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.

Reversible polyglutamylation of alpha- and beta-tubulin and microtubule dynamics in mouse brain neurons.

The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons. Selective posttranslational labeling with [3H]glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. Nocodazole markedly inhibited incorporation of [3H]glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain. Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. It thus appears that the slow rate component of the deglutamylation reaction offers to neurons the possibility to maintain a basal level of glutamylated alpha-tubulin in the soluble pool independently of microtubule dynamics. Finally, some differences observed in the glutamylation of alpha- and beta-tubulin suggest that distinct enzymes are involved.

Astroglial connexin43 contributes to neuronal suffering in a mouse model of Alzheimer's disease.

In Alzheimer's disease (AD), astrocyte properties are modified but their involvement in this pathology is only beginning to be appreciated. The expression of connexins, proteins forming gap junction channels and hemichannels, is increased in astrocytes contacting amyloid plaques in brains of AD patients and APP/PS1 mice. The consequences on their channel functions was investigated in a murine model of familial AD, the APPswe/PS1dE9 mice. Whereas gap junctional communication was not affected, we revealed that hemichannels were activated in astrocytes of acute hippocampal slices containing Aß plaques. Such hemichannel activity was detected in all astrocytes, whatever their distance from amyloid plaques, but with an enhanced activity in the reactive astrocytes contacting amyloid plaques. Connexin43 was the main hemichannel contributor, however, a minor pannexin1 component was also identified in the subpopulation of reactive astrocytes in direct contact with plaques. Distinct regulatory pathways are involved in connexin and pannexin hemichannel activation. Inflammation triggered pannexin hemichannel activity, whereas connexin43 hemichannels were activated by the increase in resting calcium level of astrocytes. Importantly, hemichannel activation led to the release of ATP and glutamate that contributed to maintain a high calcium level in astrocytes placing them in the center of a vicious circle. The astroglial targeted connexin43 gene knocking-out in APPswe/PS1dE9 mice allowed to diminish gliotransmitter release and to alleviate neuronal damages, reducing oxidative stress and neuritic dystrophies in hippocampal neurons associated to plaques. Altogether, these data highlight the importance of astroglial hemichannels in AD and suggest that blocking astroglial hemichannel activity in astrocytes could represent an alternative therapeutic strategy in AD.

[Gap junction-mediated intercellular communication in astrocytes and neuroprotection]. / Communication jonctionnelle entre astrocytes et neuroprotection.

Neuroglial interaction represents a concept that is now more and more integrated in the attempts to understand who does what and how in neuronal processing and survival, in normal as well as in pathological situations. The purpose of the review is to provide an overlook about the role of glial cells, mainly astrocytes, in neuroprotection. Since a typical feature of glia is to be connected by gap junctions that allow them to be organized as a communicating network(s), we will focus this review on what is known about the contribution of astrocyte gap junctions (AGJ) in neuronal survival. As neuroglial interaction and AGJ are both affected during neurodegenerative diseases, we will also consider the above mentioned glial properties in a pathological context with a special interest in Alzheimer's disease.

Neurotropism of rabies virus. An in vitro study.

The relative susceptibility of neurons and glia, grown as monolayers in vitro, to rabies virus infection was explored. Established cell lines of neuronal or glial phenotype and primary cultures of cells derived from mouse dorsal root ganglia (DRC) or brain were used as homologues of the targets of rabies virus in the nervous system. Fixed rabies virus (CVS) strain was used in most experiments; other fixed rabies strains (PV, HEP, ERA) and a street rabies virus isolate were used in some. Virus-cell tropism was determined by immunofluorescence assay for rabies nucleocapsid antigen and cell permissivity was assessed by titration of virus yields. Neuronal cells always exhibited a much greater susceptibility to infection and a greater propensity to sustain viral growth. By immunofluorescence, 90-100% of neurons commonly had viral inclusion bodies, while doses of the virus three to four orders of magnitude higher still left greater than 99% of astrocytes, in brain cell cultures and 90 +/- 5% of the non-neuronal cells in DRG cultures without any obvious signs of rabies virus. Neuroblastoma cells (95 +/- 5% with viral antigens) produced viral yields about four orders of magnitude higher than glioma cells (10 +/- 5% with viral antigens). Though the overall infectivity of street virus was lower than that of fixed virus strains, a significantly higher viral tropism for neurons than for glia was maintained. Thus, primary neuronal cultures offer a means of exploring molecular events in rabies virus infection and their role in pathogenesis.

Expression of the transcripts initiated in the 62nd intron of the dystrophin gene.

The pattern of expression of two distal transcripts initiated in the 62nd intron of the dystrophin gene was investigated under different circumstances; (i) during the development of different rat tissues these transcripts and Dp71, a protein encoded by one of them, increased with brain development and decreased with muscle development; (ii) in cultured glial and neuronal cells, the distal promoter was coactivated with tissue-specific upstream promoters, the muscle-type promoter in glial cells and the brain-type promoter in neuronal cells, which suggests that activity of the upstream promoter does not interfere with activity of the distal promoter; (iii) in lymphoblasts of DMD patients with various deletions of the dystrophin gene, the most distal of which included the 56th intron, the production of the distal transcript was not perturbed.

A correlation between the appearance and the evolution of tetanus toxin binding cells and neurogenesis.

The ontogenesis of cells expressing surface membrane binding sites for tetanus toxin (Tt) was studied in the mouse nervous system. Cells were labeled shortly after the tissue dissociation and the toxin bound was revealed by immunofluorescence. In the brain, spinal cord and dorsal root ganglia the toxin binding cells (TBC) are found as of very early stages of nervous system organogenesis, i.e. at 10 days of gestation. There is a close temporal correlation between the pattern of emergence and accumulation of TBC and the known pattern of appearance of post-mitotic neurons in mouse cerebral cortex, cerebellum and spinal cord. The curves of TBC abundance as a function of fetal age in various nervous system areas are different. They show regional fluctuations in the proportion of TBC that reflect the cumulative changes in the dynamics of neuronal subpopulations. The results indicate that Tt can be used as an ontogenetically early marker of neuronal differentiation and that the acquisition of Tt receptors may represent one of the earliest detectable characteristics of the developing neurons.

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

Neurotoxins as probes in the study of neuronal development.

We have investigated the expression of surface membrane binding sites for tetanus toxin and alpha-scorpion toxin (AaHII) on cells of the in vivo developing mouse nervous system. There is a close temporal correlation in the pattern of emergence and accumulation of tetanus toxin binding cells (TBC) and that of post-mitotic neurons. In different nervous system areas, the fluctuations in relative TBC abundance reflect regional changes in the dynamics of neuronal subpopulations. The results indicate that the acquisition of membrane tetanus toxin binding sites may represent one of the earliest detectable characteristics of nascent neurons. The Na+ channel-associated scorpion toxin become detectable in fetal mouse brain two days after the appearance of TBC. Their density increases with fetal age without change in receptor properties. At all stages, scorpion toxin binds to a single class of noninteracting sites with a KD = 0.1 - 0.5 nM. The affinity of binding is voltage-dependent. Studies on brain cells and various cell lines grown in vitro suggest a selective association of the high affinity scorpion toxin receptors with neuronal phenotype. In culture, as in vivo, there is a time dependent increase in receptor density. These results indicate that both tetanus toxin and scorpion toxin can be used as qualitative markers of neuronal differentiation; moreover, estimates of the density of scorpion toxin binding sites provide a quantitative index of neuronal maturation.

Astroglial connexin immunoreactivity is specifically altered at ß-amyloid plaques in ß-amyloid precursor protein/presenilin1 mice.

Activation of astrocytes surrounding amyloid plaques is a hallmark of Alzheimer disease (AD) with consequences yet poorly understood. Astrocytes are characterized by a high level of intercellular communication mediated by two gap-junction forming proteins, connexin-43 and connexin-30. As astroglial connexins (Cxs) are involved in neuronal dysfunctions and death, we have analyzed their expression pattern in two murine models of AD, that is two different ß-amyloid precursor protein (APP)/presenilin1(PS1) mice, using western blot and immunohistochemistry analyzed in confocal microscopy. In young mice at 2 months, before the emergence of ß-amyloid (Aß) deposits, the distribution of both Cxs was similar to that of control mice. In older animals≥4 months, local modifications in connexin immunostaining pattern were observed in the microenvironment of dense core Aß plaques. In a majority of plaques, an elevated immunoreactivity was detected for both Cxs contributing to the overall increase in connexin expression detected in 18 month old APP/PS1 mice. Activated microglial cells did not contribute to the elevated connexin immunoreactivity that was concentrated in astroglial processes infiltrating the plaques. In a small proportion of plaques (≤15%) a depletion of immunoreactive connexin puncta was also found. As astroglial Cxs participate in neuroglial interactions, their remodeling may contribute to neuronal alterations observed at the periplaque area.

Cultured neurons from mouse brain reproduce the muscarinic receptor profile of their tissue of origin.

These studies investigate the regional variations in the muscarinic acetylcholine receptor (mAChR) profiles in neuron populations of the CNS using primary neuron cultures derived from three areas of the mouse brain--the cerebral hemispheres, the mesencephalon and the medulla-pons--that have distinct mAChR systems. We first assessed the extent to which neurons reproduced their in vivo properties in culture by monitoring the binding capacity, the pharmacological profiles and the levels of mAChR transcripts in neuron cultures and their tissues of origin. We showed that the primary neuron cultures accumulated mAChRs with initial rates similar to those in vivo, had pharmacological profiles very close to those of their area of origin, and accumulated m1, m2, m3, m4 and m5 receptor transcripts according to patterns resembling those in the tissues. We conclude that most of the characteristics of the mAChRs in a given area are proper to the neuron population of that area, that the pattern is established early in ontogenesis, and that it is reproduced in vitro. We also show that the stimulation of phosphoinositide turnover is mediated by mAChRs with distinct pharmacological profiles in neuron cultures from the three brain areas.

Muscarinic receptor profiles of mouse brain astrocytes in culture vary with their tissue of origin but differ from those of neurons.

The two main cell populations in brain tissues are neurons and astrocytes. Cultures of both bear muscarinic acetylcholine receptors (mAChRs). Available data indicate that astrocyte mACRs are heterogeneous, but the particular subtypes on these cells are not known, nor is there any information as to whether there is a regional variation in the mAChR profile of astrocytes. This paper describes the mAChR profiles of cultured astrocytes derived from the cerebral hemispheres, mesencephalon and medulla-pons, and is a continuation of our study on cultures of neurons from these same tissues. Pharmacological studies showed that astrocytes accumulated small amounts of mAChRs with distinct pharmacological profiles which, for a given area, differed from those of neurons in culture. Northern blot analyses showed transcripts for m1 and m3 mAChRs only. Their concentrations differed from one cell population to another. Astrocyte cultures from the mesencephalon contained m1 mRNA amounts close to those in the tissue. Thus, at least part of the mAChR profile in vitro might be a true reflection of the cell's properties in vivo. Functional studies showed that mAChRs mediate the stimulation of phosphoinositide turnover in all three astrocyte cultures, that the amplitude of this response varies greatly with the origin of the cell, and that two pharmacological subclasses, M1 and M1-2-, are involved in these responses, but to different extents. Thus the CNS contains discrete astrocyte populations which in culture differ in their mAChR profiles at the molecular, the pharmacological and the functional levels.

Neuronal acquisition of tetanus toxin binding sites: relationship with the last mitotic cycle.

In an earlier study on the developing nervous system, the existence of a temporal correlation between the appearance of tetanus toxin-binding cells and neurogenesis was reported (A. Koulakoff, B. Bizzini, and Y. Berwald-Netter (1982). Dev. Brain Res. 5, 139-147). Using a combined approach of immunocytochemistry and [3H]thymidine autoradiography it is shown that, in the fetal mouse central nervous system, dividing cells do not express membrane binding sites for tetanus toxin. A time-course quantitative autoradiography revealed that the toxin-binding sites become apparent within 7 +/- 1 hr, following the last S phase, on cells undergoing the conversion from dividing to postmitotic state. The acquisition of surface binding sites for tetanus toxin may thus be an early property of nascent central neurons, marking the transition from cycling precursor neuroblasts to postmitotic neuronal cells. Parallel studies on in vivo-developing dorsal root ganglia disclosed that at least some peripheral nervous system cells are endowed with tetanus toxin-binding capacity while still capable of DNA synthesis and undergo one or more divisions.

Carbenoxolone blockade of neuronal network activity in culture is not mediated by an action on gap junctions.

Spontaneous activity in the central nervous system is strongly suppressed by blockers of gap junctions (GJs), suggesting that GJs contribute to network activity. However, the lack of selective GJ blockers prohibits the determination of their site of action, i.e. neuronal versus glial. Astrocytes are strongly coupled through GJs and have recently been shown to modulate synaptic transmission, yet their role in neuronal network activity was not analysed. The present study investigated the effects and site of action of the GJ blocker, carbenoxolone (CBX), on neuronal network activity. To this end, we used cultures of hippocampal or cortical neurons, plated on astrocytes. In these cultures neurons display spontaneous synchronous activity and GJs are found only in astrocytes. CBX induced in these neurons a reversible suppression of spontaneous action potential discharges, synaptic currents and synchronised calcium oscillations. Moreover, CBX inhibited oscillatory activity induced by the GABAA antagonist, bicuculline. These effects were not due to blockade of astrocytic GJs, since they were not mimicked nor occluded by endothelin-1 (ET-1), a peptide known to block astrocytic GJs. Also, these effects were still present in co-cultures of wild-type neurons plated on astrocytes originating from connexin-43 (Cx43) knockout mice, and in neuronal cultures which contain few isolated astrocytes. CBX was not likely to exert its effect through neuronal GJs either, as immunostaining for major neuronal connexins (Cx) as well as dye or electrical coupling, were not detected in the different models of cultured neurons examined. Finally while CBX (at 100 microM) did not modify presynaptic transmitter release and postsynaptic responses to glutamate, it did cause an increase in the action potential threshold and strongly decreased the firing rate in response to a sustained depolarising current. These data demonstrate that CBX does not exert its action on network activity of cultured neurons through astrocytic GJs and suggest that it has direct effects on neurons, not involving GJs.

Developmental regulation of polyglutamylated alpha- and beta-tubulin in mouse brain neurons.

Polyglutamylation is an important posttranslational modification of tubulin that is very active in nerve cells, where it accounts for the main factor responsible for tubulin heterogeneity. In the present work, we have analyzed quantitative and qualitative changes in glutamylated alpha- and beta-tubulin occurring during neuronal differentiation in culture. Glutamylated alpha- and beta-tubulin both markedly accumulate during this process with a time course remarkably similar to that observed in vivo during brain development. However, the characteristics of the glutamylation of the two subunits are not exactly the same. Glutamylated alpha-tubulin is already abundant in very young neurons and displays, at this stage, a wide range of its degree of glutamylation (1 to 6 glutamyl units present in the lateral polyglutamyl chain), which remains unchanged during the entire period of the culture. Glutamylated beta-tubulin is present at very low levels in young neurons and its accumulation during differentiation is accompanied by a progressive increase in its degree of glutamylation from 2 to 6 glutamyl units. Posttranslational incorporation of [3H]glutamate into alpha- and beta-tubulin decreases during differentiation, as well as the rate of the reverse deglutamylation reaction, suggesting that accumulation of glutamylated tubulin is accompanied by a decrease in the turnover of glutamyl units onto tubulin. Neuronal differentiation is also accompanied by an increase of other posttranslationally modified forms of tubulin, including acetylated and non-tyrosinatable alpha-tubulin, which can occur in combination with polyglutamylation and contributes to increase the complexity of tubulin in mature neurons.

Na+-channel-associated scorpion toxin receptor sites as probes for neuronal evolution in vivo and in vitro.

Purified neurotoxin II of the scorpion Androctonus australis Hector (ScTx) has previously been shown to bind specifically to the Na+-ionophore-associated, voltage-sensitive receptor sites of excitable cells. We have conducted binding studies, using high-specific-activity 125I-labeled ScTx, to detect and quantify the Na+-channel receptors on cells of the developing fetal mouse brain. In vivo, the onset of detectable specific binding is at 12 fetal days. The rate of receptor appearance is initially slow but increases sharply as of the 16th day of mouse ontogenesis. The mean number of receptors at 12 and 19 days is 120 and 20,000 per cell, respectively (i.e., 0.5 and 80 per square micrometer). When corrected for the fraction of cell population corresponding to putative neuroblasts and neurons, identified by immunofluorescence as tetanus toxin binding cells, these values are, respectively, 1040 and 33,900 ScTx receptors per tetanus toxin binding cell or 4.2 and 136 per square micrometer. At all stages, the toxin binds to a single class of noninteracting sites; Kd = 0.1-0.5 nM. Similar findings in terms of ScTx-receptor properties and quantitative evolution were obtained in vitro. Specific 125I-labeled ScTx binding the presence of tetanus toxin binding cells. In cultures of central nervous system glia without neurons, only nonspecific low-level ScTx binding was detected. These results suggest that the high-affinity scorpion toxin receptors may be used as quantitative markers of neuronal differentiation.

Dystrophin gene transcribed from different promoters in neuronal and glial cells.

It has been shown that the dystrophin gene, which is defective in patients with Duchenne and Becker muscular dystrophy (reviewed in ref. 1), is transcribed in brain from a specific promoter that is different from the one used in muscle, and so the two types of transcripts differ at least in their first exon. We recently found that the dystrophin gene is expressed at a higher level in primary cultures of neuronal cells than in astro-glial cells derived from adult mouse brain. Here we investigate the use of two different promoters in each cell type. Our results demonstrate that the brain-type promoter of the dystrophin gene is highly specific to neurons, in which there is a significant increase in the amount of brain-specific messenger RNA during the course of in vitro maturation. By contrast, the muscle-type promoter is active in a wider range of cell types, including not only striated and smooth muscle, but also glial cells to a lesser extent, and probably neurons.

Posttranslational modifications of tubulin in cultured mouse brain neurons and astroglia.

Posttranslational modifications of tubulin were analyzed in mouse brain neurons and glia developing in culture. Purified tubulin was resolved by isoelectric focusing. After 3 weeks of culture, neurons were shown to express a high degree of tubulin heterogeneity (8 alpha and 10 beta isoforms), similar to that found in the brain at the same developmental stage. Astroglial tubulin exhibits a less complex pattern consisting of 4 alpha and 4 beta isoforms. After incubation of neuronal and glial cells with 3H-acetate in the presence of cycloheximide, a major posttranslational label was found associated with alpha-tubulin and a minor one with beta-tubulin. The acetate-labeled isotubulins of neurons were resolved by isoelectric focusing into as many as 6 alpha and 7 beta isoforms, while those of astroglia were resolved into only 2 alpha and 2 beta isoforms. The same alpha isoforms were also shown to react with a monoclonal antibody recognizing selectively the acetylated form(s) of alpha-tubulin. Whether acetate-labeling of alpha-tubulin in these cells corresponds to the acetylation of Lys40, as reported for Chlamydomonas reinhardtii, is discussed according to very recent data obtained by protein sequence analysis. Tubulin phosphorylation was analyzed by incubation of cell cultures with 32PO4. No phosphorylation of alpha-tubulin isoforms was detected. A single beta-tubulin isoform (beta'2), expressed only in neurons, was found to be phosphorylated. This isoform is similar to that previously identified in differentiated mouse neuroblastoma cells.

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

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.

Neurotoxin-sensitive sodium channels in neurons developing in vivo and in vitro.

Fetal mouse brain cells were investigated by 22Na+ flux assays with the aim to determine the ontogenetic time course of appearance of functional voltage-sensitive sodium channels. Their pharmacological properties were assessed by measurement of the response to known neurotoxins, acting at site 1, 2, or 3 of the Na+ channel. Brain cell suspensions, prepared at 11-19 d of prenatal development in vivo, and fetal brain neurons in culture were explored. In vivo neurotoxin-sensitive Na+ influx becomes detectable at 12 d of gestation, in concordance with the time of appearance of saturable binding sites for alpha-scorpion toxin (alpha-ScTx) and saxitoxin. Progression in fetal age or in time in vitro is accompanied by an increase in the initial rate and in the amplitude of Na+ uptake stimulated by batrachotoxin or veratridine. The general pharmacological properties of developing Na+ channels are very similar to the known properties of voltage-dependent Na+ channels in adult nerve: Batrachotoxin acts as a full channel agonist and veratridine as a partial agonist. Their respective apparent affinities are increased in presence of alpha-ScTx, in agreement with the known positive cooperativity of toxins acting at sites 2 and 3 of the Na+ channel. alpha-ScTx alone induces a small increase in Na+ permeability; its effect is greatly amplified in the presence of batrachotoxin or veratridine. The apparent affinity of alpha-ScTx is reduced by cell depolarization. Tetrodotoxin and saxitoxin block the increase in Na+ permeability induced by batrachotoxin, veratridine, and alpha-ScTx.(ABSTRACT TRUNCATED AT 250 WORDS)