation and microscopy studies that Doublecortin is associated with microtubules (MTs) and postulate that it is a novel MAP. Our data suggest that the cortical dysgeneses associated with the loss of Doublecortin function might result from abnormal cytoskeletal dynamics in neuronal cell development.

A Novel CNS Gene Required for Neuronal Migration and Involved in X-Linked Subcortical Laminar Heterotopia and Lissencephaly Syndrome

X-SCLH/LIS syndrome is a neuronal migration disorder with disruption of the six-layered neocortex. It consists of subcortical laminar heterotopia (SCLH, band heterotopia, or double cortex) in females and lissencephaly (LIS) in males, leading to epilepsy and cognitive impairment. We report the characterization of a novel CNS gene encoding a 40 kDa predicted protein that we named Doublecortin and the identification of mutations in four unrelated X-SCLH/LIS cases. The predicted protein shares significant homology with the N-terminal segment of a protein containing a protein kinase domain at its C-terminal part. This novel gene is highly expressed during brain development, mainly in fetal neurons including precursors. The complete disorganization observed in lissencephaly and heterotopia thus seems to reflect a failure of early events associated with neuron dispersion.

Astroglial Metabolic Networks Sustain Hippocampal Synaptic Transmission

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.

Astroglial networks: a step further in neuroglial and gliovascular interactions

Dynamic aspects of interactions between astrocytes, neurons and the vasculature have recently been in the neuroscience spotlight. It has emerged that not only neurons but also astrocytes are organized into networks. Whereas neuronal networks exchange information through electrical and chemical synapses, astrocytes are interconnected through gap junction channels that are regulated by extra- and intracellular signals and allow exchange of information. This intercellular communication between glia has implications for neuroglial and gliovascular interactions and hence has added another level of complexity to our understanding of brain function.

Gap junctions and connexin expression in the normal and pathological central nervous system

Summry— Gap junctions are widely expressed in the various cell types of the central nervous system. These specialized membrane intercellular junctions provide the morphological support for direct electrical and biochemical communication between adjacent cells. This intercellular coupling is controlled by neurotransmitters and other endogenous compounds produced and released in basal as well as in pathological situations. Changes in the expression and the function of connexins are associated with number of brain pathologies and lesions suggesting that they could contribute to the expansion of brain damages. The purpose of this review is to summarize data presently available concerning gap junctions and the expression and function of connexins in different cell types of the central nervous system and to present their physiopathological relevance in three major brain dysfunctions: inflammation, epilepsy and ischemia.

Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by β-amyloid

Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)‐astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin‐1? (IL‐1?) and tumor necrosis factor‐? (TNF‐?) were identified as being the main factors responsible for this conditioned medium‐mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimers disease, at sites of β‐amyloid (A?) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL‐1? and TNF‐? activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

Gap Junction-Mediated Astrocytic Networks in the Mouse Barrel Cortex

The barrel field of the somatosensory cortex constitutes a well documented example of anatomofunctional compartmentalization and activity-dependent interaction between neurons and astrocytes. In astrocytes, intercellular communication through gap junction channels composed by connexin 43 and 30 underlies a network organization. Immunohistochemical and electrophysiological experiments were undertaken to determine the coupling properties of astrocyte networks in layer IV of the developing barrel cortex. The expression of both connexins was found to be enriched within barrels compared with septa and other cortical layers. Combination of dye-coupling experiments performed with biocytin and immunostaining with specific cell markers demonstrated that astrocytic networks do not involve neurons, oligodendrocytes or NG2 cells. The shape of dye coupling was oval in the barrel cortex whereas it was circular in layer IV outside the barrel field. Two-dimensional analysis of these coupling areas indicated that gap junctional communication was restricted from a barrel to its neighbor. Such enrichment of connexin expression and transversal restriction were not observed in a transgenic mouse lacking the barrel organization, whereas they were both observed in a double-transgenic mouse with restored barrels. Direct observation of sulforhodamine B spread indicated that astrocytes located between two barrels were either weakly or not coupled, whereas coupling within a barrel was oriented toward its center. These observations indicated a preferential orientation of coupling inside the barrels resulting from subpopulations of astrocytes with different coupling properties that contribute to shaping astrocytic networks. Such properties confine intercellular communication in astrocytes within a defined barrel as previously reported for excitatory neuronal circuits.

Lysosomes Are the Major Vesicular Compartment Undergoing Ca2+-Regulated Exocytosis from Cortical Astrocytes

Although Ca2+-dependent exocytosis is considered to be a pathway for gliotransmitter release from astrocytes, the structural and functional bases of this process remain controversial. We studied the relationship between near-membrane Ca2+ elevations and the dynamics of single astroglial vesicles with styryl (FM) dyes. We show that cultured astrocytes, unlike neurons, spontaneously internalize FM dyes, resulting in the labeling of the entire acidic vesicle population within minutes. Interestingly, metabotropic glutamate receptor activation did not affect the FM labeling. Most FM-stained vesicles expressed sialin, CD63/LAMP3, and VAMP7, three markers for lysosomes and late endosomes. A subset of lysosomes underwent asynchronous exocytosis that required both Ca2+ mobilization from intracellular stores and Ca2+ influx across the plasma membrane. Lysosomal fusion occurred within seconds and was complete with no evidence for kiss and run. Our experiments suggest that astroglial Ca2+-regulated exocytosis is carried by lysosomes and operates on a timescale orders of magnitude slower than synaptic transmission.

The RhoGAP activity of OPHN1, a new F-actin-binding protein, is negatively controlled by its amino-terminal domain.

Recent human genetic approaches showed that mutations in three genes encoding OPHN1, PAK3, and alphaPIX cause nonspecific X-linked mental retardation. These three proteins act to modulate Rho GTPase signaling pathways and may participate in neuronal morphogenesis by regulating the actin cytoskeleton. Here we showed that the Oligophrenin-1 gene is expressed in the developing spinal cord and later in brain areas that are characterized by high synaptic plasticity. At the cellular level OPHN1 is expressed in both glial and neuronal cells where it colocalizes with actin, notably at the tip of growing neurites. This interaction seems to be direct through a novel uncharacterized domain in the carboxyl-terminal end of OPHN1. Overexpression experiments in fibroblasts showed that the OPHN1 RhoGAP domain regulates in vivo the actin cytoskeleton by inhibition of Rho pathways. Interestingly the amino-terminal domain of OPHN1 inhibits the RhoGAP activity through an as yet unknown mechanism, suggesting that OPHN1 may be tightly regulated in vivo.

Adenoviral Vector as a Gene Delivery System into Cultured Rat Neuronal and Glial Cells

Previous studies have demonstrated that a defective recombinant adenovirus can infect a wide range of postmitotic and slowly proliferating cell types such as hepatocytes, myotubes, pneumocytes and intestinal cells (Stratford‐Perricaudet et al., Hum. Gene Ther., 1, 241–256, 1990; Quantin et al., Proc. Natl. Acad. Sci. USA, 89, 2581–2584, 1992; Jaffe et al., Nature Genetics, 1, 372–378, 1992). We have used a defective recombinant adenovirus, Ad.RSVβgal, containing the Escherichia coliβ‐galactosidase gene targeted to the nucleus under the transcriptional control of the Rous sarcoma virus long terminal repeat promoter (Stratford‐Perricaudet et al., J. Clin. Invest., 90, 626–630, 1992) to infect non‐dividing neural cells in primary culture. We show that 80–100% of neuronal and astroglial cells infected with a viral titre lower than 109 p.f.u./ml express β‐galactosidase for at least 1 month without cell damage. These results demonstrate the potential usefulness of recombinant adenovirus infection for the analysis of brain‐specific gene regulation and for the transfer of genes into neural cells before their transplantation into the brain.